Ferritin As An Indices of Body Iron Stores and Acute Myocardeal Infarction in Diabetics

Wednesday, July 22, 2:00 pm - 4:30 pm
Clinical Studies/Outcomes
the plasma levels of these vitamins but most significant is the effect of maternal age on plasma level of vitamin C. Mean plasma vitamin C and E in the pregnant cases and controls Pregnant Pregnant Pregnant Non-pregnant Pwomen women women women value (N=60) (N=60) (N=60) (N=20) Plasma levels 1st 2nd 3rd Controls of vitamins Trimester Trimester Trimester Vitamin C 1.750.36 1.760.38 1.600.37 2.10.41 <0.0001 (microg/ml) Vitamin E 0.900.39 1.870.57 0.880.36 2.310.42 <0.0001 (mg/dl)
Wednesday PM, July 22

Poster Session: 2:00 pm - 4:30 pm Clinical Studies/Outcomes
D-1 The influence of nocturnal oxyhaemoglobin desaturation to fibrinolitic dysfunction in hypertensive patients D. Begovic, B. Pencic, V. Celic, M. Dekleva, Z. Caparevic, N. Kostic, R. Cvetkovic. Clinical Health Center Dr Dragisa Misovic, Belgrade, Serbia
Recent study confirmed that plasma levels of PAI-1 have positive correlation with blood pressure in both sexes. Nocturnal oxyhaemoglobin desaturation has been also recognized as potentional new risk factor for arterial hypertension. The aim of our study was to evaluate the relation between PAI-1 and nocturnal oxyhaemoglobin desaturation in hypertensive patients before and after exercise test. Study included 40 patients with essential hypertension, age< 65 without heart failure or pulmonary diseases. Patients were divided according the oxyhaemoglobin desaturation in two groups: group I - patients with oxyhaemoglobin saturation index (ODI)5/h and group II - patients with oxyhaemoglobin saturation index (ODI)< 5/h. Groups were similar in demographic and clinical characteristics.Two days before exercise test all antihypertensive medica-mentations were withdrawn. In all patients we determinated body mass index, total cholesterol,LDL- cholesterol and HDL- cholesterol. PAI-1 (U/ mL) was measured by spectrophotometric method in plasma one hour before exercise and immidiately after the recovery phase. Patients in groupI had significantly higher PAI-1 levels before exercise ( 3.281.00 U/mL ) than patients in group II ( 2.36 0.71 U/mL). After exercise PAI-1 levels were slightly, but not significantly more decreased in group II ( p= 0.41) than in group I ( p= 0.898).The final model of the stepewise multiple regression analysis showed that nocturnal oxyhaemoglobin desaturation was independent risk factor for increasing PAI-1 levels before ( B=2.806, Beta standardized coefficient = 0.227, p=0.000) and also affter exercising ( B= 0.122, Beta standardized cofficient= 0.727, p=0.030). Our results confirm that nocturnal oxyhaemoglobin is likely to be predictive for increasing PAI-1 levels in patients with essential hypertension.
D-3 Investigation of Inflammatory Markers in Cerebrospinal Fluid from Infectious and Non-Infectious Human Brain Disease L. L. Wen1, C. Liang2, J. Wang3. 1En Chu Kong, Taipei Hsien, Taiwan, 2Far Eastern Memorial Hospital, Taipei Hsien, Taiwan, 3National Defense Medical Center, Taipei, Taiwan
Inflammation in the brain was associated not only with infections but also with neurodegeneration (Alzheimers disease, Parkinsons disease, and stroke). Recent evidence suggested that protein concentration changed in cerebrospinal fluid (CSF) during central nervous system inflammation. In this study several markers were examined. The following were investigated: C-reactive protein (CRP): serum amyloid A (SAA); haptoglobin (HPT); tumor necrosis factor-alpha (TNF-); interleukin-1b (IL-1) and IL-6 due to inflammation in CSF of bacterial meningitis and stroke. CSF samples were taken from three different groups of patients, classified as the following: (1) infection (bacterial meningitis; n=11) (2) non-infection (stroke; n=17) (3) normal control group (n=18). The levels of change in concentration within the markers were assessed. SAA, CRP and HPT were determined using BN II nephelometer (Dade Behring Inc., Newark, DE, USA). The concentrations of TNF-, IL-1 and IL-6 were measured by chemiluminescence immunoassay with IMMULITE automated analyzer (Diagnostic Products Corp., Los Angeles, CA, USA). The preliminary data indicated that the relative change (folds of control) of IL-6 in CSF of meningitis was the highest compare to the other markers. Immunohistochemical staining studies also revealed elevated expression of IL-6 in activated of leukocytes of CSF were activated during cerebral infection by bacteria. Although the CRP was also increased in meningitis and stroke patients, the CRP showed no significant difference between these groups. These results suggested that TNF-, IL-1 and IL-6 in CSF could be important as useful diagnosis markers for infectious brain disease (bacterial meningitis).
D-2 THE EFFECTS OF SOCIO DEMOGRAPHIC FACTORS ON PLASMA ASCORBIC ACID AND ALPHA TOCOPHEROL ANTI OXIDANTS DURING PREGNANCY IN NIGERIA WOMEN S. E. Idogun1, P. Aikoriogie2. 1University of Benin Teach Hospital, Benin, Nigeria, 2University of Benin Teaching Hospital, Benin, Nigeria
Background: Plasma Ascorbic acid (vitamin C) and Alpha-tocopherol (vitamin E) are antioxidants known to alter during pregnancy. Objectives: Was to assess the plasma levels of vitamins C and E at the various stages of pregnancy and to correlate the plasma levels of both vitamins with some sociodemographic factors of pregnant Nigerians. Setting: University of Benin Teaching Hospital, Nigeria. Study Design: A cross-sectional study. Patients and Methods: Patients were randomly recruited from the antenatal clinic according to the gestational ages. The controls were non-pregnant women of the same age range, selected from the gynecology clinic. Blood specimens were collected from both the cases and controls for the assay of both vitamins. Ascorbic acid was analyzed using the 2,4 Dinitrophenyl hydrazine photometric method and hexane extraction method was used for Alpha-tocopherol anlysis. Results: A total of 180 pregnant cases and 20 controls were studied. The mean plasma vitamins C and E in the pregnant cases and controls are shown in table 1.The correlation of the mean plasma vitamins Cand E versus the parity of the cases was not significant; r=0.013, P>0.05; r=0.047, P>0.05, for vitamins C and E respectively.The mean plasma level of vitamin C was highest in the farmers and lowest in the house wives, although this was not significant; P>0.05. However, the correlation of vitamin C versus maternal age showed a significant negative relationship; r= -0.59, p<0.05; the mean plasma level of vitamin C declining with increase in maternal age. Conclusion: The mean plasma Ascorbic acid and Alpha-tocopherol are reduced during pregnancy and the plasma vitamin E decline from first trimester and became significantly low in the third trimester. Sociodemographic factors have mild effects on
D-4 Association of Vitamin D receptor gene polymorphisms with Multiple Sclerosis in an Indian Population S. Vivekanandhan1, M. Murali1, N. Sonali1, V. Sneha1, M. Tripathi2. 1Neurobiochemistry, All India Institute of Medical Sciences, New Delhi, India, 2Neurology, All India Institute of Medical Sciences, New Delhi, India
Purpose: Multiple Sclerosis is a chronic, immune-mediated inflammatory and neurodegenerative disease of the central nervous system (CNS), with an etiology that is not yet fully understood. The prevalence of MS is highest where environmental supplies of vitamin D are lowest. The present study was undertaken to investigate VDR gene variation using three intragenic restriction fragment length polymorphisms (Apa I, Taq I and Fok I) in an Indian MS case-control population. Methods: 40 Multiple Sclerosis patients and 40 age, sex and ethnicity matched controls were investigated for VDR variants (Taq I, Apa I and Fok I) genotypes using polmerase chain reaction -restriction fragment polymorphisms (PCR-RFLP). Allele frequencies were derived from genotypic data. Case-control comparisons were made using Chi-square tests. Deviations from the Hardy-Weinberg equilibrium were also tested. Results: The VDR Variants Taq I, Apa I and Fok I polymorphisms was seen to be in Hardy-Weinberg equilibrium and showed significant genotypic and allelic association between the case and control groups for the functional exon 9 VDR Taq I polymorphism. Genotypic: P = 0.05; OR = 0.39 (0.13-1.15), Allelic: P = 0.008; OR =2.79 (1.20-6.58). The Apa I and Fok I polymorphisms showed significant allelic association .Allelic : P = 0.0431,OR = 2.13(0.96-4.75), P= 0.05,OR= 2.15(0.93-5.04)
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CLINICAL CHEMISTRY, Vol. 55, No. 6, Supplement, 2009
but no significant genotypic frequencies between MS and controls (P=0.174, OR = 0.54). Conclusion: Our findings suggest that significant role of VDR gene polymorphisms in the risk of developing multiple sclerosis in an Indian population.

cirrhosis. In Asia, 90% of liver cancer patients are due to chronic HBV infection. Over 300 million HBV patients are in Asian and Number one cause of cancer death. Chronic liver fibrosis is a slow-developed liver injury. It represents a wound healing process responding to hepatocellular demages due to HBV, HCV infections or other causes. It will develop into liver cirrhosis or hepatocarcinoma. The time to develop these severe liver conditions takes 20 to 30 years. There are many articles indicated the disease is treatable during the first 5-15 years of development. When liver cirrhosis symptom is presented, it is often not treatable or curable any more. Therefore, early detection of chronic liver fibrosis is very significant clinically. HBV is a major infectious disease in Asia. Over 300 million HBV patients are residing in Asia. It is the number one cause in cancer death. Diagnosis of chronic liver fibrosis: Chronic liver fibrosis is traditionally diagnosed by liver biopsy then starting treatment. Due to the severe adverse effects of HCV treatment (chronic flu-like symptom), it is recommended to treat patients with liver fibrosis Stages II or above. In United States, less than 10% of patients with chronic HCV accept liver biopsy. Therefore, many HCV patients are not treated. Synopsis for current study: In recent years, several blood testing methods are developed. Fibrometer, Fibrosure (Fibrotest), APRI, Hepascore, and many more are used in US and Europe. Among them, Fibrometer has the best performance based on literature reports. Fibrometer is developed using only Caucasian HCV, HBV patients as study subjects. Our study is to evaluate results of Fibrometer performance among Asian patient populations who has chronic hepatitis B or hepatitis C, compare them to the results of Caucasian samples. Study methods: Fibrometer uses the following 9 assays- glucose, bilirubin, AST, ALT, GGT, Ferritin, Urea, Hyaluronic Acid (HA), and Alpha 2 Macroglobulin (A2M) plus Prothrombin index and platelets. Prothrombin time and platelets are done at site of sample collection. The biochemical tests use Roche Hitachi and Elecsys instruments. Fibroscan, an ultrasound based liver elasticity measuring instrument was also studied as a reference method to compare to the accuracy of Fibrometer. Results: The study results indicated that Fibrometer is an excellent test for hepatitis B induced chronic liver fibrosis measurement. The AUROC of HCV and HBV are comparable at 0.82 vs. 0.81. There are no significant differences between the two applications in diagnostic accuracy (0.87%). The diagnostic accuracy of Fibrometer is closely correlated to Fibroscan but Fibrometer has a much superior accuracy (1.5x in Index alpha ratio) for the clinically significant (Metavir Stages II and above). Conclusion: Based on this study, Fibrometer is an excellent tool to assess the staging of Chronic Liver Fibrosis/Cirrhosis in Asian Patients who has chronic liver fibrosis caused by either HBV or HCV.
D-5 Atypical cells in the morning fresh urine, a and their possible significance S. Giju1, C. Flangea2, V. Dumitrascu3, L. Petrica4, D. Vlad1, S. Ursoniu5. 1Counnty Emergency Hospital, Timisoara, Romania, 2Victor Babe University of Medicine and Pharmacy, Biochemistry Department, Timisoara, Romania, 3Victor Babe University of Medicine and Pharmacy, Pharmacology Department, Timisoara, Romania, 4Victor Babe University of Medicine and Pharmacy, Department of Nephrology, Timisoara, Romania, 5Victor Babe University of Medicine and Pharmacy, Department of Public Health, Timisoara, Romania
Objective: The main goal of our study was to demonstrate the importance of the assessment of urinary atypical cells in the urinary sediment. Furthermore, we attempted to evaluate the significance of atypical cells according to their importance. In our study, we emphasize that atypical cells, even if they are 1/lpf, have diagnostic importance. Relevance: When atypical cells are observed in the urinary sediment from spontaneous urine, it is necessary to know the conditions of their appearance, as well as their origin and significance. We proposed to us to emphasize in the urinary sediment the atypical cells, as well as the presence of other cells, which indicate various tumoral diseases (renal or non renal). To avoid any possible confusions, we used the parallelism of microscopic techniques and when necessary, the stained and unstained samples. Each photo belongs to one patient. In some circumstances we took photos (to eliminate any doubt) of the same atypical cells with different types of microscopy. We can warn the patient and recommend a pathological examination. The techniques proposed by us are not expansive and they dont last long. An experimented microscopist is required. Methodology: In these studies the urinary sediment was screened at a low power (x 100) and thereafter the suspect structures were examined at a higher power (x 400). Initially we worked on a normal microscope but with reduced light with the condenser placed at a lower position to increase the contrast. After that we used a phase contrast microscope, more suitable for the study because atypical cells are relatively difficult to identify using just the traditional bright field microscopy. The investigation was performed by using different microscopic techniques in the observation of the sediment elements in both unstained and stained samples (May-Grnwald-Giemsa stain and Sternheimer-Malbin stain). Validation: The purpose of our study is to show the diagnostic importance of atypical cells and also the high relevance of other types of cells. We studied a lot of 62 patients admitted to the hospital, most of them in different clinics of the County Emergency Hospital No.1 Timisoara. Due to the rarity of the cases, the study lasted almost three years (February 2005 - January 2008). For urinalysis we used either the first or the second morning urine specimen. The microscopic examination revealed the presence of urinary atypical cells. To confirm our findings, we took photos of the same cast with different types of microscopy. The other types of cells are diagnosis elements too. Conclusions: To emphasis the atypical cells between the slide and cover slip (stained and unstained), as well as in the smears, is a cytoprevention element, because a simple suspicion of the presence of atypical cells determine us to recommend a pathological examination. We say that because this department is the only one which can certainly confirm or not the tumoral cells presence. The microscopic study of the atypical cells is a non - invasive and accurate method. It has the advantage of decreasing costs and eliminating the risks associated with invasive methods.
D-7 Does Proteinuria Justify a Microscopic Examination of the Urine Sediment (MEUS)? C. Puza1, E. S. Pearlman2. 1South Boston Community Health Center, Boston, MA, 2Boston Medical Center, Boston, MA
A widely adhered protocol reflexes to a MEUS if urine dipstick chemistries (UDCs) are positive for hemoglobin (H), leukocyte esterase (LE), nitrite (N) or protein (P). We queried whether reflex MEUS was warranted when, of the above four analytes, only P was positive by UDC. We examined this issue by assessing data on 110 consecutive specimens from ambulatory patients seen at two community health centers in Boston. UDCs were assessed using a Status urinalysis instrument (Siemens: Tarrytown NY) and were all negative for H, LE and N but were all =/> 1+ positive for P. The population consisted of 67 M and 43 F (z score=2.27 versus equal gender representation; p<.03 [2-tailed]). The median (range) M age was 49 YO (2 mos.-91 YO) and for Fs was 24 YO (7-85 YO) (p<.005; 2-tailed median test). Review of the electronic medical record (EMR) indicated that proteinuria or renal insufficiency was part of the patient problem list in 22 instances (20%). Associated conditions (number of patients) possibly relevant to occurrence of proteinuria included hypertension (35), diabetes mellitus (21) hepatitis B/C (10), pregnancy (9) renal calculi (3) and gout (2). Relevant associated conditions were absent in 45 patients (41%). The magnitude of the proteinuria was 1+ (n=85), 2+ (n=23) and 3+ (n=2). Simultaneous urine culture (UC) was obtained in 41 patients but only one revealed =/> 10,000 CFU/mL of a single organism. Repeat UDCs were obtained in 33 patients within 6 months of the initial UDC and P reverted to negative/trace in 23 instances. A microalbumin/ creatinine ratio (MACR) was obtained within 6 months in 33 patients and was within the reference interval (WRI) in 20. Serum creatinine concentration was determined within 6 months in 64 patients and was WRI in 56 and was =/> 2 mg/dL in only 1 patient. Potentially significant microscopic findings [Ringsrud & Linne, Urinalysis and Body Fluids (1995)] were not identified in 87 patients (79%). In the remaining patients findings included 6-10 WBC/hpf (10), 6-10 RBC/hpf (5), 11-15 RBC/hpf (1), 10-30 epithelial cells/hpf (11), 10-30 hyaline casts/lpf (2), 2-5 granular casts/lpf (1)
D-6 Evaluation of Chronic Liver Fibrosis Test, "Fibrometer" In Asian Population H. H. Liu1, C. Cheng2. 1Taiwan Specialty Reference Laboratory, Taipei, Taiwan, 2National Taiwan University Hospital, Taipei, Taiwan
Disease background: Chronic liver fibrosis is a chronic liver disease caused mainly by Hepatitis B, Hepatitis C, fatty Liver, and chronic alcoholic consumptions. Over 20% of chronic HBV patients and 85% of HCV patients eventually develop liver
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and 3+ bacteria (1) [UC was negative in this case]. From review of the EMR over the six months subsequent to the initial MEUS however, it was unclear how MEUS findings specifically impacted patient management. Review of the EMR indicated that when the finding of proteinuria was identified in provider notes the usual follow-ups were repeat of the UDC, MACR or serum creatinine. No specific reference to the MEUS in clinician/provider notes was identified. We concluded after review of the above results that the yield in respect to data that would influence patient management did not justify continuation of the MEUS in P only specimens and a decision was made to reflex to an MACR in such cases.
as blank control. Dry and wet chemical methods were used to test each sample 4 times. Result: The Cr result of the serum mixture that with 0mg/L Etamsylate was 67.330.43mol/L in dry chemistry and it was 66.230.13mol/L in wet chemistry. When added 300mg/L Etamsylate(peak drug level), the Cr result was 40.780.46mol/L in dry chemistry and 17.350.39mol/L in wet chemistry. When added 150mg/L Etamsylate, the Cr result was 50.70.36mol/L in dry chemistry and 22.880.56mol/L in wet chemistry. When the drug Etamsylate that added into the serum mixture was 9.38mg/L, the Cr result in dry chemistry was 66.21.12mol/L and there was no difference between results of dry chemistry and 0mg/L group(without Etamsylate, true value, p<0.01); when the level of Etamsylate is 1.17mg/L, the Cr result in wet chemsitry was 66.130.59mol/L and it had no difference with 0mg/L group(without Etamsylate, true value, p<0.01). Discussion: Etamsylate in curable level does have inhibition effect on assaying creatinine using enzymatic methods; when presented in blood, Etamsylate decreases Cr results. But the inhibition effect of Etamsylate fades with dose-dependent. To test Cr in laboratory, it can be concluded from this study that at least 11 hours after treatment with Etamsylate, patient's blood is available for creatinine test and then its result is reliable.
D-8 Diagnostic Efficiency of Amylase and Type IV Collagen in Predicting Chronic Pancreatitis S. K. Das1, V. Balakrishnan2, D. M. Vasudevan3. 1Agartala Govt Medical College, Agartala- 799006, India, 2Amrita Institute of Medical Sciences, Cochin 682 026, India, 3Amrita Institute of Medical Sciences, Cochin 682026, India
BACKGROUND: Chronic pancreatitis, an irreversible inflammatory disease of the pancreas, is associated with the replacement of the destroyed parenchyma by extended development of fibrosis. The biological cause of fibrosis is the accumulation of excessive amounts of extracellular matrix, which leads to tissue dysfunction and organ failure. There is growing evidence that pancreatic fibrosis represents a dysregulation of the normal repair processes after injury. The severity of pancreatic fibrosis can be assessed only by direct histologic analysis of pancreatic tissue. The anatomic position and relationships of the pancreas make direct observation of pancreatic pathology difficult. OBJECTIVE: Therefore, we examined the hematological and common biochemical parameters in serum of chronic pancreatitis patients and analyzed their association, in particular with serum amylase and type IV collagen levels. METHODS: A complete clinical history, including anthropometric measurements; haematological and biochemical investigations including renal function tests, liver function tests and lipid profile were performed among forty (40) chronic pancreatitis patients within 18 to 67 yrs. Type IV collagen level in serum was measured by latex turbidometric immunoassay. RESULTS: ESR level and alkaline phosphatase (ALP) activity elevated in 40% cases. Hyperglycemia was observed in 45% patients Though serum calcium level decreased in 25% patients and serum amylase activity increased in 80% patients; it showed significant correlation with ALP (r=0.458, p=0.003), CA-19.9 (r=0.556, p<0.001), and calcium level (r= -0.472, p=0.002). Type IV collagen level in chronic pancreatitis patients also elevated (164.4 55.5 ng/ ml) and showed significant negative correlation with calcium level (r= -0.505, p=0.001). However, no significant correlation was observed between amylase activity and type IV collagen (r=0.289, p= 0.07). CONCLUSIONS: Pancreatic fibrosis is a key pathological feature of chronic pancreatitis and sometimes results in diabetes mellitus. The blood parameters such as amylase activity and type IV collagen level, and their correlations with hematocrit, calcium, alkaline phosphatase activity and CA-19.9 may be monitored in these patients. More study is necessary to identify the biomarker or combination of biomarkers for these patients.
D-10 The analysis of increasing Ca125 serum level in patients with CKD J. Gao, Y. Tian. Dept. of Clinical Biochemistry, Chinese PLA General Hospital, Beijing, China
Objective: To find out the reason of elevated Ca125 serum level in patients with chronic kidney disease (CKD). Relevance: The elevated tumor marker serum level such as CA125 in patients with CKD always confused the nephrologists. Either there was tumor exited in the patients or just an elevated level of tumor markers compared with chronic inflammation? Validation: 649 cases in our hospital were investigated retrospectively. The patients less than 70 years old with the diagnosis of CKD according to DOQI guideline were included in this study and the patients with tumor, rheumatic disease, tuberculosis, virus hepatitis or acute kidney disease were excluded. Methodology: The serum levels of 38 biochemical parameters including CA125, CA199, albumin (ALB), total cholesterol (TC), and triglyceride (TG) were detected with ROCHE modular analyzing system. Serum level of 35 U/ml was set as cutoff value of CA125, and all patients were divided into two groups. Data of two groups were collected and analyzed. Univariate analysis was performed to compare data difference of two groups. Binary logistic regression model (method was forward: conditional) was developed to assess factors affecting CA125 positive rate in CKD. CA125 serum levels were compared between IgA nephropathy and non-IgA nephropathy groups, between serousal effusion and no effusion groups and between hypoalbuminemia and no hypoalbuminemia groups. Results: The results showed there were significant differences in 24 parameters between CA125 positive group and negative group, including clinical diagnosis, pathological diagnosis, CA153, et al (P<0.01 or <0.05). Logistic regression analysis showed serousal effusion, pathological diagnosis and hypertension were main independent factors affecting CA125 serum positive rate (P were 0.001, 0.025 and 0.009; odds ratio were 21.141, 0.112 and 12.678; model log-likelihood values were 34.154, -29.562 and -31.105). CA125 serum level of IgAN was lower than non-IgA nephropathy ( 11.2 U/ml vs 26.0 U/ml,P<0.01); of hypoalbuminemia group was higher than no hypoalbuminemia group (37.0 U/ml vs 11.1 U/ml, P<0.01); of serousal effusion group was much higher than no effusion group (154.4 U/ml vs 12.7 U/ml, P<0.01). Stratification analysis was used to calculate the correlation of CA125 serum level and ALB. The correlation coefficient of ALB with CA125 was -0.391 (P<0.01) when ALB serum level was less than 35 g/L. But it was only -0.085 (P>0.05) when ALB serum level was higher than 35 g/L. Conclusion: Patients with non-IgA nephropathy are at a high risk of getting elevated CA125 serum level especially complicating hypertension or serousal effusion. However hypoalbuminemia also is an important factor on it.
D-9 Analysis of the influence of Etamsylate on creatinine using enzymatic methods Q. YANG. Chinese PLA general hospital, Beijing, China
Background: Etamsylate is widely used in clinical treatment as an antihemorrhagic. During clinical work, it has been found that patients administrated with Etamsylate had poor results of creatinine(Cr)(using enzymatic methods), which later was found to be discrepancy to clinical symptoms. The Cr results in dry chemistry isn't consistent with results in wet chemistry(both are used enzymatic methods). So it might be Etamsylate that affects tests of Cr. Objective: This study is to investigate the influence of Etamsylate on enzymatic method assaying creatinine. Method: 20 healthy people's serum were collected and made into serum mixture, and it was divided into 10 groups , added with different doses of Etamsylate as 300, 150, 75, 37.5, 18.75, 9.38, 4.69, 2.34, 1.17and 0mg/L according to the maximum blood Etamsylate level. The same groups of saline were added the same levels of Etamsylate
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D-11 Association between chronic kidney disease and metabolic syndrome M. Orisaka1, K. Kawamura1, M. Nakamura2. 1Iwate Health Service Association, Morioka, Japan, 2Osaka Medical Center for Health Science and Promotion, Osaka, Japan
Renal disorders showing a chronic course are generically termed chronic kidney disease (CKD). Recently, its incidence has rapidly increased. The number of patients undergoing dialysis has also increased. In the presence of insulin resistance, proteinuria is frequent, commonly leading to CKD via kidney hypofunction. We investigated the relationship between CKD and metabolic syndrome (MS), which is closely associated with insulin resistance. Subjects: The subjects were 18,556 patients, excluding those undergoing chronic nephritis treatment. They consisted of 11,244 males (mean age: 54.611.3 years, range: 19 to 90 years) and 7,312 females (mean age: 55.311.4 years, range: 20 to 92 years). The estimated glomerular filtration rate (eGFR, mL/min/1.73m2) was calculated using the Modification of Diet in Renal Disease (MDRD) formula: 0.741 x 175 x Age-0.203 x creatinine-1.154 (females: x 0.742). Patients showing an eGFR below 60 were regarded as having CKD. Furthermore, MS subjects were selected according to a revision of the NCEP-ATP III criteria (2005). Concerning abdominal circumference, males with a circumference of 90 cm or more and females with that of 80 cm or more were included. Results: MS subjects consisted of 2,713 males (24.1%) and 1,611 females (22.0%). We examined incidences of CKD with respect to gender, age, and the presence or absence of MS. In males younger than 30 years, the incidences were 0.0 and 1.5% in the MS and non-MS groups, respectively. Among those aged 30 to 39 years, the values were 6.2 and 2.9%, respectively. For those aged 40 to 49 years, the values were 10.0 and 6.9%, respectively. Concerning those aged 50 to 59 years, the values were 14.1 and 9.9%, respectively. For those aged 60 to 69 years, the values were 15.8 and 12.9%, respectively. Among those aged over 70 years, they were 28.5 and 16.1%, respectively. In females younger than 30 years, the incidences were 0.0% in both the MS and non-MS groups. Among those aged 30 to 39 years, the values were 15.4 and 3.8%, respectively. For those aged 40 to 49 years, the values were 4.6 and 7.6%, respectively. Concerning those aged 50 to 59 years, the values were 8.5 and 5.9%, respectively. For those aged 60 to 69 years, the values were 9.1 and 5.5%, respectively. Among those aged over 70 years, they were 14.7 and 1.7%, respectively; the incidence of CKD was higher in the MS group at all ages regardless of gender. In males aged over 40 years, there were significant differences between the MS and nonMS groups (p<0.01). In males overall, the incidences of CKD in the MS and non-MS groups were 14.7 and 9.8%, respectively. In females overall, the values were 9.8 and 6.6%, respectively. Among both males and females, the incidence of CKD in the MS group was 1.5 times higher than in the non-MS group, with a significant difference (p<0.01). Conclusion:The incidence of CKD was significantly higher in the MS group, indicating the association between CKD and MS. MS strategies must be established, considering CKD.

damage and thus to reduce the incidence of end-stage renal failure and mortality. Continuing our previous studies (Voskaridou et al, Kidney Int, 69:2037-2042, 2006), we explored the activation of tubuloglomerular feedback in 58 adult patients (age 4217years, males/females 20/58) with HbS/-thal by measuring the above mentioned parameters, along with the more classical ones such us 2-microglobulin and N-acetyl--D-glucosaminidase (NAG) in blood and urine using standard methodology. GFR values were calculated according to the recently proposed cystatin C-based prediction equation using only each concentration in mg/L: GFR [mL/min/ 1.73m2] = 76.7 x Cystatin C-1.18. The main results of the study were: a) impairment of GFR in 21/58 patients (36%); b) increased plasma concentrations of NGAL and IL-18 in 34/58 (58%) and 58/58 (100%) patients, respectively; c) increased and/or detectable urine concentrations of NGAL and IL-18 in 54/58 (93%) and 46/58 (79%) patients, respectively; d) significant negative correlations between GFR and plasma NGAL and IL-18 (r=-0.735, p<0.0001 and r=-0.350, p<0.007, respectively) and e) significant positive correlations between cystatin C and urine NGAL and IL-18 (binomial, p<0.005 and r=0.412, p<0.03, respectively). These findings suggest that tubuloglomerular feedback is activated in almost all studied patients with HbS/thalassemia. Measurements of plasma and urine NGAL and IL-18 contribute significantly to the early detection and risk stratification of renal disease in these patients. However, prior to their clinical usefulness, these biomarkers must undergo through rigorous validation in multiple cohorts.
D-13 Development of a System Check (Prozone Check) for Total Bilirubin Test (BILTS) on Roche cobas c 311 and cobas c 501 Systems H. Klima1, B. Loehr2, G. Sobczak1. 1Roche Diagnostics GmbH, Penzberg, Germany, 2Roche Diagnostics GmbH, Mannheim, Germany
Introduction: Interference of paraproteins in diagnostic tests is a well known problem in the routine clinical laboratory. Due to poor solubility of immunoglobulin proteins the samples become turbid during analysis. This turbidity interferes with the reaction kinetics of analyte determination. This interference seems to be independent from the concentration and from the type of immunoglobulin in the patient sample. It is known from the literature and from internal investigations at Roche Diagnostics that in rare cases patient samples containing paraproteins give falsely elevated results in the Total Bilirubin liquid test (BILTS) on Roche cobas c 311 and cobas c 501 analyzers. Objectives: In order to identify falsely elevated results in patient samples with untypical reaction kinetics a system check, so called prozone check, was implemented in the applications of BILTS on Roche cobas c 311 and cobas c 501. Due to difference in the applications, two different system specific checks detecting abnormal reaction kinetics in patient samples were developed. The BILTS applications with and without prozone checks are currently evaluated under routine conditions using routine clinical laboratory samples in order to detect falsely flagged samples. Study Design: In this study we analyzed whether applications for total bilirubin show falsely elevated results for Total Bilirubin in gammopathic samples. Patient samples, n = 24, with known interference in the current BILTS test on cobas c 311 and cobas c 501 systems were used for development and verification of the prozone check. Materials and Methods: The Serum I Index and the measurement with the Roche BILT2 liquid reagent were used as reference methods for verification of total bilirubin concentration in discordant samples. Immunoglobulin concentration were measured with Roche IgA, IgG and IgM assays on cobas c 501 system. Results: The measurements of samples with known paraprotein interference has proven that the prozone check on cobas c 311 and cobas c 501 analyzers allows to detect interference from gammopathic samples. A flag (>kin) occurred with all test results for BILTS application having abnormal reaction kinetics. Discordant test results in patient samples were confirmed by two independent methods for total bilirubin in all paraprotein samples. The bias in the bilirubin value measured with the BILTS assay and the reference methods was found in the range of 17.6 mol/l to 704. 3 mol/l in the patient samples. Conclusions: The reliability of BILTS applications was markedly improved with implementation of the prozone check. The check can be used for detection of patient samples with abnormal reaction kinetics. All samples tested were marked by the prozone check. Although it cannot be ruled out that these modified applications might miss patient samples with very special kinds of paraprotein. The prozone check for BILTS applications will be released in 2009 after the external evaluation has been completed.
D-12 INCREASED PLASMA LEVELS OF NGAL AND IL-18 CORRELATE WITH REDUCED GFR PROVIDING EVIDENCE OF TUBULOGLOMERULAL DEFECT IN PATIENTS WITH HbS/ THALASSEMIA I. Papassotiriou1, A. Margeli1, E. Hantzi1, E. Terpos2, E. Voskaridou3. 1Department of Clinical Biochemistry Aghia Sophia Childrens Hospital, Athens, Greece, 2Department of Medical Research, 251 General Airforce Hospital, Athens, Greece, 3Thalassemia Center, Laikon General Hospital, Athens, Greece
Promising novel biomarkers for acute kidney injury (AKI) or chronic kidney injury (CKI) include a plasma levels of cystatin C, Neutrophil Gelatinase-associated Lipocalin (NGAL) and Interleukin-18 (IL-18) and urinary levels of NGAL, IL-18 and Kidney Injury Molecule-1 (KIM-1). These biochemical indices have been reported to be useful for evaluating the duration and severity of kidney disease and for predicting progression and adverse clinical outcomes. Furthermore, these biomarkers have given promising results in differentiating the various causes of AKI or CKI. Progressive renal failure is one of the main complications in HbS/-thalassemia (HbS/-thal). Early identification of patients at high risk of developing renal failure is of great importance as it may allow specific measures to delay the progression of renal
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D-14 A Comparative Evaluation of the Clinical Utility of the Premier Enzyme Immunoassay (for IgG) and the Meridian Immunodiffusion assay (for F antigen) in the Diagnosis of Coccidioidomycosis. C. M. LaPolt1, E. I. Blackman2. 1University of California Berkeley, Berkeley, CA, 2Thousand Oaks Pathology Associates, Thousand Oaks, CA
Objective: The objective of this study is to compare the clinical utility of the Premier Enzyme Immunoassay for IgG (P-EIA) versus the Meridian Immunodiffusion assay for F antigen (M-ID) for the diagnosis of Coccidioidomycosis in a community hospital setting. Methods: We studied 472 consecutive specimens (from 447 patients) with orders for diagnostic serology for Coccidioidomycosis. P-EIA and M-ID were performed according to manufacturers specifications. Definitive testing at a specialized referral lab (SRL), unrelated to our institution, was performed for 78 of these specimens (from 78 patients), including all specimens with a discordance between the P-EIA and M-ID results. Samples were assigned a reference diagnosis based on the SRL results or on concordant P-EIA and M-ID results. SRL results were considered positive if: the original specimen was positive by Complement Fixation (CF) or Immunodiffusion for IgG (ID-CF); or the original specimen was positive by Immunodiffusion for IgM (ID-TP) and a subsequent specimen was positive by CF or ID-CF within 60 days of the original sample. Sensitivity, specificity, and positive and negative predictive values were calculated for P-EIA and M-ID based on a comparison to the reference diagnosis. Statistical significance was determined by the z test, with a significance level of 0.05. Results: The reference diagnosis was positive for 22 specimens (from 22 patients) and negative for 450 specimens (from 425 patients). Sensitivity, specificity, and positive and negative predictive values were as follows: Sensitivity Specificity Positive Predictive Value Negative Predictive Value P-EIA 21/22 (95.5%) 442/450 (98.2%) 21/29 (72.4%) 442/443 (99.8%) M-ID 18/22 (81.8%) 448/450 (99.6%) 18/20 (90.0%) 448/452 (99.1%) p-value ns ns ns ns
levels of these two groups were 81.531.6 and 9.187.6 respectively (P<0.001). Urinary NGAL has shown an inverse correlation with eGFR (R=-0.607, P<0.05) and a correlation with time from transplantation (R=0.622, P<0.05). Analytically, the Architect NGAL assay demonstrated a good precision with CVs of 4.35, 2.2%, 2% at levels of 21, 393, and 1192 ng/ml. The Architect NGAL assay correlated very well with the ELISA method: Architect=1.0213xELISA-0.454 (R=0.992, P<0.001). Conclusion: NGAL is a novel marker for chronic CsA-induced nephrotoxicity after cardiac transplantation. Monitoring urine NGAL may help adjusting dosage of CsA to prevent irreversible renal damage. The performance of the Architect NGAL assay was satisfactory for routine laboratory practice. * in development
D-16 Serum TIMP-1 and HER-2/neu levels in metastatic breast cancer (MBC) treated with either letrozole or tamoxifen P. J. Hamer1, A. Lipton2, K. Leitzel2, L. Demers2, S. M. Ali3, D. B. Evans4, H. A. Chaudri-Ross5, S. Brown-Shimer1, K. Pierce1, W. P. Carney1, V. Gaur1. 1Oncogene Science/Siemens Healthcare Diagnostics, Cambridge, MA, 2Penn State University, Hershey Medical Center, Hershey, PA, 3Lebanon VA Medical Center, Lebanon, PA, 4Oncology Research, Novartis Pharma AG, Basel, Switzerland, 5Oncology Business Unit, Novartis Pharma AG, Basel, Switzerland
Purpose: This was a combined analysis of serum TIMP-1and serum HER-2/neu levels in MBC patients treated with an aromatase inhibitor, Letrozole versus tamoxifen to determine if levels of these biomarkers were associated with clinical outcome. Patients and Methods: Five hundred twenty-two patients estrogen receptor-positive MBC were randomly assigned to receive first-line hormone therapy with letrozole or tamoxifen. Serum tissue inhibitor of metalloproteinases-1 (TIMP-1) and serum HER2/neu pretreatment levels were measured using enzyme-linked immunosorbent assays for both biomarkers. Results: Among patients with available pretreatment serum, 298 patients had normal pretreatment serum levels of both TIMP-1 and HER-2/ neu (reference group), 72 patients had elevated serum TIMP-1 and normal serum HER-2/neu, 104 patients had normal serum TIMP-1 and elevated serum HER-2/neu levels, and 48 patients had elevated pretreatment serum levels of both TIMP-1 and HER-2/neu. Median time to progression (TTP) for all three patient groups with either or both serum biomarkers elevated was significantly reduced compared with that of the reference group (normal pretreatment serum levels of both TIMP-1 and HER- 2/neu; P< .0001). In this analysis by treatment arm, patients with normal serum levels of both TIMP-1 and HER-2/neu, median TTP for letrozole was 14.4 months versus 9.2 months for tamoxifen (P< .01). In patients with elevated TIMP-1 and normal HER-2/neu, median TTP for letrozole was 9.2 months versus 3.7 months for tamoxifen (P< .02). In patients with normal TIMP-1 and elevated HER-2/neu, median TTP was 7.4 months for letrozole versus 4.6 months for tamoxifen (P< .13). Finally, in patients with elevation of both TIMP-1 and HER-2/neu, median TTP was 3.2 months for both letrozole and tamoxifen (P<.45). Elevated versus normal serum TIMP-1 was associated with a significantly increased risk of death, both within the normal serum HER-2 subgroup (median survival, 26 v 42 months; P < .0006), and within the elevated serum HER-2 subgroup (median survival, 15 v 25 months; P<.01). Patients with elevated serum levels of both TIMP-1 and HER-2 had the worst prognosis, which was significantly shorter compared with all other subgroups (median survival, 15 months; P < .0001 compared to the reference group. We next compared overall survival for the four biomarker subgroups. Elevated versus normal serum TIMP-1 level was associated with a significantly increased risk of death, both within the normal serum HER-2 subgroup (median survival, 26 v 42 months; P < .0006), and within the elevated serum HER-2 subgroup (median survival, 15 v 25 months; P<.01). Patients with elevated serum levels of both TIMP-1 and HER-2 had the worst prognosis, which was significantly shorter compared with all other subgroups (median survival, 15 months; P < .0001 compared to the reference group and p = .01 for both remaining subgroups. Conclusion: Combined analysis of both serum TIMP-1 and HER-2/neu conferred additional ability to predict significantly different clinical outcomes compared to using either biomarker alone.
Conclusion: No statistically significant differences were observed in the clinical performance characteristics of the M-ID and P-EIA assays. P-EIA may be considered as an acceptable alternative to M-ID for the diagnosis of Coccidioidomycosis.
D-15 Urine neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for chronic cyclosporine nephrotoxicity after cardiac transplantation Y. Chen1, F. Gustafsson2, J. Sheedy2, H. Ross2, M. Liao3, Y. Wang3, P. Wong3. 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, 2Heart Failure and Transplant, Toronto General Hospital, Toronto, ON, Canada, 3University Health Network, Toronto, ON, Canada
Background: Cyclosporine (CsA) has improved allograft survival and quality of life of for solid-organ recipients. However, after over two decades of CsA use its chronic nephrotoxicity remains a significant clinical problem, which is the most important cause of renal dysfunction after cardiac transplantation. Urine neutrophil gelatinaseassociated lipocalin (NGAL) is an emerging renal biomarker for a variety of clinical situations leading to acute kidney injury or to chronic kidney disease. The aim of the present study is to investigate the role of NGAL measurement in chronic CsA-induced nephrotoxicity following cardiac transplantation. Methods: Eight male and three female heart transplant recipients (mean age 548) were recruited in the study. Patients were treated with the combination of CsA (11480 mg/day) with Everolimus (n=9, 1.580.38 mg/day) or Mycophenolate mofetil (n=2, 1500 mg/day or 3000 mg/day). Urinary NGAL were measured consecutively from 0 to 12 hours after CsA administration at 6 time points by an automatic NGAL assay* on Architect i2000 analyzer and a manual ELISA method (AntibodyShop, Denmark). On the day of study, serum creatinine was also measured for the estimated glomerular filtration rate (eGFR). Results: The urine NGAL levels peaked at 2 hr (C2) post CsA treatment in 3 patients with a concentration above 70 ng/ml and then gradually decreased to baseline level at C12. This pattern correlates with the general pharmacokinetics of blood CsA that peaks 2 hr after oral administration and reach a through at 12 hr. Urine NGAL concentrations in other eight patients did not show such increase. C2 urine NGAL
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D-17 Serum HER-2/neu levels in early stage HER-2/neu positive breast cancer (HER-2+BC). Results from the NCCTG adjuvant intergroup trial N9831 W. P. Carney1, A. Moreno-Aspitia2, A. Dueck3, W. Lingle4, L. Kutteh5, K. Tenner6, N. Davidson6, E. Perez2. 1Oncogene Science/Siemens Healthcare Diagnostics, Cambridge, MA, 2Mayo Clinic, Jacksonville, FL, 3Mayo Clinic, Scottsdale, AZ, 4Mayo Clinic, Rochester, MN, 5Oncology Associates of Cedar Rapids, Cedar Rapids, IA, 6Johns Hopkins Oncology Center, Baltimore, MD
Background: Serum HER-2/neu is the circulating extracellular domain ( p97Kdap115Kda) that is cleaved from full-length, membrane-bound p185 HER-2/neu protein. Previous studies support that increased serum HER-2/neu levels are strongly associated with poor prognosis and a predictor of poor response to chemotherapeutic and hormonal treatments in metastatic breast cancer patients. The role of serum HER2/neu levels in early breast cancer patients in the adjuvant setting was evaluated in study N9831. NCCTG N9831 is a randomized, phase III clinical trial comparing three drug regimens: doxorubicin/cyclophosphamide followed by paclitaxel with trastuzumab added concurrently, sequentially, or not at all as adjuvant therapy for women with HER2-positive resected breast cancer. Methods: The aims of the study was to analyze the association between baseline serum HER-2/neu and disease-free survival (DFS) in patients randomized to Arms A (standard chemotherapy) and C (standard chemotherapy with concurrent trastuzumab) of N9831. Baseline serum samples of 1423 patients from both arms were analyzed for serum HER-2/neu levels using the ADVIA Centaur HER-2/neu test (Siemens HealthCare Diagnostics). Normal levels of serum HER-2/neu is less than 15 ng/ml. . Patients who cancelled prior to receiving therapy, ineligible patients, and patients whose HER-2/neu status was not corroborated by central review were excluded from analysis. This study used Cox models stratified by hormone receptor status and nodal status to assess the association between serum HER-2/neu levels and DFS. Results: Baseline characteristics between patients with serum HER-2/neu levels <15 ng/mL (N=1234) and patients with serum HER-2/neu levels 15 ng/mL (N=189) demonstrated differences between groups by age, menopausal status (higher serum HER-2/neu levels in pts 50 yrs/post-menopausal), and hormonal receptor status (lower serum HER-2/neu levels in hormone receptor + tumors). Hazard ratios between serum HER-2/neu groups within Arm A and within Arm C showed that pts with serum HER-2/neu 15 ng/mL had worse DFS than pts with serum HER2/neu <15 ng/mL (A: HR=1.71, p =.004; C: HR=1.42, p =.22). Hazard ratios between arms within each serum HER-2/neu group demonstrated similar benefit from trastuzumab in each serum HER-2/neu group (<15: HR=.66, p=.006; 15: HR=.60, p=.10). Results remained consistent when including menopausal status and age in the Cox models. Conclusions: 13% of HER2+BC pts of this study had high levels of serum HER-2/ neu. In the standard chemotherapy arm, pts with high serum HER-2/neu had significantly worse DFS than patients without. In the concurrent trastuzumab arm, a similar trend was observed but did not reach statistical significance, potentially due to a smaller number of events

assessment was performed at week 8, 12 and every 12 weeks thereafter. Disease status was assessed by an independent radiology committee, and response was measured using RECIST. Serum HER-2/neu levels were measured using the test from Siemens HealthCare DX. A serum HER-2/neu decrease or increase of 20% from baseline was defined as a significant change. Results: In this study baseline serum HER-2/neu levels were measured for all 138 MBC patients. Of the 138 patients, 109 (79%) % had serum HER-2/neu baseline levels > above the normal cutoff of 15ng/ml. During the first 16 weeks post therapy, serum HER-2/neu levels were measured every 4 weeks ( 4, 8,1 2 and 16) and the changes from baseline measured. Baseline serum HER-2/neu levels did not predict patient response not were baseline serum HER-2/neu levels associated with PFS, however, changes in serum HER-2/neu levels from baseline did have clinical significance. A 20% decrease from baseline of serum HER-2/neu was associated with prolonged PFS, while a 20% increase from baseline was associated with shorter PFS. Patients who experienced a 20% decrease from baseline in serum HER-2/neu levels at week 4 had a median PFS of 199 days (95% confidence interval [CI] = 145, 253) compared with 135 days (95% CI = 104, 171) for patients who did not (p = 0.016). Similarly, patients who experienced a 20% increase from baseline of serum HER-2/ neu at week 4 had a median PFS of 113 days (95% CI = 59, 135) compared with 199 days (95% CI = 142, 253) for patients who did not (p < 0.001). Similar associations were observed at weeks-8,12 and 16. Conclusions: A 20% or greater decrease from baseline of serum HER-2/neu level at weeks 4, 8, 12 and 16 was strongly associated with prolonged median PFS, whereas a 20% or greater increase from baseline was strongly associated with shorter median PFS. When adjusted for changes in tumor burden, serum HER-2/neu was an independent predictor of median PFS..
D-18 Disparity in estimated average glucose due to different hemoglobin A1c assays and variant hemoglobins Y. Zhu, L. Williams, B. Horne, M. Jenkins. Medical University of South Carolina, Charleston, SC
Background: It has been recommended that estimated average glucose (eAG) values calculated using hemoglobin A1c (A1C) results be reported in addition to A1c. However, many A1c assays based on various principles are used in clinical laboratories, and therefore, different eAG results may be obtained when the same equation is used. Also, different A1c methods may have variable susceptibility to variant hemoglobin interference, which may further increase the difference in eAG values. The objective of this study is to compare eAG results calculated based on A1c results measured with BioRad variant II TURBO (BioRad) and Beckman Coulter (Beckman) A1c assays in patients with normal and variant hemoglobins. Methods: BioRad variant II TURBO is an HPLC A1c assay, while Beckman Coulter method is an immunoturbidimetric assay. Both methods are certified by the National Glycohemoglobin Standardization Program (NGSP). Freshly collected blood samples in EDTA tubes from 129 patients with normal hemoglobin and 42 patients with heterozygous hemoglobin S were measured for A1c with both BioRad and Beckman assays within 24 hours after collection. eAG values were calculated using the following recently published equation: eAG (mg/dL) = 28.7 A1c - 46.7. T-test was used for the statistical analysis. Results: For 129 patients with normal hemoglobin, the mean eAG was 142.5 62.8 mg/dL and 133.1 57.8 mg/dL (mean SD) based on BioRad and Beckman methods respectively. The eAG bias between these two methods was statistically significant (P = 0.0000) with a mean bias of 9.5 10.2 mg/dL. The mean bias is larger for patients with A1c 7.0% (17.7 13.5 mg/dL), while it is smaller for patients with A1c < 7% (7.6 8.4 mg/dL) (P = 0.0016). For 42 patients with heterozygous hemoglobin S, the average eAGs were 187.1 76.3 mg/dL and 157.8 68.5 mg/dL respectively determined with BioRad and Beckman methods. The mean bias was 29.3 15.4 mg/ dL, and the difference was statistically significant (P = 0.000). Similar to patients with normal hemoglobin, for patients with heterozygous hemoglobin S, the mean bias was larger for patients with A1c 7.0% (37.3 16.9 mg/dL, n = 17), while smaller for patients with A1c < 7% (23.9 11.7 mg/dL, n = 25) (P = 0.009). Further analysis showed that the mean bias between patients with normal and variant hemoglobins were statistically significant (29.3 15.4 vs 9.5 10.2 mg/dL). Conclusions: eAG results derived from A1c determined with BioRad and Beckman assays are not interchangeable when the same equation is applied, although both assays are certified by NGSP. The bias is more significant in patients with elevated A1c and in patients with hemoglobins S. Since NGSP allows 1% A1c deviation from NGSP reference method, the bias in eAG is most likely due to different bias of A1c assays to NGSP reference method. This problem can be solved by further standardizing A1c assays.
D-18 Decrease in serum HER-2/neu levels at 4 to 16 weeks is associated with prolonged progression-free survival in metastatic breast cancer patients treated with lapatinib monotherapy W. P. Carney1, A. Lipton2, K. Leitzel2, S. M. Ali3, G. Platek4, L. O'Rourke4, A. Martin4, J. Maltzman4. 1Oncogene Science/Siemens Healthcare Diagnostics, Cambridge, MA, 2Penn State University, Hershey Medical Center, Hershey, PA, 3Lebanon VA Medical Center, Lebanon, PA, 4Medicine Development Center, GlaxoSmithKline, Collegeville, PA
Background: The extracellular domain of the HER-2/neu oncoprotein is shed into serum and can be quantitated using a standardized immunoassay. This study investigated if changes in serum HER-2/neu levels at weeks 4, 8, 12 and 16 after start of lapatinib monotherapy was associated with prolonged progression free survival (PFS). Methods: Study EGF20009 investigated lapatinib monotherapy in 138 HER2positive patients with metastatic breast cancer who had not previously received chemotherapy or trastuzumab. Serum was collected at baseline and every 4 weeks after initiation of oral lapatinib (1,500 mg daily or 500 mg twice daily). Disease
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D-20 Infections with Nontyphoidal Salmonella Species Producing CTX-M38 and CMY-2 -Lactamases, in China C. Jian, Z. Sun, J. Chen. Laboratory Medicine of Tongji Hospital affiliated to Huazhong University of Science and Technology, Wuhan, China
A total of 102 (0.97%,102/10,464) nontyphoidal Salmonella (NTS) were isolated from 10,464 stool cultures between 2003 and 2005 in Wuhan, China. S. enterica servoar Typhimurium (Stm), serovar Enteritidis (Sen), serovar Derby, serovar Newport, serovar Stanley and serovar Thompson accounted for 51.0% (52/102), 28.4% (29/102), 9.8% (10/102), 3.9% (4/102), 2.9% (3/102) and 1.0% (1/102), respectively. Another 2.9% (3/102) could not been typed. The percentage of NTS from <1 year of age, 1~4year of age, 4~14 year of age and >14 year of age were 61.7%, 25.4%, 2.9% and 10.0%. The susceptibility of NTS to 16 antibiotics showed that the resistance of extended-spectrum cephalosporins (ESC) was between 2.9% and 6.9% (7/102). In 7 isolates, CTX-M-38 ESBLs, CMY-2 AmpC and TEM-1 lactamase were all detected in plasmids. 4 were detected above two types of lactamases. None of -lactamase was detected in 2 isolates. In conclusion, NTS infection was mainly due to Stm (51.0%), following by Sen (28.4%). CTX-M-38 ESBLs was firstly detected in NTS, and NTS producing CMY-2 AmpC -lactamase has not been reported in China other than Taiwan.
useful, non-invasive indicator of skeletal health in preclinical species and humans. The rat is a common preclinical species utilized for assessing the responses of drug toxicities in preclinical drug development. Method: In this study, the investigational drug is intended for osteoporosis therapy and the osteocalcin biomarker is an indicator of a drugs efficacy at various doses in healthy 6-7 week old normal rats. We describe the evaluation and application of an Osteocalcin (OC) enzyme-linked immunosorbent assay (ELISA) for measuring responses to bone formation in rat serum. The Rat Osteocalcin ELISA (IDS) is an enzyme-linked immunosorbent assay for the quantitative determination of osteocalcin in rat serum and plasma. The Rat-MID Osteocalcin EIA is based upon the competitive binding of a monoclonal antibody to soluble osteocalcin or to immobilized osteocalcin. Briefly, the monoclonal antibody is raised against human osteocalcin and recognizes the mid-molecular part (amino acids 21-29) of the molecule. Results: The sensitivity of the OC ELISA was 50 ng/ml. The averaged intra- and interassay variation was 4.36% and 7.43%. Averaged spiked recovery was 108 ng/ml. In vivo validation of osteocalcin ELISA was performed in 6-7 weeks old SpragueDawley (SD) rat models treated with PTH (Para Thyroid Hormone) analog; 200 ug/ kg. In treated rats, the osteocalcin concentrations were approximately 35%, higher than the sham group and exhibited acceptable sensitivity, accuracy, and selectivity necessary to evaluate bone formation in a normal rat model. Conclusion: In conclusion, the inclusion of osteocalcin, a bone biomarker for assessing bone formation mechanisms in preclinical and clinical drug development programs for osteoporosis adds value by providing additional information about the mechanism of action of the investigational drug.
D-21 A study on the relationship between apolipoprotein E gene polymorphism and hepatitis B infection Z. Yin1, S. Yan2, J. Wang1. 1Medical Laboratory Center, Beijing Chuiyangliu Hospital, Beijing, China, 2Department of Laboratory Medicine, China-Japan Friendship Hospital, Beijing, China
Objective The rate of hepatitis B virus (hepatitis B virus, HBV) infection is currently the highest among hepatitis viruses in Han Chinese from northern China. The development of different diseases after infection is related to individual on hepatitis B infection occurred in the immune response and the virus itself factor as well. Research has shown that certain genetic polymorphisms can lead to difference on body immune function, thus affecting different clinical rehabilitation for HBV patients. Apolipoprotein E (apolipoprotein E, ApoE) gene polymorphism has multiple biological functions. This study explored the relationship between the HBV infection and ApoE gene polymorphism so as to provide evidence for the pathogenesis of hepatitis B from genetic angle. Methods Multiplex Amplification Refractiry Mutation System (Multi-ARMS) was used to detect apolipoprotein E genotype of 270 cases of hepatitis B patients with different infection models and 112 normal healthy cases, and chemiluminescence was used to detect serological indicators of hepatitis B for the samples mentioned above and HBV DNA qualitative was carried out. Results The frequency of 2 allele of patients with different models of hepatitis B virus infection and HBV DNA infection positive rate were higher than that of the normal control group, and with statistical significance (P <0.01). Compared to 3 + 4, the incidence of risk of hepatitis B in 2 was more than 1.0 with the positive correlation. And also HBV DNA infection positive rate is also higher than normal population (P <0.01) Conclusion There is correlation between Apolipoprotein E genotype and patients with hepatitis B virus infection. This shows that Han Chinese from northern China who carry 2 allele are more susceptible to hepatitis B virus.
D-23 The Ordering Patterns of Serological Tests in the Diagnosis of Celiac Disease Y. Huang, A. C. Don-Wauchope, M. Mansour, V. L. Grey. Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada
Objectives: Current guidelines for the diagnosis of celiac disease in children include recommendations to measure tissue transglutaminase antibodies (tTG). In adults there are similar recommendations. We designed a laboratory audit to review the ordering patterns of serological tests in celiac disease. Methods: The specimen number, age of patient, serological test result, and ordering physician were extracted from the Laboratory Information System over three months at McMaster University Medical Centre. The specimen number was used as the primary identifier to determine the ordering patterns. Intestinal biopsy results were retrieved for those with a positive serology test and an age matched control group with negative serology. Frequency data was analyzed by Fisher Exact Test or Chi-Square Test. Results: 1152 serological tests were ordered for 666 patients in three months (47.6% <=18y). The majority of pediatric sub-specialists ordered tTG alone , significantly different (p <0.001) from the other groups. In adult practice the ordering pattern was similar across groups with the majority ordering celiac panel (Table 1). The overall positive rate of tTG and celiac panel were 8.3% and 20.9% (tTG-Gliadin+ 15.7%). The biopsy reports in the following 6 months revealed that 75.0% tTG positive patients had an intestinal biopsy which is similar to the celiac panel group (83.3%), when all 3 tests were positive. Fewer patients had a biopsy (37.5%) with a positive tTG and a negative gliadin (IgA or IgG). A negative tTG result with a positive gliadin (IgA or IgG) reduced the biopsy rate to 24.6%, while with a negative gliadin to 16.0%.
The ordering patterns of serological tests in celiac disease Gastroenterologist Other and immunologist Pediatrician hospital sub-specialization physician Number (%) of 24(11.3) 23(10.9) physicians Number (%) of tests 422(36.6) 231(20.1) ordered Test ordering patterns% IgA Children: Anti-tTG 71.1 40.3 (p<0.001) Celiac panel * 21.0 59.0 Adults: Anti-tTG IgA 8.9 --AntiEndomysial 16.1 --IgA Celiac panel 69.6 --* Celiac panel: Anti-tTG IgA and Anti-Gliadin IgA, IgG 50(23.7) 161(14.0) 15.8 63.2 8.1 5.4 78.4 Nonhospital physician 114(54.0) 338(29.3) 10.2 77.3 3.7 1.9 80.4
D-22 Bone biomarkers significantly enhances the predictability of preclinical study outcomes and translation to first in man studies targeted towards osteoporosis K. K. Maddali, C. I. Starks, C. McDonough, J. Dharmadhikari, B. A. Litzenberger. Huntingdon Life Sciences, East millstone, NJ
Objective: Translational bone biomarkers applicable to the various preclinical species can provide important tools for studying the bone remodeling process in the preclinical studies in response to Osteoporosis drugs. Circulating osteocalcin (OC) is associated with changes in the rate of bone turnover and regarded as a specific marker for bone formation. Recent studies have indicated that serum OC may provide a
Conclusions: The pediatric sub-specialists preferentially ordered tTG for children. In adult practice, celiac panel was most common. Celiac panel appeared to influence biopsy rate when the tTG and Gliadin results did not agree. These findings need to be confirmed by a chart review.
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D-24 The clinical application of fully automated urine cell analyzer in rapid diagnosis of urinary tract infections in children Q. Wu, Y. Li, M. Wang, Z. Xia, H. Hu. Renmin Hospital of Wuhan University, Wuhan, China
Objectives: To evaluate the clinical application of fully automated urine cell analyzer in the diagnosis of urinary tract infections (UTI) in children. Methods: We considered 322 subjects, aged between 0 and 12, 139 males and 183 females. The samples were strictly collected by using the midstream technique. Each sample was subjected to microbiological evaluation bacteria culture, dipstick tests and fully automated urine cell analyzer examination. The results of the urine cell analyzer and urine bacteria culture were compared to evaluate the value of the application of urine cell analyzer in diagnosis of UTI in children. Results: Out of the total 322 subjects 105 cases was considered of UTI (account for 32.61%). The urine cell analyzer-based screening method had a sensitivity of 0.89, a specificity of 0.87, a positive predictive value of 0.72, a negative predictive value of 0.95 and a correctly classified incidence of 0.93. Conclusion: In this study, we considered that the bacteria counting cut-off value of UTI was 3000/l. The results of the urine cell analyzer-based screening method show a very good correlation with the diagnosis of UTI in children,and have a better sensitivity compared with the results (sensitivity 0.69) of dipstick tests in the same conditions.

pregnancy outcomes. We therefore conducted this study to determine this relationship if any. Women presenting at the antenatal clinic from July 1 - December 31 2008 were evaluated for risk factors for development of GDM. All women who had more than one risk factor were recruited into the study group. All patients underwent the 75g - 2 hour oral glucose tolerance test. GDM was diagnosed according to the criteria set by the American Diabetes Association. Glucose was measured by the glucose oxidase method in the hospital laboratory. After a six month period, a total of three hundred and sixty four patients were recruited into the study. OGTT was more likely to be requested if the woman was obese, had a family history of diabetes mellitus, previous history of GDM or had a history of poor fetal outcomes. The mean fasting, 1 hour and two hour blood glucose for the study population was 4.05 1.16, 6.4 1.66, 5.64 1.86 respectively. The prevalence of GDM was 6.6% while that of one abnormal value was 4.7%. The results of the study of the adverse effects of GDM on pregnancy are ongoing.
D-27 SERIAL MEASUREMENTS OF B-TYPE NATRIURETIC PEPTIDE (BNP) IN PATIENTS UNDERGOING CARDIAC SURGERY H. Turner, J. Reeve, J. McNeilly, W. Mutch, R. Peake, D. Rae, P. Gibson, G. Hillis, B. Cuthbertson, B. Croal. Aberdeen Royal Infirmary, Aberdeen, United Kingdom
Background. Patients undergoing cardiac surgery are ultimately at risk of significant morbidity and mortality in the post-operative period. While various scoring mechanisms exist to assist in the prediction of such adverse outcome, these are complex and not always reliable. We have looked at the significance of serial measurements of BNP taken before and after cardiac surgery. Methods. 331 consecutive patients undergoing cardiac surgery had samples assayed for BNP (Siemens ADVIA Centaur) prior to surgery and at 6 and 24 hours following surgery. Patients were followed up in the immediate post-operative period and for one year following surgery. Results. BNP levels (pg/L) increased from baseline values through to 6 and 24 hours (median 62.1 v 135.2 v 305.8 respectively; p <0.001). In patients who subsequently died (n=14), BNP levels were higher at baseline (108.1 v 61.4; p=0.062), 6hrs (239.9 v 134.0; p=0.002) and 24hrs (642.9 v 294.0; p<0.001) following surgery. ROC analysis for the prediction of mortality using BNP levels taken 24hrs post surgery demonstrated high diagnostic accuracy with an area under the curve of 0.841 (p < 0.001). In addition a multivariate logistic regression model, indicated, that BNP measured at 24hrs remained the best predictor of mortality (p=0.014) even when adjusted for age, sex and established risk scoring systems (Parsonnet and Euroscore). In a similar adjusted multivariate regression model, patients with a BNP level in the highest quartile at 24 hours were more than 6 times likely to die (OR 6.33; p=0.003).
D-25 Establishment of the accurate cutoff index and 95% interval for the HBsAg qualitative test in Chinese population Y. Li, H. Zhang, L. Song. Renmin Hospital of Wuhan University, Wuhan, China
Objectives: The Hepatitis B surface antigen kit (electrochemiluminescence immunoassay assay, ECLIA) has stated the borderline as 0.9-1.0 (COI) for the HBsAg qualitative test, but whether this borderline is suitable for Chinese people is unknown. Our purpose was to verify and establish the accurate cutoff index (COI) and 95% interval for the HBsAg qualitative test (ECLIA) in Chinese population. Methods: Following the NCCLS global consensus guideline, a series dilutions was made for this study such as 0.32, 0.64, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.4, 2.5, 5.0, 10.0, 16.0, 32.0 (COI). Every dilution were tested for 20 times, then determined the percentage of positive /negative results for each sample. It should be the accurate cutoff index at which concentration we can yield 50% positive results and 50% negative results. Test the concentration above / below the cutoff index, if we can yield 95% positive / negative results, the 95% interval can be determined. According to this cutoff point and 95% interval, we analyzed 100 clinical samples. All reactive results or borderline samples were investigated using an independent neutralization test (HBsAg Confirmatory Test). Negative predictive value (NPV) and positive predictive value (PPV) were calculated at last. Results: Not at 1.0 COI but at 1.4 COI, the percentages of the positive results and negative results were both 50%; and not at 0.8 and 1.2 COI, but at 0.8 and 2.0 COI, the percentages of the negative results and positive results were both 95%. NPV and PPV of the 100 clinical samples were both 100%. Conclusion: It was suggested that the accurate cutoff index for the HBsAg qualitative test (ECLIA) maybe 1.4 COI in Chinese population; the proper 95% interval maybe 0.8-2.0 COI.
D-26 THE PREVALENCE OF GESTATIONAL DIABETES AT THE UNIVERSITY OF PORT HARCOURT TEACHING HOSPITAL INTERIM RESULTS OF A PROSPECTIVE STUDY A. A. Ejilemele1, S. A. Uzoigwe2, O. N. Okike2. 1Department of Chemical Pathology, University of Port Harcourt, Port Harcourt, Nigeria, 2Department of Obstetrics and Gynaecology, University of Port Harcourt, Port Harcourt, Nigeria
Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance with onset or first recognition during pregnancy. It affects up to 14% of pregnant women and is commoner in those who are obese, older than 25 years, have a previous history of abnormal glucose metabolism or poor obstetric outcome, have first-degree relatives with diabetes, or are members of ethnic groups with high prevalence of diabetes. In this environment, there is no available data on the prevalence of GDM or the relationship between GDM, one abnormal glucose value (OAV) and adverse
Conclusion. BNP levels measured post-operatively provide unique prognostic information regarding the short and long term mortality that is independent of already established clinical risk scoring systems. Such information may allow direction of more aggressive treatment and follow up.
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D-28 Association between tumor necrosis factor- polymorphism and coronary heart disease in a Chinese population M. Wang1, W. Li2, Y. Li1. 1Renmin Hospital of Wuhan University, Wuhan, China, 2Department of Hematology, Renmin Hospital of Wuhan University, Wuhan, China
Objectives: To investigate the association between tumor necrosis factor- (TNF-) gene polymorphisms as well as its serum levels and coronary heart disease (CHD) in a Chinese population. Methods: Polymerase chain reaction- sequence specific primers (PCR-SSP) was used for the detection of TNF- C804A genotype in 210 patients with CHD and 186 controls. The serum TNF- levels were measured by enzyme-linked immunosorbent assay (ELISA). Results: The frequencies of CC, CA and AA Genotypes of C804A were 25.7% and 37.1%, 49.5% and 45.7%, 24.8% and 17.2%, patients and controls respectively. There were statistically significant differences in the distributions of the genotypes (P<0.05) and the allele frequencies (P<0.05) between two groups. The relative risk suffered from CHD of AA and CA genotypes was 1.704 times of the CC genotype (OR=1.704, 95%CI: 1.109~2.617). The serum TNF- and high sensitive C-reactive protein (hsCRP) levels of the patients were significantly different from those of the controls (both P<0.05), however, among different TNF- genotypes of patients and controls respectively, there were no significant differences. Conclusion: The single nucleotide polymorphism at position 804 in the exon 3 of TNF- gene is associated with CHD and the allele A may be a risk factor for CHD in Chinese.
Conclusion: This study demonstrates the potential usefulness of pre-operative measurements of creatinine, cystatin C and their derived estimates of GFR as risk prediction tools in patients undergoing cardiac surgery.
D-30 Renal function markers in the prognosis of patients presenting with chest pain J. L. Reeve1, H. Turner1, W. J. Mutch1, V. Mills2, J. D. McNeilly3, R. Peake1, J. Allison1, G. Hillis2, R. Soiza2, B. Croal1. 1Department of Clinical Biochemistry, Aberdeen Royal Infirmary, Aberdeen, United Kingdom, 2Department of Cardiology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom, 3Department of Clinical Biochemistry, Royal Hospital for Sick Children, Glasgow, United Kingdom
Background: We have assessed the value of several markers of renal dysfunction (cystatin C, creatinine, eGFR and Neutrophil Gelatinase-Associated Lipocalin (NGAL), a novel marker of acute kidney injury) in predicting mortality in patients presentating with chest pain. Methods: Admission levels of serum creatinine (umol/L; Siemens ADVIA 2400), cystatin C (mg/L; Siemens ProSpec) and their derived estimates of Glomerular Filtration Rate (eGFR-Cr and eGFR-CC, respectively) were measured in 210 patients presenting with chest pain to a Coronary Care Unit. Serum NGAL (ng/mL; ELISA, AntibodyShop) was measured at 12 hours, while creatinine was monitored daily until discharge. Patients were followed up for 6 months for subsequent mortality. Results: Levels of cystatin C (1.53 v 0.93; p=0.001) and peak creatinine (189 v 103; p=0.001) were higher in patients who subsequently died (n=16). Similarly, levels of eGFR-Cr (48.8 v 77.8; p=0.010) and eGFR-CC (43.8 v 102.3; p=0.001) were lower in patients who subsequently died. Serum NGAL levels were also higher in patients who died, however this was not significant (250.1 v 207.1; p=0.159). Kaplan Meier survival curve analysis demonstrated higher mortality for those patients within the higher quartiles for peak creatinine (p=0.002) and cystatin C (p=0.001), and the lower quartiles for eGFR-CR (p=0.017) and eGFR-CC (p=0.001). In a regression model, a creatinine or a cystatin C level in the upper quartile equated to a much higher risk of death by 6 months (OR 5.94, p=0.001; OR 11.79, p<0.001, respectively). Cystatin C in the upper quartile remained an independent predictor of mortality even when adjusted for the GRACE score prediction of mortality at 6 months (OR 10.35; p=0.006). Serum NGAL quartile status was significantly related to subsequent peak creatinine levels (p<0.001) (see Figure).
D-29 THE SIGNIFICANCE OF CYSTATIN C AND ITS DERIVED eGFR IN PATIENTS UNDERGOING CARDIAC SURGERY J. D. McNeilly1, J. L. Reeve2, H. Turner2, R. Peake2, W. J. Mutch2, D. Rae3, P. H. Gibson3, G. S. Hillis3, B. H. Cuthbertson3, B. L. Croal2. 1Department of Clinical Biochemistry, Royal Hospital for Sick Children,, Glasgow, United Kingdom, 2Department of Clinical Biochemistry, Aberdeen Royal Infirmary, Aberdeen, United Kingdom, 3Department of Cardiology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom
Background: Renal dysfunction, as assessed by a reduced estimated glomerular filtration rate (eGFR) is a potentially useful predictor of outcome in patients undergoing cardiac surgery. Cystatin C, a cysteine protease inhibitor, and its derived eGFR, are potential alternatives which have the added advantage of improved sensitivity, specificity and less inter/intra-individual variability. We assessed the significance of preoperative cystatin C in comparison to creatinine-based risk assessments in predicting subsequent mortality in patients undergoing cardiac surgery. Method: 973 patients undergoing cardiac surgery had serum cystatin C (Siemens, Prospec) and creatinine (Siemens, Advia 2400) measured pre-operatively. GFR was estimated by the adjusted 4-variable MDRD equation using creatinine (eGFR-Cr) and an equivalent equation using cystatin C (eGFR-CC). Patients were followed up for 1 year. Results: Pre-operative creatinine and cystatin C levels were higher in those patients who subsequently died in the first year following surgery (creatinine, 111 v 102 mol/L; p=0.004) (cystatin C, 1.13 v 0.93 mg/L; p<0.001). Similarly, both eGFR-Cr (58.9 v 69.9;p<0.001) eGFR-CC (72.6 v 100.6;p<0.001) levels were lower in patients who subsequently died. Using Kaplan-Meier survival curve analysis, patients in the higher quartile for creatinine (p<0.001) and cystatin C (Figure 1) demonstrated higher mortality Both eGFR-Cr and eGFR-CC remained independent predictors of mortality in a multivariate regression model adjusted for EuroScore, an established objective assessment of risk (eGFR-CC p=0.029, eGFR-Cr p=0.020)
Conclusions: Serum NGAL levels may predict future renal function deterioration; however, creatinine and cystatin C serve as more useful predictors of mortality.
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D-31 THE ROLE OF URINARY NGAL TO URINARY CREATININE RATIO IN THE EARLY DETECTION OF CONTRAST AGENT INDUCED ACUTE KIDNEY INJURY AFTER CORONARY ARTERY ANGIOGRAPHY K. Makris1, C. Demponeras2, F. Zoubouloglou1, S. Potamitis1, N. Kafkas2, I. Drakopoulos1, D. Rizos3, A. Nikolaou2, D. Babalis2, A. Haliassos4. 1Clinical Biochemistry Department, KAT General Hospital, Athens, Greece, 2Cardiology Department, KAT General Hospital, Athens, Greece, 3University of Athens, Aretaieio Hospital, Hormones Laboratory, Athens, Greece, 4National External Quality Assessment Scheme in Clinical Chemistry (ESEAP), Athens, Greece
Acute kidney injury (AKI), usually defined by an increase in the serum creatinine >0.5mg/dL or >25% from baseline, is a common complication of contrast agent used during percutaneous coronary interventions (PCI). This increase typically occurs 35days after contrast administration when patients are already discharged. Neutrophil gelatinase-associated lipocalin (NGAL) is highly accumulated in the human kidney cortical tubules, blood and urine after nephrotoxic and ischaemic injuries, has been proposed as an early, sensitive biomarker for AKI. The aim of our study was to evaluate the use of the urinary-NGAL/urinary-creatinine ratio in order to detect contrast-induced AKI. This ratio was used in order to normalize the urine NGAL concentrations due to the different levels of diuresis among our patients. Sixty patients undergoing either coronary angiography alone (n=21) or angioplasty with stenting (n=39) were included. According to admission serum creatrinine (>1.2mg/dL), saline and/or n-acetylcysteine (NAC) administration was decided by the cardiologist. Low-osmolar contrast agent (iodixanol) was used in all patients. Serum and urine samples obtained just before PCI (baseline), 6-hours after contrast administration and 24 and 48 hours thereafter. NGAL was measured with ELISA (Bioporto,Gentofte,Denmark). Urine and serum-creatinine were measured with Jaffe method, and cystatin-C with immunoturbidometric assay on Architect-16200 analyzer (Abbott Diagnostics, Abbott Park,Il). Table summarises our results. Group I Group II Baseline cystatine Baseline cystatine <1mg/L >1mg/L No AKI AKI NAC No NAC p value 32 10 10 8 58.7(39-81) 62.2(49-74) 68.2(52-82) 68.6(48-80) 356(90426(130290(80-740) 295(140-750) 800) 1100) 10/20 0.43(0.042.23) 2.48(0.187.20) 1.15(0.064.54) 0.77(0.073.76) p<0.001 0.86 0.0% 0.82 -2.4% 4/6 1.83(0.104.14) 9.89(1.1315.40 9.69(1.0924.24) 7.83(0.3518.01) p<0.001 0.83 28.9% 0.80 25.0% 2/8 3,75(0.028.93) 5.69(0.2717.44) 4.87(0.4312.02) 4.64(1.1011.32 p=NS 1.24 5.7% 1.23 0.0% 2/6 9,48(1.1025.08) 17.07(4.8329.87) 12.60(4.6719.26) 8.83(1.7615.08) p<0.001 0,95 27.4% 1.11 21.62% p<0.001 p<0.001 p<0.001 p<0.001 p<0.001

D-32 24-Hour Bence-Jones Protein Determinations: Can We Insure Accuracy? J. Kaplan, G. Horowitz. Beth Israel Deaconess Medical Center, Boston, MA
Current guidelines recommend that Bence-Jones proteinuria be monitored with 24hour urine collections. Implicit in this recommendation is that the collections be accurate, but this is frequently not the case. We hypothesized that, in practice, BenceJones quantitation by this technique is often misleading and that, instead, one can use random urine protein/creatinine ratios to provide better information. We reviewed data (urine creatinine (UCR), protein (UTP), total volume, and proportion of Bence-Jones protein (%BJP)) from 2003 through 2008 on all BIDMC patients with Bence-Jones proteinuria who had 24-hour urine collections for protein and creatinine. In addition, for these same patients, we found all other random urine samples on which UCR, UTP, and %BJP were reported. We focused first on those patients who had more than four 24-hour collections. There were a total of 14 patients to evaluate, with 135 24-hour urine protein collections. We found that the urine creatinine excretion values for each patient, which should be reasonably constant, varied considerably (CV range 12%-39%). We then calculated the BJP protein excretion based on the 24-hour protein (i.e., assuming an accurate collection) and based on a corrected value determined from that patients mean 24hour creatinine excretion. The differences in these values ranged from -1377 to 2374 mg/day. We looked at differences in serial 24-hour Bence-Jones excretion (mg/day) by each method. We initially defined clinically significant as values with opposite signs (i.e., an increase by one measure but a decrease by the other) or values whose magnitude was at least 2-fold different and >100 mg/day. With these criteria, of 121 events, 44 were clinically significant. We lowered this number to 37 by inspecting the data in the context of the patients overall level of BJP. Three patients had no clinically significant differences; among the other eleven patients, the proportion of samples with clinically significant differences ranged from 17% to 80%. For all 14 patients, the median was 30%. We then evaluated the reliability of random urine samples to estimate 24-hour BenceJones excretion. From our original database, there were 23 random urine samples, from 11 different patients, on which BJP was measured within 10 days of a 24-hour urine collection. In 18 of 23 cases, the random sample gave results that were consistent with the clinical scenario and the normalized BJP on the corresponding 24hour sample. In 4 of the remaining 5 cases, the random sample was probably consistent, but we did not have access to enough clinical data to be certain. We conclude that 24-hour urine collections for Bence-Jones proteinuria can, in practice, often be misleading. At a minimum, one should verify that the 24-hour creatinine excretion is accurate. In addition, it may be possible to use the protein/ creatinine ratio from random urine samples to determine 24-hour Bence-Jones excretion.
* mean (range) n Age* contrast volume (mL)* stable angina/ UA+AMI uNGAL/uCREA baseline (ug/mg)* uNGAL/uCREA at 6hrs* uNGAL/uCREA at 24hrs* uNGAL/uCREA at 48hrs* p-value sCreatinine at baseline (mg/dL) sCreatinine change (%) sCystatine at baseline (mg/L) Serum cystatine change (%)
D-33 Pulmonary Hypertension in Patients with Graves' Disease T. Sugiura, H. Kataoka, N. Morimoto, T. Hisahara, S. Yamanaka, H. Takeuchi, Y. Kumon. Kochi Medical School, Nankoku, Japan
Back ground: Thyroid hormone affects almost all organs of the body including cardiovascular system. The aim of this study was to determine the frequency and clinical correlates of pulmonary hypertension in patients with Graves' disease. Methods and Results: Fifty consecutive patients with Graves' disease were studied by echocardiography and laboratory findings. Among 50 patients with Graves' disease, 14 patients (28%) had pulmonary hypertension (pulmonary artery systolic pressure 40mmHg calculated as the sum of the trans-tricuspid pressure gradient plus 10mmHg). Although there was no significant difference in cardiac output between the two groups, patients with pulmonary hypertension had significantly higher free T3 (12.687.45 vs 8.724.85pg/ml) and thyroid stimulating hormone receptor antibody (82.798.9 vs 18.922.9lu/l) compared to those without (p=0.031 and p<0.001, respectively). Pulmonary artery systolic pressure had a weak correlations with free T3 (r=0.36, p=0.010), and a fair relation with thyroid stimulating hormone receptor antibody (r=0.52, p<0.001). Conclusion: Pulmonary hypertension was a relatively common complication in patients with Graves' disease, and was associated with more severe hyperthyroid state. Moreover, autoimmune process of the pulmonary artery seems to be another important factor causing pulmonary hypertension in Graves' disease.
p<0.001
Normal renal function observed in 42 patients at baseline (group-I) while renal impairment (cystatine>1.0mg/L) in 18(group-II). Ten patients from group-I developed AKI (mean serum-creatinine increase 28.9%), while deterioration of kidney function (mean serum-creatinine increase 27.4%) was observed in 8 patients from group-II. In AKI-patients urinary-NGAL/urinary-creatinine ratio at 6hours was significantly increased from baseline as well as compared with non-AKI patients at the same timepoint. Similar increase observed among the patients who did not receive NAC. We conclude that urinary-NGAL/urinary-creatinine ratio can be used to early identify patients that develop AKI due to contrast administration.
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D-34 The relationship between the levels of serum testosterone and bone metabolic biochemical markers in elder males Z. Zhao1, F. Wang1, X. Yang1, Z. Jiang1, Y. Li2. 1Department of Geriatric, Renmin Hospital of Wuhan University, Wuhan, China, 2Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, China
Objectives: Bone metabolic biochemical markers were associated with osteoporosis, and the levels of serum androgen also are associated with primary osteoporosis in elder males. However, a few studies have specifically addressed the relationship between the levels of androgen and bone metabolic biochemical markers in elder males. We assessed the relationship between the levels of serum testosterone and bone metabolic biochemical markers in elder males. Methods: 186 elder males were divided to three groups according to stage of age, 64 with Group A (60-69 years old), 68 with Group B (70-79 years old), 54 with Group C (80 years old), respectively. The levels of serum testosterone (T), bone glaprotein (BGP), procollagen I C-terminal peptides (PICP) were determined with ELISA method. Alkaline phosphatase (ALP) and urinary deoxypyridinoline (DPD) were determined with chemical luminescent technique, respectively. Results: As increasing age in elder males, the levels of serum T, BGP, PICP and ALP decreased, but the levels of urinary DPD increased (P<0.05 or P<0.01).The correlation analysis showed that the levels of serum T were positive correlation with the levels of serum BGP (r=0.57, P<0.05) and that were negative correlation with the levels of urinary DPD (r= -0.49, P<0.05). Conclusion: Our study suggests that the levels of androgen are closely related to bone metabolism in elder males. The change of levels of androgen plays an important role in primary osteoporosis in elder males.
with different pathway demonstrated the similar effects on the treatment of MG. To date, use of the CYP3A5 genotype to guide the drug choice of CNI and predict appropriate dose of tacrolimus is the most promising option for individualization of drug therapy in MG.
D-36 Evaluation of clinical application of HBsAg confirmatory test for samples with poor reactive HBsAg J. Shi, Y. Li, J. Yang. Renmin Hospital of Wuhan University, Wuhan, China
Objectives: To Evaluate the clinical application of the HBsAg confirmatory test for samples with poor reactive HBsAg. Methods: Out of 24281 serum samples routinely tested by electrochemiluminescence immunoassay (ECLIA) method there were 200 samples of poor reactive HBsAg. The results of these samples were from 0.9 to 10.0 COI. Both HBsAg Confirmatory Test and Enzyme Linked Immunosorbent Assay (ELISA) were performed on these 200 samples, and the results of both were compared. Results: Total reactive result samples by HBsAg confirmatory test have 197, the coincidence rate was 98.5%(197/200); The COI value in 0.9~1.5 sample have 19, the confirmatory coincidence rate was 89.5%(17/19); The COI value in 1.5~2.0 sample have 33, the confirmatory coincidence rate was 97%(32/33); The COI> 2.0 sample confirmatory coincidence rate was 100%; The reactive results samples by the ELISA test have 86 only because the sensitivity limit, the coincidence rate is 43.7%(86/197)and the undetected rate was 56.3%(111/197). Conclusion: For the samples with poor reactive HBsAg, undetected rate of ELISA test is very high, and the sensitivity is not good. Therefore, patient and blood donor HBsAg screening should be performed using an automated immunoassay. Moreover, it was suggested that HBsAg confirmatory test should be performed for poor reactive HBsAg samples that COI rang from 0.9 to 1.5.
D-35 Personalized Medicine in the Treatment of Myasthenia Gravis K. Nakatani1, Y. Sakamoto2, J. Nishioka2, N. Kawaguchi3, T. Nobori1. 1Mie University Graduate School of Medicine, Tsu, Mie, Japan, 2Mie University Hospital, Tsu, Mie, Japan, 3Chiba University Graduate School of Medicine, Chiba, Chiba, Japan
[Background] Myasthenia gravis (MG) is a prototypic antibody-mediated neurological autoimmune disorder. Thus, a wide array of immunosuppressive treatments has been established. In addition to the most popular drug, glucocorticoid, the calcineurin inhibitors (CNI), tacrolimus and ciclosporin, became also useful immunosuppressive drugs for the treatment of MG. CNI are widely used after organ transplantation but has a narrow therapeutic range and its pharmacokinetic variability complicates its daily dose assessment. CNI are metabolized by cytochrome-P4503A (CYP3A) enzymes and CYP3A5 gene polymorphism has been shown to influence tacrolimus blood concentration. In this study, it was investigated whether appropriate administration of tacrolimus for each subject was determined using CYP3A5 genotype. [Subjects] 74 Japanese subjects with MG were recruited in Chiba University Hospital. This study was approved by the ethics committee of both Mie and Chiba Universities, and written informed consent was obtained from each subject. [Methods] Tacrolimus administration was started at 3 mg/day and the maintenance dosage was adjusted to target trough levels. DNAs were extracted from pieces of nail obtained from each subject, and CYP3A4 (A-392G, *1B) and 3A5 (A6986G, *3) were genotyped by single primer extension method. To evaluate the effects of CNI and the possibility of substitution of CNI for predonisolone (PSL) with strong side effects, MG activities of daily living (MGADL) scale and the dose of PSL were assessed at baseline and 4 months later. [Results] Among 74 recipients, CYP3A4 *1B allele was not observed. The genotype frequencies of CYP3A5 were 10.8% for *1/*1, 29.7% for *1/*3 and 59.5% for *3/*3. As 3 allele is known to possess no metabolic activity for tacrolimus, recipients without *1 allele showed significantly higher plasma concentration of tacrolimus than those having CYP3A5 with 1 at trough level. Moreover, recipient with CYP3A5 *1 allele needed the higher maintenance dose of tacrolimus than those without *3 allele. Therefore, the maintenance dose of tacrolimus was strongly influenced by CYP3A5 genotype but ciclosporin showed no infuence of CYP3A5 genotype. PSL dose could be decreased at 4 months after administration of both tacrolimus and ciclosporin. Moreover, MGADL scores were similarly improved by both CNI. Thus, two CNI showed similar effects on MG treatment. [Conclusion] The great influence of CYP3A5 on the pharmacokinetics and pharmacodynamics of tacrolimus in recipients with MG suggests the need for pretreatment screening of this polymorphism to improve tacrolimus therapy. Two CNI
D-37 Hyperadiponectinemia predicts mortality in hemodialysis (HD) patients J. Racek1, M. Vostry1, D. Rajdl1, J. Eiselt1, L. Malanova2, T. Ladislav1. 1Charles University, Faculty of Medicine, Pilsen, Czech Republic, 2B. Braun Avitum, Pilsen, Czech Republic
Introduction and aims. Low adiponectin (ADPN) level is associated with adverse cardiovascular outcome and increased mortality in a variety of clinical conditions. Nevertheless, studies in HD patients addressing the ADPN value for survival and outcome prediction give inconsistent results. Methods. We performed a prospective cohort study with 204 HD patients (median [IQR] of age = 68 [59.8-74] years, dialysis duration = 16 [5-38] months, BMI = 26.5 [23.9-30.1] kg/m2, 76 males). Fasting serum total ADPN levels were measured using commercial ELISA kit. Results. During the follow-up period (28 [10-36] months) 92 patients died (45%). Non-survivors had significantly higher ADPN levels than patients who survived (22.76 [15.87-35.03] vs. 16.65 [23.9-30.1] mg/l, 95% CI for difference 2.68-9.00 mg/ l, p<0.001). Survival analysis was performed by Kaplan-Meier plot and by a Cox model. In a multivariate Cox regression model (adjusted for age, sex, BMI, HD duration, albumin and CRP level) ADPN emerged as significant independent predictive factor for all-cause mortality (relative risk per tertile 1.39, 95% CI 1.041.85, p = 0.025). Conclusions. Our results indicate that higher ADPN independently predicts all-cause mortality in HD patients. This study is not the first to show such association and the results support the hypothesis that in high-risk conditions such as uremia ADPN may not mediate its protective effects and rather reflects the disease severity. In this context, ADPN could promote and also mirror the progressive wasting of HD patients.The study was supported by research project MSM 0021620819.
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D-38 Effects of heart operations under mild and profound hypothermal cardiopulmonary bypass on red-cell immunity. J. HU, Y. TIAN. Department of Clinical Biochemistry, PLA General Hospital, Beijing, China
Siegel raised the theory of red-cell immune system according to many research results of predecessors and those of themselves in 1981. It is suggested from the theory that red cells have an immune function as well as a respiratory one. There are many materials on surface or in cytoplasm of erythrocytes associated with red-cell immunity, among which complement receptor type 1 (CR1, also named CD35), membrane attack complex inhibition factor (MACIF, also named CD59) and Duffy antigen receptor for chemokines (DARC, also named ECKR) are especially important. CR1 mediates the binding of immune complexes(IC) to erythrocytes, which is the passage of ridding IC from the circulation. DARC can regulate plasma levels of chemokines. While the binding of CD59 on erythrocytes to CD2 on T lymphocytes can activate and modulate T lymphocyte immunity. As cardiopulmonary bypass (CPB) is not a physiological circulation, many complications happen in single or multiple organs, which are mainly due to the damage of hematological system. The fact that RBCs are severely damaged in morphology and respiratory function after operation under CPB provokes our interests to know how about red-cell immunity in that condition. Eighteen patients with heart operation under CPB, 10 with mild hypothermia and 8 with profound hypothermia, were included in this study. Venous blood was drew to tube with EDTA at pre-CPB, minimum body temperature, end of CPB, and 1 day, 3 days, 7 days after operation. The level of CD35 and CD59 were measured with flow cytometry by fluorescence-activated cell sorter, and the results were showed as geometric mean fluorescence intensity ratio (GMFIR). The results showed that there were the same tendencies in mild hypothermal CPB (MHCPB) group and profound hypothermal CPB (PHCPB) group that CD35-GMFIR and CD59-GMFIR decreased at time of minimum body temperature and end of ECC compared to that at pre-CPB, and then turned back on 1 day, 3 days, 7 days after operation. But the descend extents of CD35-GMFIR and CD59-GMFIR in MHCPB group were bigger that those in PHCPB group, while the recovery period were longer. We conclude that the immune function of RBCs is damaged by heart operations under CPB, and the extent of red-cell immunity damage in MHCPB group is more serious than that in PHCPB group.

Results Reference range [mg/L] Overall analysis: (n=259) 0.45 to 0.85 Gender subgroups: Male (136) 0.45 to 0.93; Female (123) 0.44 to 0.88 Age subgroups: <50 years (137) 0.44 to 0.76; >50 years (122) 0.44 to 0.93 Best x-validated eGFR equations among adults (n=445) New equations Compact PETIA: 71.0 x CysC- 1.28 PETIA: 848.8 x x 0.857 (if female) Existing equations MDRD-IDMS Rule1: 66.8 x Cys C- 1.30 Mean of MDRD-IMDS and Rule 1 CysC- 0.796 x Crea- 0.468 x Age-0.089 P30% (95%CI) 82 (78 - 85) 92 (89 - 94) 78 (74 - 82) 83 (79 - 86) 89 (86 - 92) APE%
15 11
15 16 11
eGFR can further be enhanced by adding creatinine. More complex equations offered no significant improvement in accuracy. Conclusion: Reference range established with the new PETIA on the Abbott Architect cSystem confirmed published PENIA data (0.53 to 0.93 mg/L). Prediction of eGFR by compact PETIA and Rule1 was essentially equivalent.
D-40 Comparison of Urinary Catecholamins Results for Specimens Collected with Preservative Added at the Beginning or at the End of the Collection P. P. Chou1, W. Price2, K. Sisco1, N. Sherman1. 1Quest Diagnostics Nichols Institute, Chantilly, VA, 2Carilion Labs, Roanoke, VA
Introduction: Catecholamines in urine are very labile thus need to be preserved with acid while the 24-hour urine is being collected. Although acetic acid and/or boric acid can be used as the preservative, most laboratories prefer to use 25 mL 6N HCl as the preservative. The use of 6N HCl as preservative presents a challenge because patients may inadvertently be exposed to the strong acid. Method: In this study, five (5) volunteers were recruited to collect 24-hour urine specimens. Special urine containers (P-splitter) were used to collect the specimens. This container has two compartments (A and B). This container divides all urine fractions evenly into both compartments. Before the collection, 12.5 mL 6N HCl was added to compartment A. At the end of the collection, another 12.5 mL 6N HCl was added to the compartment B. Both aliquots were analyzed by HPLC with electrochemical detector. Results: ID 1 Analyte Norepinephrine Epinephrine Dopamine Norepinephrine Epinephrine Dopamine Norepinephrine Epinephrine Dopamine Norepinephrine Epinephrine Dopamine Norepinephrine Epinephrine Dopamine Before (g/24h) 11 36 214 4 42 217 13 53 264 14 59 242 22 57 257 After (g/24h) 10 32 210 5 41 198 12 56 309 13 46 156 22 66 294 Diff % -9 -11 -2 25 -2 -9 -8 6 17 -7 -28 -35 0 16 14 CV % 6.7 8.3 1.3 15.7 1.7 6.5 5.7 3.9 11.1 5.2 17.5 30.6 0 10.4 9.5
D-39 Clinical evaluation of a new Cystatin C Assay on the Abbott Architect cSystems and on AEROSET Clinical Chemistry Systems: Commutability and eGFR. F. Rota1, G. Borsani1, V. Maffulli1, R. Lucini1, J. Bjoerk2, M. Fritz3, J. Herzog3, H. Troonen3. 1Sentinel CH., Milan, Italy, 2Competence Centre for Clinical Research, Lund University Hospital, Lund, Sweden, 3Abbott GmbH & Co. KG, Diagnostics, Delkenheim, Germany
Objective: To introduce the new Cystatin C assay on the ABBOTT Clinical Chemistry Systems. This is primarily based on robust assay standardisation. We have generated clinical data documenting commutability of results and equivalency of reference range with the nephelometric immunoassay (PENIA). New eGFR equations were derived and compared with those published. Materials/Instruments: The new Cystatin C assay, SENTINEL CH., is a particle enhanced turbidimetric immunoassay (PETIA). Abbotts ARCHITECT cSystems and AEROSET Systems are random-access analyzers. Assay commutability: Review of published references ranges (13 PENIA, 4 PETIA, 4 not specified) supported standardization of the new PETIA. Using PENIA as basis target values were assigned to a panel of 8 human sera pools (HSP), ranging from 0.7 to 5.8 mg/L in three external laboratories, which is used to assign target values on commercial calibrator lots. Reference range study: 259 plasma samples from healthy blood donors were assayed on the Architect cSystem in duplicate. The reference range was defined at the 95% interval. eGFR derivation [GFR in ml/min per 1.73 m2]: 522 plasma samples from referred patients (445 adults, 77 children <18 yrs) were assayed on the Architect cSystem in duplicate. New and existing eGFR-prediction equations were derived and accuracy within 30% (P30%) and absolute % error (APE%) vs. reference iohexol assay measured GFR compared.
Conclusions: With a few exceptions, the variations are within the inter-assay variations for each analyte. Therefore, using limited number of specimens, we have demonstrated that the preservative used for urinary catecholamines can be added either at the beginning or at the end of the collection. More specimens need to be collected and analyzed to confirm this finding. Once confirmed, the potential risk for patients being exposed to caustic acid can be avoided.
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D-41 UVA Clinical Evaluation of Abbott Next Generation Calcium on the ARCHITECT ci8200 S. Colberg1, L. Legendre2, M. Edwards2, D. Armbruster1, Y. Lemma1. 1Abbott Laboratories, Irving, TX, 2University of Virginia Medical Laboratory, Charlottesville, VA
Objective: To compare assay performance of Abbott Clinical Chemistry Next Generation Calcium (Next Gen Ca), on-market Abbott Clinical Chemistry Calcium (Ca), and Randox Ca (R-Ca) assays for Imprecision and Method Correlation. Background: The Next Gen Ca is a liquid, ready-to-use, concentrated reagent that is automatically diluted on-board the instrument allowing for less operator intervention and instrument down time. The Next Gen Ca is available in multiple kit sizes to suit the testing needs of varying size laboratories. The University of Virginia Medical Laboratories (UVA) evaluated the Next Gen Ca assay for use on the ARCHITECT ci8200 analyzer during one week in which a large population of renal patients was scheduled for dialysis. Sporadic falsely increased Ca results for dialysis patients prompted specific evaluation of factors that might affect Ca values in this patient population. Method: A 5-day (N=20) precision study was conducted to evaluate the total imprecision of the Next Gen Ca using a normal and abnormal control material as samples. Method correlation was performed to compare the Next Gen Ca, Ca, and RCa reagents. A total of 199 serum and plasma samples were collected. 103 of these samples were from renal patients (pre and post dialysis) including 25 geriatric samples. The remainder of the samples consisted of 25 pediatric, 32 geriatric, 22 samples with additions of high concentrations of magnesium (a common potential interferent), and 17 normal patient samples. All samples were run on the three reagents simultaneously. Results Study Results Mean 11.3 mg/dL Total % CV 1.4 Mean 11.3 mg/dL Total % CV 1.7 Mean 10.8 mg/dL Total % CV 1.4 R = 0.994, Slope = 0.97, Int = 0.4 mg/dL R = 0.998, Slope = 1.02, Int = 0.3 mg/dL R = 0.995, Slope = 1.00, Int = 0.6 mg/dL Mean: 8.7 mg/ Next Gen dL Ca Total % CV 1.0 Mean: 8.6 mg/ dL 5-Day Precision Ca Total % CV 2.3 Mean: 8.2 mg/ dL R-Ca Total % CV 1.5 Next Gen Ca vs. Ca Method Correlation Next Gen Ca vs. R-Ca R-Ca vs. Ca
sensitivity 56.5%, PPV 37.3%, NPV 89.3%, accuracy 75.6% of urine leukocyte parameter. Accuracy value was 80.8 % when using bacteriuria and leukocyteuria parameters together. The highest specificity (99 %) and accuracy (82.3 %) level was established with urine leucocyte and bacteria and LE and, NO2 combination. The sensitivitity of this combination was 2.8%. Conclusion: Although the outcome of the Urised analyzer results specificity was in accaptable limits we observed the sensitivity value as low. Thus, Urised urinalysis results do not accurately predict the outcome of cultures.
D-43 Comparison of Aqueous sCD44 Level in Patients with Degenerative Myopia and Primary Open-Angle Glaucoma Y. U. Budak1, M. Akdogan1, K. Huysal2. 1Sevket Yilmaz Devlet Hospital, Bursa, Turkey, 2Yksek htisas Training Hospital, Bursa, Turkey
The transmembrane glycoprotein CD44 is a major hyaluronic acid (HA) cell surface receptor widely distributed in eye tissues and fluids. The shed ectodomain of CD44 is known as soluble sCD44 and is toxic to human TM cells and retinal ganglion cells in cell culture. sCD44 is released from cell surface by metalloproteases in response to metabolic stress or hypoxia. Glaucoma, one of the world's leading causes of visual impairment and blindness, is characterized by excavation of the optic nerve head and selective apoptotic loss of retinal ganglion cells (RGCs), resulting in a progressive decline in visual function. There are several types of glaucoma (e.g., primary open-angle, normal tension, and early onset-to name a few). Patients with degenerative myopia (spherical equivalent at least -6.0 D) are more susceptible to glaucoma . The purpose of this study was to investigate the concentration of soluble sCD44 in the aqueous humor of normal subjects and patients with primary open-angle glaucoma (POAG) and degenerative myopia without glaucoma to understand if the presence of this molecule may represent a protein marker of POAG. For this case control study, aqueous samples were collected from normal patients (n=16), patients with POAG (n=8) and patients with degenerative myopia (n=8) who underwent phacoemulsification surgery for mature or immature cataract. Aqueous specimens from anterior chambers were obtained during phacoemulsification surgery in all patients. The aqueous concentration of sCD44 was measured using a commercial ELISA kit (Bender MedSystems, Wien, Austria). In normal aqueous samples the sCD44 concentration was 5.40 1.21 ng/ ml, in degenerative myopia patients the sCD44 concentration was 5.76 1.15 ng/ ml. There was no statistical significant difference between these two groups (p >0.05). The aqueous sCD44 concentration of cases with POAG (12.2 10.1 ng/ ml) was higher than control group (p< 0.05). sCD44 concentration in aqueous is a possible protein biomarker of visual field loss in POAG.
Conclusion: The Abbott Next Generation Calcium Assay was very precise, delivers good correlation on serum and plasma samples, including samples from dialysis, pediatric and geriatric patients and also lacks interference from high magnesium. Lastly, the kit size for Next Gen Ca meets the workflow needs of large volume laboratories such as UVA, allowing for less operator intervention.
D-44 COMPARISON OF THREE AUTOMATED SYSTEMS FOR URINE CHEMISTRY AND URINE SEDIMENT ANALYSIS Y. U. Budak1, K. Huysal2. 1Sevket Yilmaz Devlet Hospital, Bursa, Turkey, 2Yksek htisas Training Hospital, Bursa, Turkey
D-42 CAN THE RESULTS OF THE URISED AUTOMATED ANALYZER BE USED IN EARLY PREDICTION OF URINARY TRACT INFECTIONS Y. U. Budak1, K. Huysal2. 1Sevket Yilmaz Devlet Hospital, Bursa, Turkey, 2Yuksek Ihtisas Training Hospital, Bursa, Turkey
Aim: The aim of this study is to evaluate if its possible to predict the positive cultures by analyzing the Urised (77 electronica, Budapest, Hungary) automated urine analyzer results. Methods:Leucocyte and bacteria counts were estimated by the analyzer. Urine cultures were performed by using conventional methods based on colony counting technique. Results: 386 patients attended at the laboratory for urinalysis and culture at the same day were included in the study. 68 % (263/386) of the patients were female, 32% (123/386) were male. 317 of the cultures did not give bacterial growth; culture positivity was found in 69 of the patients. Among the individual measures analyzed with Urised analyzer to discriminate culture positivity, we found a specificity 79.8%,
Urine analysis is an essential component of patient assessment, which is used for screening, diagnosing and planning care. Its assessment includes evaluation of physical characteristics, biochemical parameters (urine pH, blood, glucose, ketones, bilirubin, urobilinogen, and protein) and microscopic sediment evaluation (RBC, WBC, organisms, epithelial cells, crystals, and casts). Automated urinalysis systems can save labor and time and is more feaseble for high-volume laboratory workload. Aim of this study is to compare the diagnostic performance of 3 automated urinalysis systems (and the chemical units of these systems); namely the Iris iQ200 (AX-4280) (Iris Diagnostics, Chatsworth, CA), Sysmex UF-1000i (Urisys 2400) (Sysmex Corporation, Kobe, Japan) and Urised (LabuMAt) (77 electronica, Budapest, Hungary) and determine the convenience of use in the routine hospital laboratory. A total of 412 consecutive fresh urine samples, which were sent to the laboratory for urinalysis were analyzed by three automated urinalysis systems. The performance of the systems was evaluated and the differences between the instruments were converted to same scalas before data analysis. The pairwise concordance rate between the 3 instruments was defined by the percent of results within +/-1 grading from the best fit line. Correlation between the 3 instruments represented as within 1 grading difference was
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better between the LabUMat and AX-4280 chemistry systems for pH, blood, bilirubin, glucose and nitrite (>90 %). For protein, LE and nitrite, better correlation was observed between the Urisys 2400 and LabUMat (> 87 %), while the AX-4280 and Urisys 2400 conveyed better corralation for blood, glucose, urobilinogen and ketone (>90 %). When we compared the microscopic units; correlation between the 3 instruments was better between the iQ200 and Urised (92 %) for red blood cell data. For white blood cell data (86 %) and epithelial cell data(90 %) UF1000i and iQ200 conveyed better correlation. Bacteria counts showed the greatest differences by the 3 systems. Casts were difficultly differantiated by all of the systems. The automated urinalysis systems demonstrated acceptable correlations with each other in urine chemistries. On the other hand, automated microscopy units could be used as a screening procedure but some manual microscopy was still necessary. We suggest that measurements by urinalyzers may be reliably employed as a screening method to discard negative urine samples,while sediment examination may be employed as confirmation tests.This approach would increase the throughput and standardisation of laboratories.

significantly raised in study subjects (P<0.03) in comparison to controls. Serum iron, ferritin and erythrocyte lipid peroxidation statistically correlated positively while negatively with total iron binding capacity. However, there were no significant changes in these parameters between diabetic and non-diabetic acute myocardial infarction patients. The interplay of serum ferritin, total iron, total iron binding capacity and erythrocyte lipid peroxidation activity in potentiating and progression of diabetes and of its complication, an acute myocardial infarction has been discussed in the light of the observation that iron-catalyzed oxidative stress mediate apoptosis of pancreatic islets with a resultant decrease in insulin secretory capacity and the vascular endothelial dysfunction and ischemic myocardial damage.
D-47 Correlation between homocysteine and biochemical markers of renal function in the general population. G. Lippi1, G. Targher2, M. Montagnana1, G. L. Salvagno1, G. C. Guidi1. 1Laboratorio di Biochimica Clinica, University of Verona, Italy, 2Sezione di Endocrinologia e Malattie del Metabolismo, University of Verona, Italy
Context. Despite the well-known influence of renal function on homocysteine metabolism, there is contradictory information on the biological interrelationships between homocysteine and traditional or innovative markers of renal function. Material and method. Results of homocysteine, creatinine, cystatin C and folate, which were performed on consecutive outpatients referred by general practitioners to our laboratory for routine testing over the previous 12 months. Results. Cumulative results were retrieved for 140 adults >35 years old. After stratifying plasma homocysteine values according to the thresholds of creatinine, estimated glomerular filtration rate (e-GFR), and cystatin C, significant differences were observed in subjects with values of these biochemical markers suggestive for reduced renal function. Accordingly, the prevalence of homocysteine values 15 mol/L was significantly higher in subjects with biochemical markers suggestive for reduced glomerular filtration rate. Although plasma homocysteine was strongly associated with serum cystatin C, creatinine and e-GFR (all p<0.001) in multiple linear regression analysis, the only biological interrelationship remaining statistically significant after adjustment for age, sex and folate status was that with cystatin C (p<0.001) (table 1, Data are expressed as means and 95%CI or percentages.).
D-45 Uniparental Disomy Analysis using Linkage Mapping Set Markers J. Lee, A. Chhibber, B. Johnson, C. Davidson, D. Rodriguez, A. Pradhan, R. Padilla, R. Fish, S. Berosik, S. Hung, L. Joe, A. Felton, R. Petraroli. Life Technologies, Foster City, CA
In a departure from classical Mendelian genetics, there are a few imprinted genes that are only expressed from the allele inherited from one parent. Uniparental disomy (UPD) occurs when an individual inherits both copies of a chromosome pair from only one parent and no copies from the other parent. Individuals with UPD that have a deficiency in the expression of imprinted genes on Chromosome 15 are subject to two genetic disorders, Prader-Willi syndrome (PWS) and Angelman syndrome (AS). One method used to determine the presence or absence of the maternal or paternal chromosome is to interrogate the DNA from the individual and both parents by microsatellite or STR (Short Tandem Repeat) analysis with fluorescently labeled primers. We demonstrate new methods to compare the DNA via fragment analysis by capillary electrophoresis (CE). Such methods could help in producing consistent results across a wide range of laboratory environments. We also present a software analysis procedure to rapidly generate results and draw conclusions.
D-46 Ferritin an indices of body iron stores and acute myocardial infarction in diabetics A. M. Jarari, M. M. Abdelmoneim Farag, H. Hyder, J. R. Peela, R. Pathak. Department of Biochemistry,Al-Arab Medical University, Benghazi, Libyan Arab Jamahiriya
Body keeps most of the iron in the bound form by sequestering it in transport and storage proteins as a defence against oxidants. It is the catalytic form of iron which is responsible for the generation of free reactive oxygen radicals. Speroxide and other oxidants have also been involved in the release of ionic form of iron from ferritin. Iron is a transition metal that can catalyze toxic redox reactions, and it has been suggested to be involved in many harmful biological processes and diseases in the human body such as diabetes and cardiovascular diseases. The role of tissue iron and elevated body iron stores play in causing type 2 diabetes and or the pathogenesis of its important complications, particularly diabetic nephropathy and cardiovascular disease is lately under extensive investigations. A causative link with iron overload is suggested of the improvement in insulin sensitivity and insulin secretion with frequent blood donation and decreased iron stores. Viewing the participation of the free radicals in potentiating the pathogenesis of diabetes and its complications such as cardiovascular disease, it was fascinating to investigate the body iron status and lipid peroxidation activity in type 2 diabetics at the onset of acute myocardial infarction. The present study investigated 30 acute myocardial infarction patients with or without diabetes for the serum levels of ferritin, total iron, total iron binding capacity and erythrocyte lipid peroxidation activity by employing authentic analytical methods and compared with age and sex matched 30 healthy controls. A statistically significant elevated levels of serum iron (P<0.02), ferritin (P<0.001) and a decrease in total iron binding capacity (P<0.03) were observed in diabetics and non-diabetics also showed similar trends at the onset of acute myocardial infarction as compared with healthy controls. The erythrocyte lipid peroxidation activity was Conclusions. These results suggest that the measurement of cystatin C might be warranted to improve the clinical usefulness of homocysteine testing for assessing the thrombotic risk in the general population
D-48 Simple,rapid and sensitive detection of Toxoplasma gondii by loopmediated isothermal amplification J. Zhang1, M. Jiang2. 1Department of Biochemistry,Wuhan University School of Medicine, Wuhan, China, 2Department of Etiological Biology,Wuhan University School of Medicine, wuhan, China
Objective: The traditional clinical diagnosis of Toxoplasma gondii (T. gondii ) infection requires time-consuming cell culturing,mouse inoculation,which are not very sensitive and specific.Polymerase chain reaction(PCR)-based detection methods including nested PCR and real-time PCR need sophisticated equipments for amplification.In this study,a sensitive,specific, rapid and simple loop-mediated isothermal amplification(LAMP) method was developed for the detection of T. gondii DNA in blood and amniotic fluid from patients with suspected PCR diagnosed positive. The sensitivity and specificity of the LAMP were compared with those conventional and nested PCR.
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Methods: DNA from clinical samples was extracted with conventional phenol/ chloroform extraction.4 primers specific for 6 distinct regions of the T. gondii B1 gene(GeneBank AF179871) were designed.After amplifying DNA under isothermal conditions(65C) within 1 h by Bst DNA polymerase with strand-displacement activity. The LAMP reaction products can be simply judged with eye inspection by a white turbidity of magnesium phrophosphate or a colour change of a mixture with SYBR Green 1. In addition,LAMP and PCR products can be confirmed by 1.5% agarose gel electrophoresis and SYBR Green 1 staining. T. gondii tachyzoites and some parasites were used as positive,negative controls,respectively. Results: The LAMP assay showed higher sensitive than the conventional PCR and as sensitive and specific as nested PCR with a detection limit of 3 T. gondii tachyzoites per reaction.All the nested PCR positive samples were positive by LAMP.There was no cross-reaction with DNA of other parasite controls. Conclusions:The results demonstrated that the LAMP assay is a sensitive,specific,simple and rapid diagnostic method for T. gondii infection.One of the most important advantages of LAMP may be its simplicity and convenience requiring only a heating block or water bath for the reaction and can satisfy clinical and field applicability.
of hospital admittance. eGFR was estimated using the full MDRD equation containing six variables (MDRD 6) (n=8611) and the MDRD 4 (4 variables) equation (n=14658). Laboratories using a creatinine method calibrated to be traceable to IDMS used the IDMS-Traceable MDRD Study equation. Agreement between MDRD 4 and MDRD 6 classification in the present study was evaluated utilizing a Kappa statistic. Results: A correlation coefficient r of 0.9557(CI 95% 0.9559-0.9594) (P < 0.0001 ) (n = 8611) was found between eGFR MDRD 4 and eGFR MDRD 6. The strength of agreement between MDRD 4 and MDRD 6 CKD NKF/KDOQI stages 3-5 was very good, with a Kappa statistic 0.868 (CI 95%: 0.857-0.879).In a group of patients (n = 3032) with hypoalbuminemia (serum albumin <3.5 g/l) there was also a good MDRD 4 to MDRD 6 correlation: r = 0.9675 (CI 95%: 0.9652-0.9657); P < 0.0001. Conclusions: To our knowledge, this is the first study demonstrating a very good strength of agreement between MDRD 4 and MDRD 6 for the distribution of CKD NKF/KDOQI stages 3-5 in a large population of hospitalized patients belonging to different medical and surgical departments. Although a previous study states that MDRD equations are not reliable measures of actual level of renal function and may be unsuitable for clinical application in this population, their value is limited due to the fact that inpatient selection was based on the individual nephrologists perception of laboratory values which is not reflective of actual GFR and nearly half of the patients included in the investigation were at intensive care units. In our study only 4.6% of the study population was in the intensive care unit. The strength of agreement was similar even when the study was performed only in malnourished patients with decreased serum albumin concentration. However, limitations of this study include the fact that the MDRD equations have not been thoroughly validated in a large number of ill hospitalized patients, for whom the laboratory variables may be distorted.
D-49 The clinical value of fluorescent in situ hybridization and SYBR Green I real-time quantification PCR in rapidly detection and identification of Enterococcus faecalis in urine samples Y. Tang, Y. Li, M. Wang, Z. Xia, J. Gu, Q. Wu, H. Hu. Renmin Hospital of Wuhan University, Wuhan, China
Objectives: To evaluate the clinical value of tow rapid methods to detect and identify Enterococcus faecalis in urine samples. Methods: Using fluorescent in situ hybridization (FISH) with species specific oligonucleotide probe, fluorescently labeled with fluorescein Cy3, targeting the 16S rRNA of Enterococcus faecalis was hybridized for the urine samples. Specific primers were synthesized according to the specific gene sequence of heat shock proteins' groES of Enterococcus faecalis. To select 180 urine samples were simultaneously tested by cultivation, FISH, FQ-PCR. Results: The positive was detected by Cultivation, FISH, FQ-PCR each other in 9, 11, 11 of 180 samples, the negative was in 165 of 180 samples in common. The sensitivity of cultivation, FISH, FQ-PCR was 75% (9/12), 91.6% (11/12), 91.6% (11/12). Conclusion: Fluorescent in situ hybridization (FISH) and SYBR Green I real-time quantification PCR (FQ-PCR) are rapid, specific, highly sensitive technique for the early detection of Enterococcus faecalis. They represent a considerable advancement and a great potential for diagnosis urinary tract infection.
D-51 Multiparametric classification of fibrogenic liver diseases as exemplified by non-invasive Fibrotest/Actitest: Whom can you trust? O. A. Gressner, N. Beer, S. Stanzel, A. M. Gressner. RWTH-University Hospital, Aachen, Germany
Introduction: Non-invasive, i.e. serum-based assessment of liver fibrosis and fibrogenesis is still an important challenge although multiple single tests and multiparametric panels of biomarkers have been proposed (1). Aim: Assessment of the diagnostic validity of non-invasive biomarkers of liver fibrosis. Methods: Two approaches were used to assess the reliability of the tests: (i) Practical approach: Haptoglobin, ALT, gammaGT, alpha 2-macroglobulin, apolipoprotein A1 and bilirubin were measured in sera of 4 patients with histological proven fibrosis (staging F1-F4, grading A1-A3) were determined in 6 different quality-controlled laboratories. Inter-laboratory variations of the calculated Fibrotest Scores for staging and Actitest Scores for grading (both by BioPredictiveTM, France), and their error ratios compared to the results obtained by biopsy were calculated. (ii) Theoretical approach: The variability of obtained Fibrotest/Actitest Scores depending on 64 differential combinations of the allowed analyte-specific maximum/minimum permissible values for the parameters listed above as determined by the external quality control of the German Association of Clinical Chemistry and Laboratory Medicine (DGKL) was determined and the frequency distribution of the results calculated. Results: (i): Fibrotest and Actitest Scores were largely reproducible among the different laboratories. However, the error ratio was 77% for all Fibrotest- and 73% for all Actitest results when compared to the histological findings. (ii): Calculated scores varied among F2 (9%), F3 (31%), F3-F4 (6%), and F4 (53%) (Fibrotest), as well as A1/A2 (48%), A2 (9%), A2-A3 (5%), and A3 (38%) (Actitest). Conclusion: Despite reproducibility of Fibro- and Actitest results among the six tested laboratories, large scale investigation (n=64) displayed increasing variability of the results depending on interlaboratory variation of measured analyte concentrations that were still within the quality controlled, analytically acceptable range. Furthermore, calculated scores coincided with histological findings only in less than 25% of all cases. Thus, the diagnostic accuracy of these tests must be considered as low, if histology is accepted as gold standard. (1) Gressner, O. A. et al. Clin Chim Acta 381 (2007) 107-113
D-50 Estimation of renal function in hospitalized patients in Spain: Concordance between MDRD 4 and MDRD 6. E. Fernandez1, A. de Francisco2, J. Cruz3, M. Casas4, J. Gomez-Gerique2, A. Leon5, F. Cava6, J. Bedini7, A. Enguix8, E. Ripoll9, L. Borque10, A. Fernandez11, M. Arias12. 1Hospital Cabuenes, Gijon, Spain, 2Hospital Marques de Valdecilla, Santander, Spain, 3Departamento Estadistica. Universidad Autonoma, Madrid, Spain, 4Hospital Virgen de la Arrixaca, Murcia, Spain, 5Hospital Virgen del Rocio, Sevilla, Spain, 6Fundacion Hospital Alcorcon, Madrid, Spain, 7Hospital Clinic, Barcelona, Spain, 8Hospital Virgen de laVictoria, Malaga, Spain, 9Hospital Ramon y Cajal, Madrid, Spain, 10Hospital San Pedro, Logroo, Spain, 11Hospital Mexoeiro, Vigo, Spain, 12Hospital Marque de Valdecilla, Santander, Spain
Background: More than 500 million people worldwide have some form of chronic kidney damage (CKD). Despite this high prevalence, there has been relatively little attention focused on the prevalence of CKD among hospitalized patients, a population who generally is receiving potentially nephrotoxic drugs, exposed to major surgery or to radio contrast agents. It has been suggested that improved estimation of Glomerular Filtration Rate (eGFR) may be possible in sick by incorporating albumin and BUN levels. Objective: The aim of this study was to assess the agreement between MDRD 4 and MDRD 6 formulae in hospitalized patients. Materials and methods: Patients (n = 14 658) were recruited into this study from a population of adults (18 years) hospitalized at 10 centers in Spain since May to June 2007. Patients belonging to Obstetrical and Nephrology Departments were excluded. Serum samples were taken for the analysis of hemoglobin, creatinine, albumin and urea nitrogen upon at the time
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D-52 Fecal Calprotectin Levels in Patients with Colonic Polyposis A. Barassi1, R. Pezzilli2, A. M. Morselli-Labate2, S. Finazzi3, L. Fantini2, G. Gizzi2, M. Lotzniker3, V. Villani2, R. Corinaldesi2, G. Melzi d'Eril1. 1University of Milan, Milan, Italy, 2SantOrsola-Malpighi Hospital, University of Bologna, Bologna, Italy, 3Central Laboratory, Legnano Hospital, Legnano, Italy
Introduction. Calprotectin is a cytoplasmic antimicrobial compound prominent in granulocytes, monocytes, and macrophages. It accounts for approximately 60% of the total protein of the cytosol. Calprotectin can inhibit bacterial proliferation as a component of the innate immune response and through its iron-binding capacity (1).The usefulness of stool calprotectin determination in diagnosis of inflammatory disease of the colon has been reported; information about its usefulness for patientswith polyposis are scarce, however. Aim. To evaluate the significance of stool calprotectin concentrations for patients affected by colonic polyposis. Material and methods. Sixty-three consecutive patients (35 males, 28 females, mean age 60.3 years, range 3978 years) were enrolled: 26 patients (41.3%) with polyps, 17 patients (27.0%) with asymptomatic diverticular disease, and 20 subjects (31.7%) with normal endoscopic appearance of the colon. Calprotectin concentrations were measured by use of a commercial ELISA kit (CalprotectinELISA Kit, Immundiagnostik, Bensheim, Germany) based on a two-site sandwich technique with two selected monoclonal antibodies which bind to human calprotectin. Two 100-mg samples of faeces from a single stool sample from each participant were assayed, and the mean of the two measurements was recorded. According to the manufacturer, the reference range was <15 g/g of faeces. The within run %CV (n = 20) was 9.1 at 10.7 g/g and 4.2 at 22.2 g/g and the between-run %CV (n = 12) was 12.3 at 9.9 g/g and 6.1 at 22.5 g/g; the detection limit of the test was 2.9 g/g. Results. Stool calprotectin concentrations were 17.4 24.5 g/g for patients with colonic polyposis, significantly higher than concentrations for patients with diverticulosis (7.1 5.7 g/g; p = 0.026) or for patients with normal appearance of the colon (6.0 5.8 g/g; p = 0.003). For patients with a single polyp, stool calprotectin concentrations were similar to those for patients with multiple polyps. Calprotectin fecal concentrations for patients with sessile polyps and those with flat polyps were not significantly different (p < 0.05). Calprotectin concentrations were not significantly related to the size of the polyps. Conclusion. This study demonstrated that the presence of colonic polyps may increase stool calprotectin concentrations, which suggests that these colonic lesions should be suspected in cases of elevated stool calprotectin concentrations. Further studies are needed to confirm this initial promising observation. Reference. 1) DInca R, et al. Calprotectin and lactoferrin in the assessment of intestinal inflammation and organic disease. Int J Colorectal Dis 2007 ;22:429-37.

5 had the severe form of the disease. In the patients with acute pancreatitis, blood samples were taken on admission to the emergency room and afterwards daily, at the same time, for the following 2 days. All serum samples were frozen immediately after collection and stored at -20 C until analysis. VCAM-1, ICAM-1, E-selectin, P-selectin, and L-selectin were quantified by a biochip array analyzer (Randox Laboratories). The biochip used to measure all 5 molecules simultaneously consists of a 9.9-mm substrate on which discrete test regions have been constructed. After a simple enzyme-linked immunosorbent assay procedure, each spot is imaged to capture the chemiluminescent signals generated at each spot on the array. The light signal is captured by a chargecoupled device camera as part of an imaging station and converted by image-processing software to provide results compared with calibration curves for each location on the biochip. Intraassay imprecision was 8.2% to 10.0% and interassay imprecision was 8.6% to 11.0%. Results. Acute pancreatitis patients had vascular cell adhesion molecule 1 and P-selectin concentrations significantly lower and L-selectin concentrations significantly higher than the healthy subjects. Only E-selectin was significantly higher in severe than in mild disease (P = 0.029); a value of E-selectin ranging from 3.83 to 3.92 ng/mL was the best cutoff value for differentiating severe from mild acute pancreatitis (sensitivity: 60.0%, specificity: 90.0%, cases correctly classified: 80%). Eselectin and P-selectin entered the multivariate logistic regression analysis, and a score was calculated showing a sensitivity of 93.3% and a specificity of 86.7% in identifying the patients with severe pancreatitis. Conclusions. This score seems to be useful for the early assessment of the severity of acute pancreatitis.
D-54 Genetic Variations in CYP2B6 and Risk of Cyclophosphamide Toxicity in Patients Undergoing Myeloablative Hematopoietic Stem Cell Transplantation S. E. Melanson1, K. Stevenson2, H. Kim2, J. H. Antin2, M. H. Court3, V. T. Ho2, J. Ritz2, F. C. Kuo1, J. Longtine1, P. Jarolim1. 1Brigham and Womens Hospital, Boston, MA, 2Dana Farber Cancer Institute, Boston, MA, 3Tufts University School of Medicine, Boston, MA
Introduction: Genetic variations in cytochrome P450 2B6 (CYP2B6) may predict toxicity and survival in patients receiving high dose cyclophosphamide (CPA) prior to allogeneic stem cell transplantation (SCT). CPA requires biological activation to 4-OH CPA, primarily by cytochrome P450 2B6 (CYP2B6), to exert its effects. Patients with high enzyme activity, as reported for the *4 allelic variants of CYP2B6, may have an increased risk for CPA-associated toxicity, while patients with low enzyme activity, as reported for the*6 allelic variants of CYP2B6, may have a decreased risk of toxicity and improved survival. Methods: A total of 392 DNA samples were obtained from patients with hematologic malignancies who received CPA as part of a myeloablative SCT. Genetic polymorphisms in CYP2B6 were analyzed by a pyrosequencing method, which detected three SNPs, 516A>G, 785A>G and 1459C>T, associated with four allelic variants: *4 (785A>G), *5 (1459C>T), *6 (785A>G, 516G>T) and *7 (785A>G, 516G>T, 1459C>T). Outcomes were veno-occlusive disease (VOD) and CPA-related cardiac toxicity from 0 to 100 days post-transplant and overall and progression-free survival. Results: Fifty-six (14%) patients experienced toxicity (cardiac = 8; VOD = 48). The prevalence of the *4, *5, *6 and *7 alleles was 5%, 12%, 36% and 4%. Patients with allelic variations in CYP2B6 had similar rates of toxicity with those patients without allelic variation of CYP2B6 (i.e. *1/*1). When grouped by phenotype, patients with low protein activity (i.e. slow metabolizers), *6/*6, *6/*7, did not have a lower rate of toxicity compared to those with intermediate or high protein activity (i.e. intermediate and high metabolizers), *1/*1, *1/*4, *1/*5, *1/*6, *1/*7 and *5/*5, (p=0.72). However, slow metabolizers had a trend towards improved overall and progression free survival (p values of 0.22 and 0.16) (Figure).
D-53 Serum adhesion molecules in acute pancreatitis: time course and early assessment of disease severity A. Barassi1, R. Pezzilli2, M. M. Corsi3, A. M. Morselli-Labate2, A. D'Alessandro4, G. Dogliotti3, L. Fantini2, A. Malesci5, R. Corinaldesi2, G. Melzi d'Eril1. 1University of Milan, Milan, Italy, 2SantOrsola-Malpighi Hospital, University of Bologna, Bologna, Italy, 3Institute of General Pathology, University of Milan, Milan, Italy, 4Department of Gastroenterology, San Bortolo Hospital, Vicenza, Italy, 5Humanitas Hospital, Milan, Italy
Introduction. Inflammatory mediators play an important role in acute pancreatitis and in the development of distant organ complications of the disease. However, in acute pancreatitis, there is also microcirculatory derangement; the substances capable of determining these alterations and released during the inflammation have been characterized and they are called adhesion molecules (VCAM-1, ICAM-1, E-selectin, P-selectin, and L-selectin). Aim. We designed the present study to evaluate the behavior of adhesion molecules in the early phases of acute pancreatitis and to explore the possibility that these proteins are also useful in assessing the severity of the disease. Material and methods. Fifteen consecutive patients with acute pancreatitis (7 males, 8 females; average age 63.114.5 years) were studied. The patients were admitted to the emergency room within 6 hours after the onset of pain. Diagnosis was based on a history of upper abdominal pain associated with at least a 2-fold increase in serum lipase and was confirmed by contrast-enhanced computed tomography. The pancreatitis was of biliary origin in 6 patients, and of unknown origin in the remaining 3. According to the Atlanta criteria, the patients were classified into 2 subgroups: 10 had the mild form and
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Conclusion: Patients with allelic variants of CYP2B6 that underlie the slow metabolizer phenotype may have improved survival.
limit is as low as 20%. Gestational age specific reference values are also recommended to be use for fibrinogen, factor VII, VIII & IX, while the usefulness of measuring D-dimer during pregnancy is doubtful. Nearly all women from gestation week 20 had D-dimer values above the conventional cut-off point of 0.5 mg/l with increasing levels peaking at delivery. We had too few cases with thromboembolism, but it is unlikely that D-dimer levels can differentiate between normal and pathological cases.Most values fluctuated somewhat more around the delivery, and were similar independent of delivery method.
D-55 Validation of a Sex Hormone-Binding Globulin (SHBG) Immunoassay on the ADVIA Centaur Immunoassay System from Siemens Healthcare Diagnostics C. Ahnadi1, J. Pomerleau1, M. Leonard2, R. Ludewig3, S. Tan3, J. Fan3, W. Canfield3, J. Martel3, A. Grant1. 1Collaborative Reseach for Effective Diagnostics, CHUS, Sherbrooke, QC, Canada, 2White Plains Hospital, White Plains, NY, 3Siemens Healthcare Diagnostics, Tarrytown, NY
Sex hormone-binding globulin (SHBG) is a glycoprotein, produced by the liver, with a high binding affinity for steroid hormones such as estradiol, dihydrotestosterone, and testosterone. SHBG measurement is a useful indicator of androgen disorders, including hirsutism, when it is combined with testosterone measurement to calculate a free testosterone index (FTI). The FTI is determined by calculating the ratio of testosterone to SHBG. Siemens is developing a fully automated SHBG immunoassay* for testing plasma or serum samples on the ADVIA Centaur platform. The objective of this study was to evaluate the analytical performance of the SHBG assay on this platform. The ADVIA Centaur SHBG assay is a quantitative, two-site sandwich immunoassay. Relative light units (RLUs) detected by the system are directly proportional to the amount of SHBG present in the test sample. The assay range is 0-180 nmol/L. The FTI is automatically calculated and reported by the ADVIA Centaur system. Imprecision was assessed at two clinical sites (US and Canada) on repeated measurements using calibrators, control material and prepared pools. The within-run CVs ranged from 2.7% to 4.0% and total CVs from 3.2% to 4.3% on samples with SHBG concentrations of 9.62-52.15 nmol/L. The distribution of within-run CVs for 250 patient samples showed that more than 90% of samples had a CV of 5%. A method comparison against the Roche Elecsys 2010 was performed at one site on 250 samples, including 50 pediatric samples, covering the range of the assay. Two lots of SHBG reagent were tested; their correlation coefficients were 0.992 and 0.993. In conclusion, preliminary assessment of the results of this clinical evaluation indicate that the ADVIA Centaur SHBG immunoassay is a precise method for measuring SHBG in serum across a wide range of clinically relevant concentrations and shows equivalent performance to the Roche Elecsys 2010 SHBG assay. * In development. The performance characteristics of this product have not been established. Not available for sale.
D-57 Analytical Performance of the Enhanced Liver Fibrosis (ELF) Assays on the ADVIA Centaur Immunoassay Systems from Siemens. Comparison of ADVIA Centaur and Immuno1 ELF Scores Using Patient Samples. P. W. Dillon1, R. Payne1, H. Leipold1, R. Cross2, B. Bossen1, C. DiPasquale1, L. Halik1, C. Krumm1, L. Oppenheimer1, A. Yarovoy1. 1Siemens Healthcare Diagnostics, Tarrytown, NY, 2iQur, Ltd, Southampton, United Kingdom
Background: The Enhanced Liver Fibrosis (ELF) score is calculated from the noninvasive test results of three direct markers of liver fibrosis: hyaluronic acid (HA), N-terminal propeptide of type III collagen (PIIINP), and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1). The ELF score has performed well clinically (see, e.g, Rosenberg et al. Gastroenterology. 2004;127:1704-1713). It is CE marked and available in Europe on the Immuno1 Immunoassay System. The assays and the score function are under development for use on the ADVIA Centaur Immunoassay Systems.* Objectives: Determine if the individual assays have acceptable precision on the ADVIA Centaur systems. Determine the correlation of the ADVIA Centaur ELF assays and score with the Immuno1 ELF assays and score. Methods and Materials: Remnants of routine serum samples from a reference laboratory, iQur, Ltd., were assayed in duplicate on 1 day at Siemens Tarrytown, NY, laboratory with the ELF assays on an ADVIA Centaur XP Immunoassay System. The samples had been assayed at iQur over a period of 13 months (with recalibrations at least every 2 months) on the Immuno1 system. Patient sample correlations were calculated for each assay (and their logarithms) and ADVIA Centaur ELF score coefficients estimated from the Immuno1 coefficients and the correlation parameters. The resultant ADVIA Centaur ELF score was correlated with the Immuno1. PassingBablok regressions (Analyse-it software) were used exclusively. The ADVIA Centaur ELF score within-run precision profile was estimated from the sample duplicates. Precisions of the individual assays were estimated from assays of single-analyte controls, calibrators and standards in CLSI EP-5A-like protocols run on ADVIA Centaur XP and, for PIIINP and TIMP-1, on ADVIA Centaur CP systems. Results: A total of 136 patient samples were included in the correlations. The Immuno1 score equation includes a term proportional to the logarithm of each markers concentration and a constant. It is straightforward to adjust its coefficients to ADVIA Centaur using the parameters from the fitted correlations of the three individual methods. The resultant ADVIA Centaur score correlated well with the Immuno1 score: (Centaur)=0.991(Immuno1) + 0.0811, r=0.96, range=6.4-13.8 The slope and intercept were not significantly (95% confidence) different from 1 or 0. The samples ranged from negligible fibrosis to cirrhosis. Discounting one outlier, patient sample within-run score SDs ranged from 0.00 to 0.14 (CVs are inappropriate for ELF scores). For the individual assays, within-run precisions with controls, etc. were less than 11% (HA), 6% (PIIINP) and 5% (TIMP-1). The corresponding total precisions were less than 15% (HA), 10% (PIIINP) and 6% (TIMP-1). Conclusions: The ADVIA Centaur and Immuno1 ELF scores have excellent correlation. The ADVIA Centaur ELF scores and the individual assay concentrations have acceptable precisions. * The ADVIA Centaur assays are not commercially available, and the Immuno1 assays are not available in the US.
D-56 Gestation Age Specific Reference Intervals for Coagulation Tests During Uncomplicated Pregnancy Delivery/Caesarians and Early Postpartum Period P. B. Szecsi, M. Jrgensen, A. Klajnbard, N. P. Colov, M. R. Andersen, A. Barfoed, K. Haahr, B. Bjrngaard, S. Stender. Gentofte Hospital, Hellerup, Denmark
The physiological changes occurring during pregnancy may affect biochemical parameters. Most reference values are based upon samples from non-pregnant women not necessarily useful for clinical decision in pregnant women. Eight hundred and one women were recruited among 2147 women attending first trimester screening. Among these, 391 women with a totally uncomplicated pregnancy, delivery/caesarians and puerperium were identified. Plasma were obtained at gestational week 13-20, 21-28, 29-34, 35-42, at active labor and one and two days postpartum. All tests were assayed on the STA-R Evolution coagulation analyzer with reagents from Stago. Reference ranges (2.5th and 97.5th percentiles) were calculated for each test and gestational period using RefVal ver. 4.11 as recommended by IFCC. Free protein S decreased slightly (20%) while protein S activity decreased substantially (50%). In contrast, Factor VII (50%), VIII (100%), IX (50%), Ddimer (400%) and fibrinogen (50%) increased during pregnancy. Quick prothrombin time, aPTT, antithrombin, protein C, coagulation factors II, V, X, XI, & XII did not change significantly. Protein S deficiency during pregnancy is not easily revealed, however measurement of free protein S antigen might be indicative. If protein S activity is measured, gestational specific reference intervals is mandatory, however, the lower reference
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D-58 The Clinical Performance of the Immuno1 Enhanced Liver Fibrosis (ELF) Score Is Robust to the Biopsy Staging System and Dichotomization Level Used to Fit Its Coefficients P. W. Dillon1, J. Parkes2, S. Harris2, R. Cross3, A. Burt4, W. Rosenberg5. 1Siemens Healthcare Diagnostics, Tarrytown, NY, 2The University of Southampton, Southampton, United Kingdom, 3iQur, Ltd., Southampton, United Kingdom, 4School of Clinical and Laboratory Sciences, University of Newcastle Upon Tyne, Newcastle Upon Tyne, United Kingdom, 5Centre for Hepatology, University College London & iQur, Ltd., London & Southampton, United Kingdom
Background: The gold standard for assessment of liver fibrosis is liver biopsy, a procedure with many drawbacks. IVD markers, as well as other noninvasive substitutes, are the subject of great interest. IVDs may include both direct and indirect markers of fibrosis. Indirect markers reflect liver function, whereas direct markers are directly involved with fibrogenesis and/or fibrolysis. The ELF score is calculated from three direct markers: hyaluronic acid (HA), N-terminal propeptide of type III collagen (PIIINP), and tissue inhibitor of matrix metalloproteinase 1 (TIMP-1).* The score coefficients have been fitted using logistic regression of Ishak stage 4-6 vs. 0-3 (one of many biopsy staging systems). Objectives: To determine the score performance over the entire range of fibrosis as well as in differentiating the categories of fibrosis used to set its coefficients. Determine if performance depends on the biopsy staging system used to set its coefficients. Methods and Materials: Sera were collected at 13 European sites from 921 patients undergoing liver biopsies. Each was evaluated against the Ishak (0-6) and Scheuer (04) systems. Patients had different etiologies. Fibrosis ranged from none to cirrhosis. Immuno1 HA, PIIINP and TIMP-1 assays were run on each sample. Score coefficients were defined by a logistic regression of the probability that the Ishak score was greater than I (for I=0 to 5) vs. the sum of ln(Concentration) terms: Logit[Prob(Ishak > I)]=AI + 3M=1 BI,Mln(ConcM) The commercial ELF score was evaluated with I=3. An ordinal regression that simultaneously fits all six dichotomizations with fixed BMs but variable AIs was done (as a score, the six AIs were averaged). Similar calculations were done vs. Scheuer staging. The regressions were done using SAS PROC LOGISTIC. The clinical performance of each score equation is estimated by the area under an ROC curve (AUROC). A curve for discriminating between, say, Ishak 0-1 and 2-6 may be calculated using any score (e.g., that fitted vs. Scheuer 0-3 vs. 4). Is best performance achieved using the same dichotomization for both fitting and discriminating? As seen below, the AUROC for any discrimination is effectively independent of the score chosen. ROC curves were calculated, using Analyse-It software, for all combinations of score equations and discriminations. The data used to set scores was also used to calculate AUROCs. Results: The AUROC depends on the discrimination chosen, increasing from milder to more severe fibrosis, but is effectively independent of score equation chosen. The AUROCs for discriminating Ishak stages: 0/1-6: 0.714-0.722, 0-1/2-6: 0.764-0.768, 0-2/3-6: 0.784-0.787, 0-3/4-6: 0.828-0.832, 0-4/5-6: 0.855-0.860, 0-5/6: 0.874-0.876. For Scheuer: 0/1-4: 0.714-0.722, 0-1/2-4: 0.781-0.784 0-2/3-4: 0.829-0.833, 0-3/4: 0.869-0.872. Conclusions: The AUROC of the Immuno1 ELF score to differentiate any particular level of fibrosis is independent of the level (or staging method) of fibrosis used to estimate its coefficients. A single score equation may be used over the entire range of fibrosis. * The Immuno1 ELF markers are CE marked and available in the UK through iQur, Ltd. They are not available for sale in the US.

D-59 Erythropoietin therapy leads to a reduction in hepcidin in chronic renal disease patients M. Busbridge1, D. Ashby1, D. P. Gale2, R. Chapman1, M. Patrick1, S. Bloom1. 1Imperial College Healthcare NHS Trust, London, United Kingdom, 2University College London, London, United Kingdom
Background: The hepatic hormone hepcidin prevents iron overload by inhibiting iron absorption and transport, but its increase in inflammatory states restricts iron available for erythropoiesis contributing to the anaemia of chronic disease. Levels are also high in renal failure, preventing the absorption of dietary iron, and possibly underlying erythropoietin resistance. However, using a recently developed immunoassay to measure circulating hepcidin in haemodialysis patients, we found that high erythropoietin requirements were associated with low, rather than high, hepcidin levels. Methods: Using a competitive radioimmunoassay, plasma hepcidin was measured in stable chronic kidney disease patients with moderate anaemia, before and after starting treatment with intravenous iron or erythropoietin. Results: Following a single intravenous dose of 200mg iron sucrose in 4 patients, hepcidin was markedly increased (59.3+/-18.6 vs 18.1+/-9.6ng/ml, p=0.047). In 7 patients hepcidin levels were significantly reduced after a single dose of 20mcg darbepoitein alfa (60.7+/-6.0 vs 71.0+/-4.7ng/ml, p=0.045) remaining at the lower level after several weeks of continued therapy (60.0+/-7.2ng/ml). There were no concurrent changes in iron indices, or in circulating levels of the putative hepcidin regulator GDF15 (growth differentiation factor 15). Conclusion: Erythropoietin therapy leads to a reduction in circulating hepcidin. The resulting improvement in iron transport may be an important component of its efficacy in renal anaemia. Studies characterising this effect in healthy controls are planned.
D-60 Hepcidin levels in inflammatory bowel disease compared with healthy controls M. Busbridge1, J. Arnold2, A. Sangwaiya2, R. Chapman1. 1Imperial College Healthcare NHS Trust, Clinical Biochemistry, London, United Kingdom, 2Ealing Hospital NHS Trust, Gastroenterology Department, London, United Kingdom
Introduction: Hepcidin is the principle regulator of iron homeostasis and has been considered as a key player in refractory anaemia in chronic inflammatory diseases, such as inflammatory bowel disease (IBD). We report the concentrations of hepcidin in IBD, measured using a competitive radioimmunoassay (RIA) Aims & Methods: Hepcidin, prohepcidin and ferritin along with haemoglobin (Hb), mean corpuscular volume (MCV) and iron were measured in 61 patients with IBD. Iron Deficiency Anaemia (IDA) was defined as Hb <12.5g/dL, MCV <80fL and iron <10.6 mol/L. Patients with IDA (n=18), IBD-IDA group. Patients with normal Hb, normal or high ferritin, normal or high iron and normal MCV (n=41), IBD-non anaemic group. Mean age of patients with IBD was 44.2yrs (18-88). Compared with healthy controls (n=25), mean age 36.04yrs (20-62) Results: Mean hepcidin in IBD-IDA was 4.14 3.06ng/mL. The mean prohepcidin 267.1 457.59ng/mL. The mean ferritin 15.28 19.35 g/L. Hepcidin correlation with ferritin (r=0.693, p=<0.001). The mean hepcidin level in IBD-non anaemic group was 6.81 7.68ng/mL, mean prohepcidin 104.56 76.8ng/mL and mean ferritin 110.62 138.34 g/L. The mean hepcidin level in healthy control group was 15.3 15.71ng/ mL, mean prohepcidin 236.88 83.68ng/mL and mean ferritin 109.8 128.08 g/L. Conclusion: The mean hepcidin level in IBD-IDA group and IBD-non anaemic group were significantly lower (p<0.05) when compared with healthy controls. There was also a clear positive correlation between ferritin and hepcidin (p=<0.001). The low hepcidin in patients with IBD maybe due to loss of hepcidin from the intestinal mucosa and may perpetuate inflammation by reduction in the natural protective effect as part of innate immunity.
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D-61 The use of an automated 12-part dipstix for routine urinalysis T. C. Aw, K. Teo, W. R. Lim, P. Q. Leow, Q. Liu, S. P. Tan. Changi General Hospital, Singapore, Singapore
Background: Microscopic urinalysis is labor-intensive and operator-dependent. Some laboratories advocate the use of negative dipstix via chemical screening to obviate microscopy. However, manual dipstix evaluation is affected by wide observer variability and subjectivity. As such a manual 10-parameter dipstix is included as part of our routine urinalysis. Objective: We evaluated the use of a newly available automated 12-part dipstix analyzer (Clinitek Atlas, Siemens) for urinalysis. Methods: The Clinitek assesses 12 parameters: color, clarity, glucose, Bilirubin, ketone, specific gravity, hemoglobin (blood), pH, protein, urobilinogen, nitrite (N), and leukocyte esterase (LE) in 16 seconds (225 samples per hour) from as little as 2 mL of urine. A roll of 490 test strips can be loaded at one time. The Clinitek can accommodate 20 sample racks containing 10 samples each. Freshly collected urine samples without preservatives submitted to our laboratory for diagnostic urinalysis were studied; 605 dipstix normal samples were independently examined by microscopy and urine culture. Results: Microscopy and dipstix concurred in 81.1% (491/605) of the cases; 71 of these 491 urines (14.5%) had positive culture results; 114 of the dipstix negative cases (18.9%) had abnormal microscopy reports including 30 culture positive cases. It is disconcerting that 48/101 (47.5%) culture positive samples contained mixed bacteria flora i.e. contaminated collections, underscoring the need for proper sample procurement. In this cohort of 605 samples microscopy as a diagnostic test had a sensitivity of 29.7% (30/101), false positive rate of 73.7% (30/114), specificity of 83.3% (420/504) and negative predictive value (NPV) of 85.5% (420/491); NPV for dipstix was 83.3% (504/605). In another 1601 samples submitted for urine culture the performance of the Clinitek LE, N were evaluated. When LE, N were both negative 83.4% of 1371 samples were also culture negative; 14.1% of the samples were contaminated. When LE, N were both positive 79.5% of 230 samples were culture positive; 23% of the samples were contaminated. Conclusion: The improved precision, elimination of personnel variability, ease of use, automation, and high throughput of the Clinitek has encouraged us to use it to triage our urinalysis samples. When all 12 Clinitek parameters are normal microscopy is not performed except for patients from the nephrology service. Our average turnaround time for urinalysis from sample receipt to result release is now 15 minutes.
Samples in each panel were randomized and blinded to the user prior to testing. Validation: The study generated a total of 2250 results for each assay. For the CTQ assay, the percentage correct for the panel members at 0x, 2x, and 5x the analytical LOD is 99.6% (538/540), 100.0% (540/540), and 100.0% (270/270) respectively, across days and sites. For the second panel members, the percent positive at 0.1x the LOD was 67.7% (303/450) across days and sites, and the percent positive for the panel members at 0.01x the LOD was 10.4% (47/450) across days and sites. For the GCQ assay, the percentage correct for the panel members at 0x, 2x, and 5x the LOD is 99.2% (536/540), 100.0% (540/540), and 100.0% (270/270) respectively, across days and sites. For the second panel members, the percent positive for the panel members at 0.1x the LOD was 91.8% (413/450) across days and sites, and the percent positive for panel members at 0.01x the LOD was 26.7% (120/450) across days and sites. There was no statistical difference in performance between sites. Conclusions: For the CT/GC negative simulated urine and swab samples and those spiked with organisms at either 2x or 5x the specified analytical LODs of the CTQ and GCQ assays, there was >99% agreement with the expected results. As expected, lower levels of agreement were obtained with samples spiked below the analytical LODs of the two assays.
D-63 Report on detecting Cystatin C concentration in serum and pleural effusions W. Chen, J. Tu*, H. Hu, M. Lv. Department of Laboratory Medicine, Center for Genetic diagnosis, Zhongnan Hospital,Wuhan University, Wuhan,Hubei, China
Objective: To study the correlation between Cystatin and traditional renal biomarker Creatinine and BUN, we analyzed Serum BUN, Creatinine, also measured Systatin C concentration with samples from pleural effusions. Materials and methods: 695 (male 352, female 342) serum samples from hospitalized patients had been collected and used to detect concntration of BUN, Creatinine and Cystatin C. 48 pleural effusion sampls were used to determin CysC level on on automatic biochemical analyzer (Aeroset, Abbott). Results: Out of 353 male cases, mean age: 4511, BUN: 5.916.52mmol/L (40 cases off range, 11.33%), Cr: 103107mol/L (31 cases off range, 8.78), Cysc: 1.080.74mg/L (63 cases off range, 17.85%), 342 felmale cases, mean age: 4311, BUN: 3.963.11mmol/L (8 off range, 2.34%), Cr: 67.918.3mol/L (2 off range, 0.58%), Cysc: 0.860.33mg/L (36 off range, 10.53%), Among 48 pleural effusion cases, mean age: 6117, BUN: 6.735.30mmol/L (14 cases off range, 29.1%), Cr: 10387mol/L (6 off range, 12.5%), serum Cysc: 1.330.92mg/L (18 off range, 37.5%), pleural effusion CysC: 2.161.05 (44 cases over serum reference range, 37.5%). Average pleural effusion CysC conc. is about 1.6 folds of serum Cysc conc. Conclusions: Pleural effusion has higher CysC level than serum.less than 20% patients has abnormal CysC level in male and less than 10% in female patients. When use Cr as cut off standard, less than 10% patients found Cr off range in both male and female which indicate CysC is more sensitive than Cr. 1=Department of Laboratory Medicine, Center for Genetic diagnosis, Zhongnan Hospital, Wuhan University * To Whom correspondence should be addressed znyyjyk@sina.com
D-62 Analytical Reproducibility of the BD Viper System with XTR Technology (extracted mode) for the Detection of Chlamydia trachomatis and Neisseria gonorrhoeae C. Welborn1, W. LeBar2, S. Taylor3, E. W. Hook4. 1Becton Dickinson Diagnostic Systems, Sparks, MD, 2Hospital Consolidated LaboratoriesProvidence Hospital, Southfield, MI, 3LSU Health Sciences Center, New Orleans, LA, 4University of Alabama at Birmingham, Birmingham, AL
Objective: The objective of this study was to determine the reproducibility of qualitative test results obtained with a sixteen panel member panel of simulated Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) positive and negative urine and swab specimens using the BD Viper System with XTR Technology (BD Viper System with XTR) and BD ProbeTec CTQ and GCQ Amplified DNA Assays. The study included analysis of within run and between run reproducibility in addition to instrument to instrument reproducibility across three viper instruments. The target levels tested were designed to span above and below the analytical limits of detection (LODs) of the CTQ and GCQ assays. Methods: Three clinical sites were each provided five identical reproducibility panels, each consisting of 90 simulated urine and swab samples. The 90 panel members were prepared using BD ProbeTec CT/GC Qx Swab Diluent (Qx Swab Diluent) and were either left unspiked (0x) or spiked with known quantities of CT serovar H and/or GC strain ATCC 19424 at 2x or 5x the specified analytical LOD for each analyte. Samples that were not spiked with one or both organisms were considered to be negative for the respective analyte(s). Additionally, simulated swab panels contained a clean endocervical swab. Panels were run once a day at each facility over five consecutive days. A second series of 90 panel members was prepared in Qx Swab Diluent at target levels of 1:10 (0.1x) and 1:100 (0.01x) of the specified analytical LOD for each organism. The levels for these panel members were selected to fall within the dynamic range of the analytical LOD curves of the assays.
D-64 Validation of the Brahms Kryptor Procalcitonin Assay in Surgical ICU and Trauma ICU Patients E. K. O'Keeffe, C. K. Lee, H. G. MacNew, J. M. Jenkins, A. K. May, A. Woodworth. Vanderbilt University Medical Center, Nashville, TN
Background: Sepsis is a major cause of death among critically ill patients. Early diagnosis is essential for patient management and outcome. Measurement of serum procalcitonin (PCT) in Intensive care unit (ICU) patients helps identify patients with sepsis earlier than bacterial cultures. However, PCT is elevated after surgery or trauma in the absence of sepsis. The diagnostic utility of PCT to identify early sepsis may be different among patients with recent surgery or trauma. Objective: To validate the BRAHMS KryptorPCT assay for use in trauma and surgical ICU patients. Methods: All patient samples were left-over serum or plasma specimens collected from Surgical or Trauma ICU physician ordered CRP or prealbumin analysis performed in the Vanderbilt University Core Laboratory. Sepsis was determined based upon criteria defined by the Society of Critical Care Medicine. Procalcitonin assays
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were performed on the BRAHMS Kryptor, a Time Resolved Amplified Cryptate Emission assay. For stability studies, known concentrations of pooled patient samples were incubated at 25C, 4C and -20C for up to 72 hours. 44 corresponding plasma and serum samples were analyzed for specimen type comparisons. The utility of PCT to predict sepsis was determined by ROC analysis. Results: Precision, linearity and recovery for the KryptorPCT assay were previously validated. PCT was stable in serum samples stored at 4 and -20C for up to 72 hours. PCT concentrations decreased by 12% after 24 hours in samples stored at 25C. PCT was significantly elevated in patient samples with high calcitonin. Patients with calcitonin concentrations of ~300 and 6200 pg/mL yielded PCT results of 7.5 and 274.4 ng/mL respectively. PCT concentrations were consistently higher in plasma compared with serum from matched patient samples, where plasma=1.24(serum)-0.2 (r2=0.99). 113 samples from 56 patients were collected from the TICU (n=42 samples, 18 patients) and SICU (71 samples, 38 patients). Of these, 34 patients (61%) developed sepsis; 10 (56%) were from the TICU and 24 (63%) were from the SICU. The areas under the ROC curves were 0.7 (95% CI, 0.59-0.8), 0.84 (0.71-0.96) and 0.62 (0.48-0.76) for the combined population, TICU and SICU respectively. Using ROC curves, an optimal cutoff with maximum sensitivity (74%) and specificity (65%) was 0.5ng/mL for the total population, 0.5ng/mL for the TICU (sensitivity= 56%, specificity=94%) and 15 ng/mL for the SICU (sensitivity=27%, specificity= 6%). Odds ratios for developing sepsis were 0.2 (p<0.0001), 20.3 (p=0.001), and 3.3 (p=0.044) for the total population, TICU and SICU respectively. Conclusions: The BRAHMS KryptorPCT assay is a precise, rapid method for detecting PCT in serum and plasma of patients from the SICU or TICU. The difference in measured PCT between serum and plasma suggests that different specimen types should not be used interchangeably. PCT is unstable in serum at 25C after 24 hours. Different optimal PCT cut-offs for evaluating sepsis in TICU and SICU patients were observed, likely due to non-septic elevation of PCT after surgery or trauma. Individual reference ranges for PCT must be determined for unique patient populations in the context of clinical picture.

prognosis in SICU and TICU patients. PCT should be used as a tool in combination with clinical parameters (including days post trauma or surgery) and other laboratory values in order to evaluate infection and sepsis in these patients.
D-66 Validation of the IMMULITE 2000 Syphilis Screen Assay from Siemens Healthcare Diagnostics N. M. Ureda1, A. Wu1, Z. Bostanian2, M. Edwards3, L. Silverman3, J. Martel4, C. M. Conarpe4, W. Gentzer4, Y. Luna4, A. Zhao4, M. Ghadessi4, M. Mahue4, R. Streefkerk5, M. van Westreenen5. 1University of California, San Francisco General Hospital, San Francisco, CA, 2Northridge Diagnostic Laboratory Inc, Northridge, CA, 3University of Virginia, University Hospital, Charlottesville, VA, 4Siemens Healthcare Diagnostics, Tarrytown, NY, 5Erasmus University MC, Rotterdam, Netherlands
Syphilis is caused by the spirochete Treponema pallidum. The disease is transmitted primarily via sexual contact but can also be transmitted from mother to fetus or through transfusion of blood and blood components. Syphilis that is untreated and progresses to the tertiary stage may cause irreversible and potentially lethal damage to neurologic, cardiovascular, hepatic and bone tissues. Therefore, early diagnosis of syphilis is important. Diagnosis of syphilis is primarily made through a combination of either nontreponemal and/or treponemal diagnostic techniques. Siemens is developing an assay* on the IMMULITE family of instruments for the detection of T. pallidum antibodies in human serum and heparinized plasma. In-house precision results (CVs) were 5.1%-7.9% (within-run) and 6.3%-12.6% (total) for lot A; CVs for lot B were 5.0%-6.9% (within-run) and 6.7%-8.7% (total). External clinical trials were conducted at two US sites and one European site to validate the performance of the IMMULITE 2000 Syphilis Screen* compared to the performance of the DiaSorin LIAISON Treponema Assay. Clinical enrollment included representation from high-risk populations which included HIV-positive patients. These results demonstrated good sensitivity and specificity. Lots A and B Combined Site Northridge Diagnostic Laboratory Inc. University of Virginia Erasmus Medical Center All Sites Combined n Positive Negative Sensitivity Specificity Total Predictive Predictive (95% CI) (95% CI) Agreement Value Value 99% 99% (97%-100%) (97%-100%) 100% (n/a) 99% (98%-100%) 99% 96% 100%
D-65 Clinical Utility of Procalcitonin in ICU Patients after Major Trauma or Surgical Event E. O'Keeffe, C. K. Lee, H. G. MacNew, J. M. Jenkins, A. K. May, A. Woodworth. Vanderbilt University Medical Center, Nashville, TN
Background: Detection of Procalcitonin (PCT) in the serum of critically ill patients aids in the diagnosis and monitoring of sepsis. PCT concentrations are associated with disease severity and can be used to guide antibiotic therapy and predict prognosis. Non-septic elevations of procalcitonin occur post trauma and surgery, therefore it is not regularly used as a sepsis marker in surgical or trauma intensive care units (ICU). PCT concentrations in serum rise within 4 hours of a bacterial insult, trauma or surgery. PCT stays elevated in patients with infection and/or sepsis while the infection persists. PCT concentrations decrease within 48 hours of a trauma in the absence of infection. Monitoring the change in PCT over time may allow for its routine use as a sepsis marker in the trauma and surgical ICU patients. Objective: To evaluate the diagnostic and prognostic utility of PCT in trauma and surgical ICU (SICU and TICU) patients. Methods: All patient samples were left-over plasma specimens collected from SICU or TICU physician ordered basic metabolic panels performed in the Vanderbilt University Core Laboratory. Samples were collected daily from 100 patients (51 SICU, 49 TICU) for 7 consecutive days (572 samples total). Infection was determined based upon the results of blood, urine, or bronchial cultures. Procalcitonin assays were performed on the BRAHMS Kryptor, a Time Resolved Amplified Cryptate Emission assay. Significances were determined using student ttests. Results: Out of the 100 patients, 19 died during or shortly after their ICU admission. The maximum change and total change in PCT over the 7 day collection period was calculated for each patient, no significant correlation with overall survival was observed (p=0.22 and 0.051 for max and total PCT change/7 days respectively). There was also no correlation between the highest PCT value and patient outcome (p=0.47). The mean, median and maximum PCT values over the 7 day collection period were determined for each patient and none was a significant predictor of death (p=0.88, 0.85, and 0.46 for mean, median and maximum respectively). PCT was also a poor predictor of bacterial culture positivity in the SICU and TICU (Relative Risk=1.0, p = 1.0). Patients PCT values were separated according to number of days post surgery or trauma when collected. A significant difference was observed in mean PCT concentrations between samples collected less than (mean SD = 4.50.6) or greater than (mean SD = 1.20.4) 48 hours of a trauma or surgery (p=0.0007). Conclusion: PCT is a poor predictor of infection and prognosis when evaluated singly or over 7 consecutive days in both Trauma and Surgical ICU patients. Procalcitonin values were good predictors of number of days post trauma or surgery. Thus, single or consecutive measurements of PCT should not be used alone to predict infection or
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99% 99% 99%
97% 98% 97%
100% 100% 100%
98% 99% (96%-100%) (98%-100%) 99% 99% (98%-100%) (98%-100%)
Results of the preliminary evaluations indicate that the Siemens IMMULITE 2000 Syphilis Screen is a precise immunoassay for the detection of antibody to T. pallidum in human sera. * CE marked. In the US, for investigational use only. The performance characteristics of this product have not been established.
D-67 Analytical Performance of the VITROS 5600 Integrated System Y. Qiu, K. A. Madden, J. Petersen, A. Mohammad, A. O. Okorodudu. University of Texas Medical Branch at Galveston, Galveston, TX
Background: The VITROS 5600 System integrates clinical chemistry and immunoassay testing to increase laboratory productivity for more than 100 different chemistry, immunoassay and infectious disease assays on a single system. The system uses a sample-centered processing approach that allows individual samples to be accessed independently and in parallel for chemistry and immunoassay testing. Objective: The purpose of this study was to evaluate the overall system performance, ease-of-use, and analytical performance of the MicroSlide, MicroTip, and MicroWell assays on the new VITROS 5600 Integrated System. Results from the VITROS 5600 System were compared to those obtained from VITROS ECi Immunodiagnostics System and the VITROS 5,1 FS Chemistry System.
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Methods: Forty two assays (50 samples per assay) were evaluated using samples selected to cover the entire analytical range. CLSI EP9-A2 and EP12-A protocols were followed for the method comparison evaluations for quantitative (EP9) and qualitative (EP12) assays respectively vs VITROS 5,1 FS Chemistry System and VITROS ECi/ECiQ Immunodiagnostic System. Each sample was tested in triplicate from one cup with the exception of %hemoglobin A1c samples, where the testing included one replicate per cup. The means of the triplicate results were compared to the results with either the VITROS 5,1 FS Chemistry System or the VITROS ECI/ ECiQ Immunodiagnostic System. The evaluation also included within run and day to day precision testing, using an abbreviated CLSI EP5-A2 design. Two replicates of two precision runs (AM and PM) were performed per day using appropriate precision control fluid on the VITROS 5600 System. The means, standard deviations and %CV were calculated for each assay. Results: The regression analyses for the entire menu of 42 assays had slopes that are within 1.00 + 0.10 (except for PSA with y = 0.84x + 0.185) with most slopes within 1.00 + 0.05. All the assays tested had clinically negligible intercepts. The overall within and between run precisions were less than 5%. Conclusion: The analytical performance of the VITROS 5600 Integrated System is equivalent to that of the VITROS 5,1 FS Chemistry System and the VITROS ECi/ ECiQ Immunodiagnostic System. The VITROS 5600 Integrated System provides an additional feature of workstation consolidation for testing done on routine clinical chemistry and immunoassay systems.
D-68 VITROS 5600 Integrated System Testing: Throughput/Turnaround Time Study K. A. Madden, Y. Qiu, J. Peterson, A. Mohammad, A. O. Okorodudu. University of Texas Medical Branch, Galveston, TX
Background: Sample through-put as indicated by turn around time (TAT) for patient samples that require both immunoassay and routine chemistry is often prolonged because of multiple sample handling requirements. Different laboratories have handled this problem by protocols that include pre-analytical aliquotting requiring creation of daughter tubes to be run simultaneously on different analyzers. This approach may improve the TAT intermittently. The VITROS 5600 Integrated System is designed to optimize TAT and enhance productivity by intelligently accounting for variable sample and test mixes, eliminating the need to split the sample or move sample trays between modules. The System also offers an increased menu capacity with 150 reagent positions that allow over 100 assays to be on-board at once. Objective: The purpose of this study was to assess the efficiency of handling our laboratorys workload on the new VITROS 5600 Integrated system. This was evaluated by comparing a sample workflow between the VITROS 5,1 FS Chemistry System, VITROS ECi Immunodiagnostic System and VITROS 5600 Integrated System. The results from the VITROS 5600 System were analyzed and compared to the results from the VITROS 5,1 FS System and VITROS ECi System combined. Methodology: A workflow test design was created and preprogrammed exactly the same on each of the three analyzers: VITROS ECi System, VITROS 5600 System, and VITROS 5,1 FS System. The test design consisted of 9 trays, each of which was timed and recorded according to when the tray was placed on the specific analyzer, when the tray was first sampled from, when all tests were completed for that tray, when the tray was removed from that analyzer and when all results were completed for all 9 trays. A side-by-side comparison of the 9 individual tray completion times, across the 3 analyzers, was used to determine the efficiency of the VITROS 5600 System, as well as the total tray completion time. The tray completion time was determined by measuring the time that the analyzer first started sampling to completion of all results for that particular tray. Results: The VITROS 5600 System proved to be much more efficient, with the average difference for each tray time (delta time), between the VITROS 5600 System and VITROS 5,1 FS/VITROS ECi Systems, being 17.57+4.31 minutes. When assessing the total time of completion for all 9 trays, based on the time the analyzer started the first tray to when the last tray was completed, the VITROS 5600 had a 56 minutes TAT advantage relative to the comparative analyzers, thus proving much more efficient than the comparative analyzers combined. Conclusion: Based on these customized workflow studies, the VITROS 5600System was found to be more efficient than the combination of the VITROS 5,1 FS and the VITROS ECi Systems running in parallel, thus making the VITROS 5600 System a valuable asset to laboratories that have substantial sample workloads. The VITROS 5600 System should make the laboratory TAT more reliable in terms of percentage of samples meeting defined TAT goals.
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Endocrinology/Hormones

glycated hemoglobin, and C represents irreversibly glycated hemoglobin. Our objective was to determine parameters for the kinetics of A1c formation according to the standard model that were most consistent with the recently established linear relationship between % hemoglobin A1c (%A1c) and estimated average glucose (eAG) (Nathan et al., Diabetes Care 2008;31:1473-8). Methods: Species A, B and C were designated as fractions of total hemoglobin (A+B+C=1). The model assumed a reversible equilibrium relationship of B to A as a function of glucose concentration, G, according to a single dissociation constant, K: B/(A+B) = G/(G+K). The rate of formation of C was assumed to be proportional to B: dC/dt = kB. This model has a simple solution C(t) over the lifetime of a red cell under conditions of constant G: C(t)=C(0)+(1-C(0))(1-exp(-t)) (Eqn. 1), where = kG/(G+K). Values of (G) were determined such that average C(t) for the entire red cell population (0-120 days age) for a constant value of G was equal to A1c(G) given by the eAG equation: eAG (mM) = 1.59 A1c (%) - 2.59 (Eqn. 2). The model differs from previous models in that 1. it is not assumed that dB/dt=0; 2. it is not assumed that C(0)=0; and 3. A1c(eAG) per Eqn. 2 is targeted. Results: Values for (G) were linear with G, as calculated based on %A1c(eAG) in the range of A1c = 4-14 % (G = approximately 4-20 mM): (1/day) = 0.000119 G (mM), r2 > 0.999 (Eqn. 3). A model constraint was that (G=0) should be equal to 0. This constraint determined a finite value for C(0)=0.0126 (A1c=1.26%). A non-zero value for C(0) was consistent with literature data (nascent red blood cells do not have %A1c = 0), and with the non-zero x-intercept for the eAG(%A1c) correlation (A1c(G=0) = 1.63%, Eqn. 2). The observed linear function (G)=k'G, in comparison to the nonlinear function (G)=kG/(G+K) predicted by the glycation formation model, means simply that K is a large number relative to the operative range of G (K >> 20 mM), such that k' k/K. Determination of the function (G) (Eqn. 3) consistent with the eAG vs. A1c correlation (Eqn. 2) is useful in particular because %A1c(t) may be predicted by step-wise model simulation (using Eqn. 1), accounting for cell turnover, for any arbitrary input function G(t). For instance, model calculations for A1c(t) after step changes in G predicted boundaries for the maximum possible delta %A1c(t) over a 120-day interval that were highly consistent with maximum observed delta %A1c's observed among patient data for a given initial %A1c. Conclusions: Using the standard kinetics model for formation of irreversibly glycated hemoglobin, model parameters consistent with the known relationship of %A1c to average glucose were successfully determined. Model simulations are useful in evaluating predicted %A1c(t) for varying inputs G(t).

Poster Session: 2:00 pm - 4:30 pm Endocrinology/Hormones
D-69 Reclassifications of thyrotropin (TSH) results using free thyroxine (FT4)-dependent TSH reference ranges for paired TSH-FT4 measurements with normal FT4 K. D. Perry, J. D. Landmark, D. F. Stickle. University of Nebraska Medical Center, Omaha, NE
Background: Results combining abnormal TSH (reference range: 0.4-5.0 mIU/L) with normal FT4 (reference range: 0.8-2.0 ng/dL) are the subject of frequent inquiries. Despite the known linear relationship of log TSH to FT4, by convention a FT4independent reference range for TSH is used even in the context of paired TSH-FT4 measurements. Objective: We evaluated the extent to which TSH measurements in abnormal TSH-normal FT4 pairs would be reclassified when using a FT4-dependent reference range for TSH. Methods: Primary data were paired TSH-FT4 patient measurements made during a 6-month interval for which FT4 was normal (n = 6426). Median values for TSH were determined as a function of FT4. A conservative estimate of the FT4-dependent reference interval was calculated by assuming a lognormal distribution and width (1.1 log units) of the standard TSH reference interval, which width was then centered on the median TSH for each value of FT4. The classifications of TSH (low, normal, high) with and without FT4-dependent TSH reference intervals were compared. Results: 24.5% of TSH concentrations were abnormal using the conventional TSH reference range (12.5% low, 12.0% high). Median TSH concentrations were log-linear with respect to FT4: log TSH (mIU/L) = -0.4783 FT4 (ng/dL) + 0.8463 (r2 = 0.963). Designating this correlation as f1(FT4), FT4-dependent TSH reference ranges were estimated as f2(FT4) = f1(FT4)0.55, where 0.55 = one-half of 1.1 log units. Using the TSH reference range f2(FT4), 23.5% of TSH measurements were abnormal (13.3% low, 10.2% high), with reclassification (from either low, normal or high) of 7.8% of TSH measurements. Areas of reclassification (a) for TSH are shown in Figure.
D-71 High molecular weight / total adiponectin ratios are decreased in HIVinfected women receiving protease inhibitors F. Omar1, J. A. Dave2, J. A. King1, N. S. Levitt2, T. S. Pillay1. 1Division of Chemical Pathology, University of Cape Town & National Health Laboratory Service, Cape Town, South Africa, 2Division of Endocrinology, University of Cape Town, Cape Town, South Africa
Relevance: We use two Highly Active Anti-Retroviral Therapy (HAART) regimens for the treatment of HIV. Regimen 1 contains 2 nucleotide reverse transcriptase inhibitors (NRTIs) (stavudine and lamivudine) and a non-NRTI (efavirenz), while regimen 2 contains a protease inhibitor (PI) (liponavir/ritonavir) and 2 NRTIs (zidovudine and didanosine). HAART (including PIs and NRTIs) is associated with the lipodystrophy syndrome, with insulin resistance as an important feature. Furthermore, total adiponectin (TA), an insulin sensitizing hormone is also decreased in these patients. Recently, the high molecular weight (HMW) form of adiponectin has been shown to be the active form and the HMW:TA ratio is known to be a better marker of insulin resistance than either form individually. Objectives: To establish whether HMW:TA ratios are lower in HIV patients receiving PIs, compared with those not on PIs and to establish whether the HMW:TA correlates with other markers of insulin resistance in these patients. Methodology: 66 HIV-infected African females were recruited: 22 on regimen 2 (PI group) for at least 6 months, 22 on regimen 1 (non-PI group) for the same period and 22 treatment nave (TN). All the groups were matched for BMI and age. Patients with overt diabetes, renal failure or cardiovascular disease were excluded. Waist hip ratios (WHR) were measured. Adiponectin levels (TA and HMW) were analysed on fasted serum samples using the Alpco DiagnosticsTM Adiponectin (Multimeric) enzyme immunoassay. Glucose (fasting and 2h post 75g oral glucose) and fasting insulin were measured and mathematical models of insulin resistance (HOMA-IR, HOMA-% and QUICKI) calculated. Data were analysed non-parametrically. Results: The PI and non-PI groups had significantly lower HMW adiponectin levels than the TN group (median 2.23 and 3.49, respectively, vs 5.29mg/l; p<0.005), but did not differ significantly from each other. Similarly, TA was significantly lower in both treatment groups compared with the TN group (median 5.64 and 7.3, respectively, vs 9.03mg/L; p<0.05), but did not differ significantly between the PI and non-PI groups.
Conclusions: Approximately 8% of TSH measurements are reclassified when using estimated FT4-dependent reference ranges for TSH. In practice, the calculations and Figure have proven useful for discussion with clinicians regarding association of abnormal TSH with normal FT4.
D-70 Determination of kinetic model parameters for glycated hemoglobin formation consistent with the relationship of estimated average glucose (eAG) vs. %hemoglobin A1c D. F. Stickle, J. D. Landmark. University of Nebraska Medical Center, Omaha, NE
Background: In the standard model for hemoglobin glycation, formation of irreversibly glycated hemoglobin occurs according to the following scheme: ABC, where A represents nonglycated hemoglobin, B represents reversibly
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In contrast, the HMW:TA ratio was significantly lower in the PI group than in both the NPI (p<0.05) and TN (p<0.0001) groups, and was also lower in the non-PI than in the TN group (p<0.05). Insulin, glucose, HOMA-IR, HOMA-% and QUICKI did not differ significantly amongst the groups. The HMW:TA ratio correlated negatively with WHR (p<0.005) and fasting insulin (p<0.005). A negative, though not statistically significant, correlation was also found between HMW:TA ratio and fasting and 2h glucose levels, as well as age and BMI. Conclusion: These data demonstrate that both PI- and non-PI containing HAART regimens significantly lower the HMW:TA ratio in HIV patients, with the ratio more significantly decreased in the PI-containing regimen, implying that PIs and NRTIs have an additive effect on the HMW:TA ratio. Although the HMW:TA ratio correlated negatively with indirect markers of insulin resistance, no overt insulin resistance was demonstrated. HMW:TA ratio may therefore be a more sensitive marker of insulin resistance in these patients. (Funding was provided by the NHLS, UCT and NRF.)
hydroxide in acetonitrile, dried under nitrogen and reconstituted in water. An aliquot of the reconstituted extract was chromatographed on an Atlantis HILIC Silica column using a water gradient (10% to 55%) against acetonitrile and with formic acid concentration maintained at 10mM. Free plasma MG-H levels were measured in 40 complication-free T1DM patients (DM group), ages 6 to 21 years, and 11 individuals without diabetes (ND group), ages 6 to 22 years. Methylglyoxal was measured using a LC-MS/MS method previously developed by our laboratory. A1C was measured by a Tosoh G7 automated high-performance liquid chromatograph. Results: The developed method showed high recovery (101 0.8%), sensitivity (69 nmol/L) and a short run-time (6 minutes) for the measurement of free MG-H in plasma. Plasma free MG-H (nmol/L) was significantly higher (p < 0.001) in DM group (1318 569; mean standard deviation) as compared to ND group (583 419). Within the DM group, free MG-H in plasma did not correlate with plasma methylglyoxal (r = -0.046, p = 0.779) or A1C (r = 0.06, p = 0.720). Conclusions: We have developed a novel LC-MS/MS method for measurement of free MG-H in human plasma. This method may be useful for future clinical application. Plasma MG-H levels are elevated prior to the appearance of complications of diabetes. The increased levels of free MG-H observed in individuals with diabetes, are not merely the result of short term changes in glucose or methylglyoxal, but may reflect long-term alterations to tissue proteins.
D-72 The effect of hypothyroidism, hyperthyroidism, and their treatment on parameters of oxidative stress and antioxidant status. H. Erdamar1, H. Demirci2, H. Yaman3, K. Erbil3, T. Yakar4, B. Sancak2, S. Elbeg2, G. Biberoglu2, I. Yetkin2. 1Tunceli Government Hospital, Tunceli, Turkey, 2GAZI University Faculty of Medicine, Ankara, Turkey, 3Gulhane Military Medical School, Ankara, Turkey, 4Beytepe Military Hospital, Ankara, Turkey
BACKGROUND: Free radical-mediated oxidative stress has been implicated in the etiopathogenesis of several autoimmune disorders. Also, there is growing evidence supporting the role of reactive oxygen species in the pathogenesis of thyroid disorders. The aim of this study was to investigate the influence of hypothyroidism, hyperthyroidism, and their treatments on the metabolic state of oxidative stress, and antioxidant status markers. METHODS: A total of 20 newly diagnosed patients with overt hypothyroidism due to Hashimoto's thyroiditis, 20 patients with overt hyperthyroidism due to Graves' disease, and 20 healthy subjects as the control group were enrolled in the study. Fasting blood samples (12 h), taken at the initiation, after the 30th and 60th day of therapy were analyzed for malondialdehyde, nitrite, vitamin E, vitamin A, beta-carotene, ascorbate, and myeloperoxidase and superoxide dismutase activity. No patient presented additional risk factors for increased reactive oxygen species levels. RESULTS: Malondialdehyde, nitrite, vitamin E, and myeloperoxidase activity increased in patients with hypothyroidism. After 2 months, the levels of nitrite and vitamin E were reduced to control levels by treatment. The patients with hyperthyroidism had increased levels of malondialdehyde and myeloperoxidase activity in comparison with the controls. Treatment with propylthiouracil attenuated these increments after 1 month. CONCLUSIONS: Our results reveal an increased generation of reactive oxygen species and impairment of the antioxidant system in patients with hyperthyroidism, and particularly in patients with hypothyroidism. These findings indicate that thyroid hormones have a strong impact on oxidative stress and the antioxidant system.
D-74 HbA1c and variant haemoglobins L. Owen, S. J. Lockhart, B. G. Keevil. University Hospital of South Manchester, Manchester, United Kingdom
Many laboratories report HbA1c in patients with variant haemoglobin and append a comment stating that variant haemoglobin was detected and to treat the results with caution. However, reporting the HbA1c value may give misleading low results. Some laboratories prefer to obtain a total glycated haemoglobin (GHb) result using affinity chromatography as this will give an indication of overall glycation status. We analysed data from patients with variant haemoglobin obtained by our HbA1c assay and compared this to a DCCT aligned total glycated haemoglobin affinity chromatography assay to assess if the HbA1c assay can be used to give a more reliable estimate of glycation when a GHb assay is not available for routine use. Data was collected over a 29 month period. During this time 52, 442 samples were sent to the laboratory for HbA1c analysis. Analysis was performed on an in-house Mono-S ion exchange method calibrated to give results that are aligned to the Diabetes Control and Complications Trial. Of these samples any with S, C or F carrier status were selected for data analysis. Also any new or rare variants that did not interfere chromatographically with HbA1c or HbAo measurement were included. Exclusions included homozygotes, compound heterozygotes and carriers of D, E and any rare haemoglobins that can not be resolved chromatographically. HbA1c values obtained in the variants were compared to GHb result obtained from an affinity chromatography method (Primus). An estimate of glycated variant was calculated using %HbA1c, %HbAo and %variant. This was then added to HbA1c to give an estimate of GHb and this was compared to measured GHb. Of the 52,442 samples sent, 564 data sets were analysed. Of these 121 were A/C, 387 were A/S, 20 were A/F and 36 were rare variants. Approximately 50 other data sets were excluded as the peaks could not be resolved on the chromatogram. A PassingBablock regression analysis of HbA1c and measured GHb gave the equation, measured GHb = 1.49 x HbA1c - 0.54 with a R value of 0.87. The Passing-Bablock of estimated GHb against measured GHb was, measured GHb = 0.97 x HbA1c - 0.87 with a R value of 0.90. There were no significant differences when each of the variants were compared individually. Reporting the HbA1c results in patients with variant carrier status can give misleading results for the patient and the clinician, especially when used to compare to DCCT targets. We have shown that estimating the total glycated haemoglobin can give results much closer to DCCT aligned results obtained using an affinity chromatography method for the vast majority of patients. Furthermore, the equation above can be applied to the data to give a derived DCCT aligned value. The small percentage of patients with variant haemoglobin that cannot be resolved chromatographically may be monitored using an alternative marker of glycation status such as fructosamine or glucose profiles when a GHb assay is not available.
D-73 Plasma advanced glycation endproduct, methylglyoxal-derived hydroimidazolone, is elevated in young, complication-free patients with Type 1 diabetes Y. Han1, E. Randell1, S. Vasdev1, V. Gill1, M. Curran1, L. Newhook2, M. Grant2, D. Hagerty2, C. Schneider1. 1Memorial University of Newfoundland, St. John's, NL, Canada, 2Eastern Health, St. John's, NL, Canada
Objectives: Elevated advanced glycation end-products (AGEs) are implicated in complications of diabetes. Methylglyoxal-derived hydroimidazolone (MG-H) is one of the most abundant AGEs in vivo in body fluids and cellular proteins. The purpose of this work was to develop a novel time-saving and specific method for measurement of free MG-H in human plasma and to determine the levels in complication-free young individuals with Type 1 diabetes (T1DM) compared to young individuals without T1DM. The relationship of plasma free MG-H to hemoglobin A1C (A1C) and plasma methylglyoxal levels was also determined. Design and methods: A liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was developed to measure plasma MG-H levels. The method involved solid phase extraction of 100l of acidified plasma using an Oasis MCX mixed mode ion exchange column. The bound extract was eluted using 5% ammonium
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D-75 Comparison of clinical measurements with Elecsys PTH STAT and PTH Y. Gao. Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai, China
Objective: To study if there are some differences in clinical measurements of intact parathyroid hormone (iPTH) with Elecsys PTH STAT assay and PTH assay. Method: Plasma samples from 113 patients with or without parathyroid gland disorders were collected and determined simultaneously with 9-minute PTH STAT assay and 18-minute PTH assay on Elecsys 2010 immunoassay analyzer (ECLIA, Roche Diagnostics GmbH, Mannheim, Germany). Wilcoxon and McNemar test for possible differences in the two iPTH assays were performed and their correlation was analyzed with SPSS 13.0 soft ware (SPSS, Inc., Chicago, Ill.,USA). Results: The median (P5~P95) values of iPTH were 51.7 (9.9~477.0) pg/mL and 54.6 (9.8~497.0) pg/mL for PTH STAT assay and PTH assay, respectively. Though a slight difference in the measurements was seen statistically (Z=-6.440, P<0.001), but a high correlation (Spearman r=0.993, P<0.001) and a total of 94.7% (107/113) agreement of sample classification were achieved (Kappa=0.906, P<0.001) with the two assays (Figure 1).

samples with HbA1c values determined by an NGSP Secondary Reference Laboratory (SRL) were also analyzed on the Test method. Intra-day precision was determined on the Test method by running the high and low controls 20 times in one day. Inter-day precision was determined by running the controls in duplicate for two runs per day over 10 days. Interference of HbA1c analysis by CHb and HbF was investigated using non-diabetic and diabetic blood samples with 5 levels of CHb and HbF. These samples were analyzed in quadruplicate. Results: Regression analysis for the correlation of patient samples between Test (y) and Reference (x) methods showed a slope of 1.1038, intercept of -0.7677, and correlation coefficient of 0.9932. The Bland Altman Bias plot for the NGSP samples comparison study showed that the Test method met the 0.75% acceptance criteria for level 1 laboratory certification with a lower 95% confidence limit of -0.55 and an upper 95% confidence limit of 0.20. Intraday precision study showed a CV of 0.87% and 0.31% for the low and high controls respectively. Inter-day precision study showed a CV of 1.07% and 0.81% for the Test method, while the Reference method showed a CV of 1.31% and 1.1% for the two controls. No interference of HbA1c analysis by CHb and HbF were detected in samples with CHb levels up to 6.2% and HbF levels up to 28.5%. Conclusions: The test method exhibits better precision and the increased ability to report HbA1c in the presence of higher levels of CHb and HbF. The reference and test methods correlated well in the HbA1c range of 4-13%. Recalibration and priming frequency was reduced and the need to optimize temperature was eliminated.
D-77 Development of a Second Generation Anti-Mullerian Hormone (AMH) ELISA* A. Kumar, B. Kalra, A. S. Patel, L. McDavid. DSL, Inc., a Beckman Coulter Company, Webster, TX
Objective: Development of AMH Gen II ELISA, standardized to the ImmunotechBeckman Coulter (IOT) AMH assay for the quantitative measurement of AMH in serum. Relevance: AMH is a glycoprotein dimer composed of two 72kDa monomers linked by disulfide bridges. It belongs to the transforming growth factor- family. AMH performs various physiological functions. In males, AMH is secreted by the Sertoli cells. During embryonic development, AMH is responsible for Mullerian duct regression. AMH continues to be produced by the testicles until puberty and then decreases slowly to residual post-puberty values. In females, AMH is produced in small amounts by ovarian granulosa cells after birth until menopause, and then becomes undetectable. Methodology: A two-step, sandwich-type enzymatic microplate assay has been developed to measure AMH levels in 20 L of sample in less than 3 hours. The assay measures mammalian AMH (i.e. human, bovine, mouse or rat). AMH calibrators range from 0.2-28 ng/mL. The antibodies used in the assay bind to the mature region of AMH, which is more stable against proteolysis compared to pro-hormone region. This highly characterized dual monoclonal antibody pair is specific to AMH and does not detect inhibin A, activin A, FSH and LH at 2 times physiological concentrations. Validation: The AMH Gen II assay was standardized to the IOT AMH assay. AMH Gen II, when compared to IOT using 38 serum samples in the range of 1.5-33 ng/mL yielded a correlation coefficient of >0.97 and a slope of 1.0 with an intercept of -0.29 ng/mL. AMH Gen II when compared to the current Diagnostic Systems Laboratories (DSL) assay, using 180 male and female serum samples, in the range of 0.07-18 ng/ mL yielded a correlation coefficient of >0.99 and a slope of 1.34 with an intercept of 0.01 ng/mL. The current DSL AMH patient values could be normalized to the AMH Gen II assay by multiplying the patient concentrations by 1.34. Total imprecision, calculated on 4 samples over 40 runs, 4 replicates per run, between two lots using CLSI EP5-A guidelines, was 5.7% at 3.8 ng/mL, 7.7% at 4.4 ng/mL, 5.3% at 14 ng/ mL and 5.8% at 16.4 ng/mL. The average analytical sensitivity calculated by the interpolation of the mean plus two standard deviations of 16 replicates of the zero calibrator on two independent lots was 0.75 ng/mL. Dilution and spiking studies showed an average recovery of 91-110%. Lot-to-lot comparison of two independent lots testing 38 serum samples (1.5-33 ng/mL range) yielded a slope of 1.01, intercept of -0.08 ng/mL and r2 of 0.98. When potential interferents (hemoglobin, triglycerides, bilirubin) were added at 2 times physiological concentrations, AMH concentrations were within 10% of the control. Conclusions: A highly specific and reproducible microplate AMH Gen II assay has been developed to standardize the measurement of AMH between methods. The performance of the AMH Gen II assay is ideal for investigation into the physiologic roles of AMH in men and women. *For Research Use Only. Not for use in diagnostic procedures
Conclusion: The Elecsys PTH STAT has almost identical detectivity with the PTH assay on clinical plasma samples, and is more suitable for intraoperative use to confirm the completeness of parathyroid surgery with short assay duration as well as short biological half-life of iPTH in the blood stream.
D-76 Evaluation of an Improved Bio-Rad VARIANT II TURBO HbA1c Kit C. B. Tapply1, F. H. Yu2, H. K. Lee3. 1Dartmouth-Hitchcock Medical Center, Lebanon, NH, 2Dartmouth College, Hanover, NH, 3Dartmouth Medical School & Dartmouth-Hitchcock Medical Center, Lebanon, NH
Background: The Bio-Rad VARIANT II TURBO HbA1c kit - 2.0, an improved version of the current Bio-Rad VARIANT II TURBO HbA1c kit, was evaluated. The new kit utilizes a reduced particle size cartridge, new prefilters and enhanced buffers to improve the separation of HbA1c from potentially interfering forms of hemoglobin such as Carbamylated Hb (CHb) and Labile A1c (LA1c). The improved chemistry also presents several workflow advantages, such as reduced priming and calibration frequency, and eliminating the need to optimize temperature. The current VARIANT II TURBO HbA1c kit uses an analytical and guard cartridge format for analyzing HbA1c. Calibration and priming needs to be performed after every new analytical or guard cartridge change, and temperature optimization is required to assure that the HbA1c retention time is within limits. The interference threshold for Hemoglobin F (HbF) and CHb concentrations are also lower on the current kit, resulting in some HbA1c results not being reportable. Objectives: This study aimed to validate the improved VARIANT II TURBO HbA1c kit - 2.0 (Test method) using the current kit as the Reference method. Methods: Correlation of patient samples between the two methods was performed using 200 samples with HbA1c values between 4-13%. Forty
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D-78 Development of a Simplified Second Generation Inhibin B ELISA B. Kalra, A. Kumar, K. Patel, A. S. Patel, L. McDavid, M. J. Khosravi. DSL, Inc., a Beckman Coulter Company, Webster, TX
Objective: Development of a simplified and sensitive Inhibin B Gen II ELISA for the quantitative measurement of inhibin B in serum and plasma*. Relevance: Inhibins are heterodimeric protein hormones secreted by granulosa cells of the ovary in the female and Sertoli cells of the testis in the male. The current commercially available Oxford Brooks Innovation (OBI) and Diagnostics Systems Laboratories (DSL) inhibin B assays require pre-treatment of samples with hydrogen peroxide to oxidize two methionines in the epitope to the sulfoxide for full immunoreactivity. Both the assays require overnight incubation of the sample with cumbersome steps. Publications have assessed inhibin B levels in Sertoli cell function, ovarian reserve and granulosa cell tumors. Methodology: We have developed a two-step, sandwich-type enzymatic microplate assay to measure inhibin B levels in less than 3.5 hours. Sample pre-treatment is not required. The assay measures inhibin B in 50 L of serum or Li-Hep plasma samples. Inhibin B calibrators range from 10-1000 pg/mL. The highly characterized dual monoclonal antibody pair is specific to inhibin B and does not detect inhibin A, activin A, activin B, activin AB, AMH, FSH, LH and follistatin 315 at 2 times their physiological concentrations. Validation: The Inhibin B Gen II assay was compared against two commercially available assays using 60 male and 60 female samples, ranging in age from 20-50 years. The assay showed significant positive linear correlations to OBI and DSL assays (r=0.98; P<0.0001; & r=0.94; P<0.0001, respectively). Method comparison to OBI and DSL resulted in the following slope and intercept (Gen II =1.03OBI - 6.77 pg/mL & Gen II = 1.57DSL + 11.29 pg/mL), respectively. Matched serum and Li-Hep plasma samples (n=120) showed a correlation coefficient of >0.99 and a slope of 0.97 with zero intercept. Total imprecision calculated on 3 samples and 2 controls over 40 runs, 3 replicates per run, between two lots using CLSI-EP5-A guidelines was 6.8% at 19.3 pg/mL, 4.4% at 76.0 pg/mL, 4.3% at 275.3 pg/mL, 5.4% at 99.9 pg/mL and 5.7% at 363.9 pg/mL. The average analytical sensitivity calculated by the interpolation of the mean plus two standard deviations of 16 replicates of the zero calibrator on two independent lots was 1.6 pg/mL. The functional sensitivity of the assay at 10% and 15 % CV were 9.3 and 5.6 pg/mL, respectively. Dilution and spiking studies showed an average recovery of 90-110%. Lot-to-lot comparison of two independent lots testing 120 serum samples (5-600 pg/mL range) yielded a slope of 0.98, intercept of 0.13 pg/ mL and r2 of 0.99. When potential interferents (hemoglobin, triglycerides, bilirubin and human serum albumin), were added at 2 times their physiological concentration, inhibin B concentrations were within 10% of the control. Conclusions: A highly specific, sensitive, simplified and reproducible microplate Inhibin B assay has been developed to measure inhibin B in serum and plasma. The performance of the assay is ideal for investigation into the physiologic roles of inhibin B in men and women. * For Research Use Only. Not for use in diagnostic procedures
bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent (a substituted acetanilide) increases the level of light produced and prolongs its emission. The light signals are read by the system. The bound HRP conjugate is directly proportional to the concentration of Intact PTH present. The VITROS Intact PTH assay under development has a reportable range of up to 5000 pg/mL. Limit of Blank (LoB) was measured in line with CLSI EP17-A and shown to be <0.7 pg/mL. A precision study based on CSLI EP05-A2 was conducted over 5 days. Between run imprecision at 16, 200 and 825 pg/mL was <7.5%. Within run imprecision was <2.0%. Method comparison, carried out based on CLSI EP09A2, versus a commercially available assay using 88 patient samples covering a range of 0.15 to 1308 pg/mL shows a good correlation (r = 0.980; intercept = 12.3 pg/mL, standard error = 3.94 pg/mL; Slope = 0.89, standard error 0.014). No high does hook effect was observed with PTH concentrations up to 1,000,000 pg/mL. Cross-reactivity versus PTH fragments 1-34, 39-68, 53-84, 44-68 and 39-84 was assessed based on CLSI EP07-A2 as < 0.01%. Linearity across the assay range was assessed based on CLSI EP06-A with Intact PTH spiked into normal human serum. Due to the wide range linearity was assessed in 4 experiments using overlapping concentration ranges and these studies show that the assay is linear from 7.04 to >3000 pg/mL. The time to first result is 18 minutes. In conclusion, the VITROS Intact PTH assay combines good analytical performance with the advantages of rapid automated continuous random access immunoassay systems. * Under Development
D-80 Unravelling The Diagnostic Challenges Of Polycystic Ovary Syndrome (PCOS): Should We Do More And Can We Do Better? O. A. Mojiminiyi, F. Safar, H. Al Rumaih, T. Al Rammah, M. Diejomaoh. Faculty of Medicine, Kuwait University, Kuwait, Kuwait
PCOS, the most common endocrine disorder in women of reproductive age, arises during puberty and results in infertility and increased risk of type 2 diabetes and cardiovascular disease. However, despite the recognition of a major role for metabolic disorders in the pathogenesis of PCOS, no metabolic feature is included in the definition of the syndrome. In this study we explore the hypothesis that PCOS is a predominantly metabolic disorder and evaluate if we could use metabolic indicators for screening and early diagnosis. We measured fasting follicular phase LH, FSH, estradiol, androstenedione, DHEA-S, testosterone, SHBG, free androgen index (FAI) (calculated from testosterone and SHBG levels), fasting lipid profile, adiponectin, leptin, leptin receptor, glucose, insulin and insulin resistance (homeostasis model assessment (HOMA-IR) and Quantitative Insulin-Sensitivity Check Index (QUICKI)) in 136 women diagnosed a posteriori as PCOS (n= 92) and normal controls (n = 44) using the Rotterdam criteria. We used univariate and multivariate regression analyses to find the associations of these variables with each other and PCOS and used receiver operating characteristic (ROC) analysis to determine the diagnostic performance of the biochemical parameters. Insulin resistant (22.8%), overweight/obese (82.6%) and hyperandrogenic (clinical and/or biochemical) phenotypes (96.7%) are the predominant phenotypes of PCOS in our population. The best diagnostic tests with the highest area under the ROC curve (AUC) were: FAI (AUC = 0.868), SHBG (0.765), HDL-cholesterol (0.715), HOMA-IR (0.670), QUICKI (0.658), adiponectin (0.644), leptin:adiponectin ratio (0.658). Oligomenorrhea (53.3%) and polycystic ovaries (68.2%) were not found in all PCOS and high serum androgens showed variable correlations with clinical indicators (acne (68.5%), hirsutism (77.2%), and alopecia (38%)) of hyperandrogenism. Binary logistic regression analysis showed that odds ratio (OR) of the significant associations with PCOS were: age (0.89), testosterone (3.44), FAI (2.70), HDL-Cholesterol (0.10), SHBG (0.98), adiponectin (0.89), HOMA-R (2.56) androstenedione (1.62) and DHEA-S (1.42). Whereas the clinical presentation of Oligomenorrhea, chronic anovulation and hyperandrogenism are stressed as the major diagnostic criteria for PCOS, our data show that some simple metabolic parameters have comparable diagnostic performance and should be useful adjuncts for screening. As early diagnosis and treatment may improve the reproductive sequelae and reduce cardio-metabolic risks, should we do more and can we do better to screen for PCOS? Yes, we can.
D-79 Development of an Intact PTH* assay for the VITROS ECi/ECiQ Immunodiagnostic Systems, the VITROS 3600 Immunodiagnostic System and the VITROS 5600 Integrated System E. Lindhout1, M. Sieswerda1, M. Martens1, A. Vacca Smith2, D. Montague3, S. Edwards2. 1Future Diagnostics, Wijchen, Netherlands, 2Ortho-Clinical Diagnostics, Rochester, NY, 3Ortho-Clinical Diagnostics, High Wycombe, United Kingdom
Parathyroid hormone (PTH) is involved in the maintenance of calcium homeostasis, and its measurement plays an important role in the diagnosis and management of calcium metabolism disorders. The Intact PTH assay is performed using the VITROS ECi/ECiQ Immunodiagnostic Systems, VITROS 3600 Immunodiagnostic System and VITROS 5600 Integrated System using Intellicheck Technology. An immunometric immunoassay technique is used, which involves the reaction of Intact PTH present in the sample with a biotinylated antibody (goat polyclonal anti-Intact PTH) and a horseradish peroxidase (HRP)-labeled antibody conjugate (goat polyclonal anti-Intact PTH). The antigenantibody complex is captured by streptavidin on the wells. Unbound materials are removed by a washing step. The bound HRP conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent, is added to the wells. The HRP in the
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D-81 Serum Aminotransferase Estimation Should Be An Integral Part Of Cardio-Metabolic Risk Assessment O. A. Mojiminiyi, T. Al Rammah, N. Abdella. Faculty of Medicine, Kuwait University, Kuwait, Kuwait
Increasing prevalence of obesity has made non-alcoholic fatty liver disease (NAFLD) a major health problem that is found in association with increased cardio-metabolic risk. Aminotransferase (aspartate aminotransferase (AST), alanine aminotransferase (ALT), concentrations have been shown to be associated with indices of obesity and may reflect NAFLD. This study explores the associations of ALT and AST with indices of obesity, adipokines, insulin resistance and components of the metabolic syndrome in an alcohol free normoglycemic population with negative medication history and hepatitis screen. Anthropometric measurements and fasting adiponectin, leptin, leptin receptor, insulin, glucose, high-sensitivity C-reactive protein (hs-CRP) and lipid profile were measured in 429 (155M, 274F) subjects. Univariate regression and multivariate logistic regression analyses were used to relate the aminotransferases with indices of obesity, adipokines as well as with the degree of adiposity (BMI25, 25.1 - 29.9 or 30), insulin resistance (homeostasis model assessment (HOMA-IR 2 or 2)) and the number of the criteria of the metabolic syndrome (MS) (International Diabetes Federation (IDF) criteria). 89 (21%) of subjects were classified as MS positive. All the study subjects had normal ALT and AST but gender dimorphism was noted with males having higher mean ALT (27.7 IU/L) and AST (23.5 IU/L) than females (17.4 and 20.0 IU/L respectively). ALT and AST showed stepwise increase with increasing categories of obesity, insulin resistance and the number of criteria of the metabolic syndrome. ALT showed significant correlations with age (r = 0.13), indices of obesity (r = 0.32 for BMI; r= 0.36 for waist circumference), beta cell function (%B) (r= 0.14) insulin (r= 0.18), HOMA-IR (r= 0.27), triglycerides (r= 0.24), hs-CRP (r= 0.25) and inverse correlations with adiponectin (r= -0.29), insulin sensitivity (%S) (r= -0.27) and HDL-cholesterol (r= -0.25). AST showed similar correlations. Adiponectin, HOMA-IR, %B and %S retained their significance after partial correlation analysis correcting for indices of obesity. In logistic regression models adjusting for age and sex, subjects in the 4th quartiles of ALT (odds ratio (OR) 3.8 (95% CI 1.3-10.9) and AST (OR 2.5 (95% CI 1.1-5.7) had significant risk of MS compared with those in the 1st quartile. We conclude that aminotransferases are significantly associated with cardiometabolic risk factors and should be included in routine laboratory risk assessment. As concern is growing about NAFLD, not only because it is a common liver disorder, but also because it is one of the leading causes of chronic liver disease, laboratories may need to lower the upper normal reference limits to facilitate earlier detection of obesity-associated increases in aminostransferases.

triglycerides and FAI despite similar obesity indices. Binary logistic regression analysis showed that the TT (OR = 0.89) and GT (OR = 0.83) genotypes of rs 2241766 and TT (OR = 0.90) and TG (OR = 0.87) genotypes of rs 1501299 were significant determinants of PCOS risk. We conclude that polymorphisms in the adiponectin gene are associated with lower adiponectin that may contribute to the pathogenesis of insulin resistance and hyperandrogenism in the patients.
D-83 Leptin, Leptin Receptor Free Leptin Index and Metabolic Phenotypes of PCOS in the Kuwaiti Population. F. H. Safar1, O. A. Mojiminiyi1, H. Al Rumaih2, T. Al Rammah1, M. Diejomaoh1. 1Faculty of Medicine, Kuwait, Kuwait, 2IVF unit, Maternity Hospital, Kuwait, Kuwait
Polycystic ovaries syndrome (PCOS) is a common complex endocrine disorder affecting women in their reproductive age with heterogeneous presentation and different phenotypes. The aim of the study was to find the association, if any, between circulating leptin, leptin receptor, free leptin index (FLI) with the metabolic phenotypes of PCOS. We measured follicular phase LH, FSH, estradiol, testosterone (T), androstenedione (D4), DHEA-S, SHBG, fasting lipid profile, leptin, leptin receptor, FLI, glucose, insulin and estimated beta-cell function (%B), insulin sensitivity (%S) and resistance using the homeostasis model assessment (HOMA-IR) and quantitative insulin check index (QUICKI). IDF criteria were used to define the metabolic syndrome (MS) status. Spearman Rank correlations, univariate and logistic regression analyses were used to find the associations of these variables with each other and with metabolic phenotypes of PCOS. PCOS patients had significantly higher leptin (p=0.031), FLI (p=0.022) and lower leptin receptor (p=0.032) than controls.Among PCOS patients 34.1% were metabolic syndrome positive compared with 24.3% controls. Leptin (p= 0.007) and FLI (p=0.009) were significantly higher in MS+ve PCOS patients while leptin receptor was significantly lower (p=0.031).The levels of leptin (p=0.004) and FLI (p=0.002) were also significantly higher while leptin receptor levels were significantly lower (p=0.008) among PCOS women with HOMAI-IR 2. However, no significant differences were detected in leptin, leptin receptor and FLI between hyperandrogenic and normoandrogenic PCOS. Spearman correlation analysis showed that leptin and leptin receptor had significant direct correlations with waist, hip circumference,BMI, TG, insulin, HOMA-IR, %B, FAI and inverse correlations with SHBG, %S, QUICKI and HDL-cholesterol. Similar significant correlations were found with leptin receptor but in the inverse direction. Logistic regression analysis show that leptin (O.R 1.040, 95% C.I 1.014-1.068, p=0.003), leptin receptor (O.R. 0.875, 95% C.I 0.779-0.984, p=0.025) and FLI (O.R. 1.318, 95% C.I. 1.072-1.619, p=0.009) were significantly associated with MS. Logistic regression analysis also show that leptin (O.R 1.048, 95% C.I 1.019-1.078, p=0.001), leptin receptor (O.R. 0.823, 95% C.I 0.708-0.956, p=0.011) and FLI (O.R. 1.459, 95% C.I. 1.149-1.852, p=0.002) were significantly associated with insulin resistance phenotypes. However, no significant associations were found with androgens. We conclude that leptin, leptin receptor and FLI are significantly associated with metabolic phenotypes and insulin resistance phenotype of PCOS but leptin does not appear to contribute to the hyperandrogenic status in PCOS patients.
D-82 Effect Of Single Nucleotide Polymorphisms In The Adiponectin Gene On Circulating Adiponectin And Cardio-Metabolic Risk Factors In Patients With Polycystic Ovary Syndrome. F. H. Safar1, O. A. Mojiminiyi1, H. Al Rumaih2, F. Al Mulla1, T. Al Rammah1, M. Diejomaoh1. 1Faculty of Medicine, Kuwait, Kuwait, 2IVF Unit, Maternity Hospital, Kuwait, Kuwait
The genetic mechanisms underlying the hyperandrogenism and metabolic disorders in polycystic ovary syndrome (PCOS) are complex. We postulate that polymorphisms in the gene for adiponectin, an adipose tissue-derived hormone, determine the adiponectin level and play a significant role. In a study on 92 patients with PCOS and 108 controls, we used real time PCR to determine the 45T>G (rs2241766) and 276G>T (rs1501299) polymorphisms in the adiponectin gene and measured follicular phase LH, FSH, estradiol, testosterone, androstenedione, DHEA-S, SHBG, fasting lipid profile, adiponectin, glucose and estimated insulin resistance using the homeostasis model assessment (HOMA-IR). Body fat% was measured by Bioimpedance. Univariate and multivariate regression analyses were used to find the associations of these variables with each other, adiponectin gene polymorphism and PCOS. Adiponectin showed significant inverse correlations with waist circumference, fat %, HOMA-IR, free androgen index (FAI) and triglycerides but showed positive correlations with HDL-cholesterol and SHBG. Multivariate regression analysis showed that adiponectin is a significant determinant of insulin, HOMA-IR, SHBG and FAI. The distributions of the genotypes of both polymorphisms were not significantly different in PCOS patients and controls but TT and GT genotypes of rs 2241766 and TT and TG genotypes of rs 1501299 were associated with significantly lower adiponectin and HDL-cholesterol and significantly higher insulin, HOMA-IR,
D-84 Evaluation of the Roche Testosterone II Assay on the MODULAR E170 Analyzer W. E. Owen1, M. L. Rawlins1, W. L. Roberts2. 1ARUP Laboratories Inc., Salt Lake City, UT, 2Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT
Background: The androgen testosterone is produced in Leydig cells of the testes in males. In females testosterone is produced principally in the ovaries. Normal concentrations of testosterone in females are approximately 10-fold less than in males. In males decreased testosterone may indicate hypogonadism, hypopituitarism, hyperprolactinemia, renal failure, hepatic cirrhosis, or Kleinfelters syndrome, while elevated testosterone can be caused by adrenal and testicular tumors. In females increased testosterone can be caused by polycystic ovary syndrome, stromal hyperthecosis, ovarian and adrenal tumors, or congenital adrenal hyperplasia. The purpose of our study was to evaluate a new generation Testosterone II assay (Testo II) from Roche Diagnostics. Methods: Testo II performed on a MODULAR ANALYTICS E 170 module is an
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automated random access electro-chemiluminometric assay. It was evaluated for imprecision over 21 days using 2 levels of control material and 5 patient pools with testosterone concentrations from 35 to 1321 ng/dL and method comparison with high performance liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) used in our facility. Method comparison samples were tested using the current generation Roche Testosterone assay (Testo I) and the Beckman Coulter Testosterone chemilumimometric assay (Access) and compared to LC-MS/MS. Serum samples from 100 men, 100 women, 50 boys (ages 0 to 17 years), and 50 girls (ages 0 to 17 years) were used for the comparison studies. Samples outside the analytic measurement range were eliminated from data analysis. Passing-Bablok regression was used for method comparison and included statistics for slope, intercept, r, and SMAD (scaled median absolute deviation), a non-parametric error estimate. Results: Repeatability (within-run) and within laboratory imprecision ranged from 1.6 to 2.6 and 2.3 to 5.1 %CV respectively. Passing-Bablok regression statistics are summarized in the table below.
Method Comparison Statistics Slope Intercept SMAD r n LC-MS/MS Range (ng/dL) 1.085 -4 42 0.972 100 60 - 1440 1.165 - 19 35 0.984 98 60 - 1250 0.971 7 41 0.955 100 60 - 1440 1.231 0 12 0.848 96 4 - 671 1.040 -3 10 0.989 93 4 - 671 1.061 6 13 0.920 88 10 - 671 1.021 1 9 0.982 47 4 - 1260 1.038 -4 10 0.979 38 5 - 1260 0.948 8 10 0.992 39 5 - 1260 1.848 -2 16 0.673 49 1 - 65 1.422 -6 12 0.739 46 4 - 65 1.600 3 10 0.768 48 1 - 65
D-86 Hemoglonin Yamagata[HBB: c.399A>T; p.Lys133Asn]: Hemoglobin variant detected by HbA1c test K. Hong1, C. Jung1, M. Lee1, W. Chung1, Y. Sung1, S. Lee2, C. Ki2. 1Ewha Womans University MokDong Hospital, Seoul, Republic of Korea, 2Samsung Medical center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
Hemoglobin (Hb) Yamagata is a rare Hb variant which has been reported only two times in Japan and Korea, respectively. It arise from LysAsn substitution due to a change of AAA to AAC or AAT at codon 133 of beta-globin gene. This study reports the third case of Hb Yamagata [HBB: c.399A>T; p.Lys133Asn] and its impact on HbA1c measurement. This variant was detected during routine follow-up of a 70year-old Korean man with diabetes mellitus (DM). The result of HbA1c by Variant ll Turbo (Bio-Rad, USA) was 47.9%, abnormally high level. It was impossible to measure HbA1c correctly by Variant ll Thalassemia Mode (Bio-Rad, USA) because of interference between variant and A1c peaks. On the contrary, the HbA1c was measured as low as 5.0% by HLC-723 G7 (Tosoh Corp., Japan) while those by Cobas Integra (Roche diagnostics, Switzerland) and NycoCard (Axis-Shield, Norway) were 8.0% and 7.9%, respectively. At the time of this evaluation, the patients fasting blood glucose value was 177 mg/dL which is in the diabetic range. His peripheral blood indices showed a Hb level of 12.9 g/dL, Hct 37.2%, MCV 87.3 fL, MCH 30.3 pg, and RDW 12.7%. The peripheral blood smear also showed normocytic normochromic red blood cells. The DNA sequence analysis revealed the presence of Hb Yamagata in which the LysAsn substitution was due to a change of AAA to AAT at codon 133 of beta-globin gene. In Capillary EP (Sebia, France), fractions of Hb Yamagata, HbA, and HbA2 were 40.9%, 54.4%, and 3.0%, respectively. This study demonstrates that Hb Yamagata can interfere accurate measurement of HbA1c in a Korean patient with DM. Considering that HPLC-based Variant ll Turbo and Tosoh G7 can be interfered by Hb Yamagata, an immunoassay or an affinity-chromatography can be the method of choice for accurate measurement of HbA1c with this variant. In conclusion, a Hb variant can be suspected when the concentration of HbA1c shows abnormally high or low level or discrepancy between the result and clinical status. Then the HbA1c values should be determined with a method based on different principles, which can improve the management of DM patients.
Men - Testo I Men - Testo II Men - Access Women - Testo I Women - Testo II Women - Access Boys - Testo I Boys - Testo II Boys - Access Girls - Testo I Girls - Testo II Girls - Access
Conclusions: The Roche Testo II assay demonstrated excellent precision and improved correlation with LC-MS/MS for samples from men, women, and girls.
D-85 Direct measurement of serum free testosterone by ultrafiltration and liquid chromatography-isotope dilution tandem mass spectrometry Y. Chen1, M. Yazdanpanah2, Y. Wang2, B. Hoffman1, E. Diamandis1, P. Wong1. 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, 2University Health Network, Toronto, ON, Canada
Background: Currently there is no reliable method for serum free testosterone (FT) measurement in routine clinical laboratory practice. The objective of this study was to develop a liquid chromatography-isotope dilution tandem mass spectrometry (LCIDMS/MS) method for the direct detection and quantification of serum free testosterone. Methods: After ultrafiltration (UF) with 0.5 ml of patient serum, an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source was used to separate, detect and quantify serum testosterone by using the method we previous developed (Clin Biochem, Epub Dec. 3, 2008, in press). Ion-transitions of m/z 289.2 109.1 and 294.2 113.2 were used to monitor testosterone and testosterone-2,2,4,6,6-d5 , respectively. This direct UF-LC-MS/MS method was validated by use of split-sample comparisons with calculated free testosterone (cFT) and equilibrium dialysis (ED) followed by LC-MS/MS. Results: Within-run and total imprecision of the method demonstrated coefficient of variations (CV) of 3.6%, 2.9% and 5.5%, 4%, at levels of 158 pmol/L and 67 pmol/L respectively (n=20 each). The method demonstrates a dynamic linear response up to at least 2500 pmol/L and the functional sensitivity was 8.7 pmol/L at CV 20%. The method comparison showed the correlation of FT by UF-LC-MS/MS with cFT based on total testosterone values measured by Architect i2000 (R=0.816) and by LC-MS/ MS (R=0.8996). UF-LC-MS/MS method correlated very well with ED-LC-MS/MS (R=0.9873). Average bias of UF-LC-MS/MS were -13.29%, -3.33% and 8.1% compared with cFT by Architect, LC-MS/MS and ED-LC-MS/MS. The accuracy of the UF-LS-MS/MS for FT was validated by spiking standard material at three levels and the recoveries were at 92-109%. Conclusion: We set up a new simple and definitive method for measuring serum free testosterone with UF-LC-MS/MS. This method is reliable and rapid as a reference method and can be easily used in routine clinical laboratories.
D-87 Establishment of a reference range for the Bone Alkaline Phosphatase (BAP) with DiaSorin Liaison in a biologically well-designed population. E. Cavalier, P. Delanaye, A. Bekaert, I. Carlisi, J. Chapelle. University Hospital of Lige, University of Lige, Lige, Belgium
Introduction: Defining a reference range is not an easy task. As requested by ISO 15189, laboratories must check the reference range published by the manufacturers of the kits, but generally, the way these values have been obtained is unclear. The concept of what is a reference range is also controversial. From our point of view, a healthy population, like young blood donors cannot be considered as a reference population. We prefer to use biological or clinical variables to establish our reference population. Indeed, by using these variables as a reference, any lab will have more or less the same values and, more important, we will know exactly on which population we will compare our patients. Materials and Methods: We determined the BAP on Liaison (BAP OSTASE, DiaSorin, Stillwater, MN) in duplicate. We selected our population using these variables: age >18 yo, sex, menstrual status, levels of 25-OH vitamin D (25VTD) >30 ng/mL, levels of intact parathormone (PTH) <58 pg/mL, calcium and phosphorous in the laboratory reference range and eGFR> 60 mL/min/1.73m (estimated by the MDRD equation). For each subclass (men/non-menopause women/post-menopause women), we included 120 individuals. We used the Kolmogornov-Smirnov test to check if the population was Gaussian. If it wasnt the case, we used a non-parametric method. We took the 95% right-sided result as the cut-off. Results: Our selected population presented a mean 25VTD level of 37 ng/mL (Lowest: 32; Highest: 58), a mean PTH of 38 pg/mL (L:18 - H: 57) and a mean age of 58 yo (L:22; H: 85). In men, the BAP cut-off was found to be 20.3 g/L (90% CI: 16.1 - 25.2 g/L), 23.6 g/L in non-menopause women (90% CI: 16.9 - 26.6 g/L) and 20.4 g/L in menopause women (90% CI: 17.8 - 25.0 g/L). We did not find any statistical difference between the three populations. We thus combined all the data. The cut-off observed in the whole population (n=360) was then 21.3 g/L (90% CI: 18.3 - 24.2 g/L).
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Discussion: We have evaluated the reference range for the BAP OSTASE in a well biologically designed population. We did not find any difference between men, nonmenopause and menopause women. This is quite new, as the reference ranges for BAP generally distinguish these populations. However, one should notice that in the studies showing that differences exist between these subclasses, 25VTD levels have never been taken into consideration. This is of importance as 25VTD deficient patients can suffer from secondary hyperparathyroidism, leading to an increase bone turnover and increased BAP values. Conclusions: We present here the reference range for BAP OSTASE (Liaison) in a biologically well designed cohort. Our population is representative of the normal population for the studied parameter. Our approach could be generalized so that laboratories and manufacturers could establish the reference ranges on biologically and clinically well designed populations, leading to a better harmonization.

D-89 Comparison of Three Methods for Sex Hormone Binding Globulin (SHBG) D. Young1, J. M. Mattke1, A. Artus1, P. Marquet1, A. Morel-Montero1, J. Guchot2, H. J. Roth3. 1Beckman Coulter, Inc., Chaska, MN, 2Hpital Saint Antoine, Laboratoire de Biochimie, Paris, France, 3Limbach Laboratory, Heidelberg, Germany
Objective: The objective of this study was to compare performance of the new Access SHBG immunoassay from Beckman Coulter with three other commercially available SHBG assays. Relevance: Sex hormone-binding globulin (SHBG) is a glycoprotein responsible for blood transport of testosterone and estradiol. SHBG is synthesized in the liver and has a high binding affinity for 17-hydroxysteroid hormones. Less than 2% of biologically active steroids are free in circulation, with the majority being bound with high affinity to SHBG and low affinity to albumin. The measurement of SHBG can be an important indicator of chronic or excessive androgenic activity where clinical symptoms indicate androgen excess, but upon measurement the androgen levels are found to be normal. Elevated SHBG levels can be seen in persons with androgen insensitivities, hyperthyroidism, cirrhosis and in patients on oral contraceptives or antiepileptic drugs. Decreased concentrations of SHBG are often seen in men with hypothyroidism and androgen replacement therapy; where women with hirsutism, virilism, polycystic ovarian syndrome, elevated androgen levels, obesity and acromegaly will also demonstrate decreased SHBG levels. Methodology: Serum samples were collected from residual samples for analysis across the range of the Access SHBG assay. We assessed the performance of the new Access SHBG immunoassay from Beckman Coulter with the Siemens Immulite SHBG immunoassay. A smaller sample set was employed for another method comparison between the Access SHBG assay, the Immulite SHBG assay and the Immunotech SHBG radioimmunoassay (RIA). An additional evaluation with another residual sample set was performed between the Access SHBG assay and the Roche Elecsys SHBG Validation: The results generated between the Access, Immulite and RIA methods demonstrated good agreement, as did the results between the Access and Elecsys methods. The Method Comparisons yielded the following Deming regression equations: Access = 1.12 (RIA) - 3.45 nmol/L, R = 0.96 (n = 41; range = 6 - 186 nmol/L) Access = 1.08 (Immulite) + 2.90 nmol/L, R = 0.97 (n=158; range = 6 - 190 nmol/L) Immulite = 0.93 (RIA) - 0.37 nmol/L, R = 0.97 (n = 41; range = 6 - 186 nmol/L) Access = 1.07 (Elecsys) -1.60 nmol/L, R = 0.99 (n = 123; range = 9 - 160 nmol/L) Conclusions: This study effectively demonstrates that the results from the automated Access SHBG assay correlate well to the Immulite SHBG, Immunotech SHBG RIA, and Elecsys SHBG assays. Laboratories transitioning between methods should expect very good analytical agreement. Disclaimer: Product is currently not available in the US.
D-88 Translating Sex Hormone Binding Globulin (SHBG) Measurements into Indices: Reference Intervals for Apparent Free Testosterone Concentration (AFTC), Bioavailable Testosterone and Free Androgen Index (FAI) D. Young, J. M. Mattke, C. Hodges-Savola, J. Xu, P. Marquet, A. MorelMontero. Beckman Coulter, Inc., Chaska, MN
Objective: The objective of this study was to establish readily available reference intervals, to be used as examples, for SHBG, Free Androgen Index (FAI) calculated free testosterone and bioavailable testosterone. Relevance: Sex hormone-binding globulin (SHBG) is responsible for transport of testosterone and estradiol. Less than 2% of biologically active sex hormones are free in circulation with the remainder being bound with high affinity to SHBG and low affinity to albumin. Free sex hormones and those weakly bound to albumin are available to the tissues. The sum of these fractions represents bioavailable hormone. Androgen status assessment in various conditions requires more information than total testosterone (TT) measurements alone. Albumin, SHBG and TT can be measured and used to calculate free and bioavailable testosterone to provide a better evaluation of androgen status. These calculated indices are recognized as the FAI, Apparent Free Testosterone Concentration (AFTC) and Bioavailable Testosterone. These calculated levels offer the convenience of automated immunoassays with results equivalent to technically difficult reference methods. Methodology: Reference intervals were established using a determination of the medians and 95% non-parametric reference intervals, with a bootstrap estimation of the 95% confidence interval of each of the intervals for 3 populations. Subjects included were: a.) 160 healthy males aged 20-50 years; b.) 149 healthy, cycling females aged 20-46 years; and c.) 141 healthy, postmenopausal females not on hormone replacement therapy, aged 47-91 years. Access Testosterone and Access SHBG assays from Beckman Coulter were performed on samples from all subjects. A constant of 4.3 g/dL was utilized for albumin. Free and bioavailable testosterone results employed the Vermeulen calculations available at http://www.issam.ch/ freetesto.htm. FAI was calculated by: FAI = (TT/SHBG)*100. Validation: Nonparametric reference intervals for: SHBG:13.2-89.5 nmol/L with a median of 36.9 nmol/L for males, 18.2-135.7 nmol/L with a median of 48.3 nmol/L for cycling females and 16.8-106.9 nmol/L with a median of 49.6 nmol/L for postmenopausal females. FAI : 22.2-110.2% with a median of 51.5% for males, 0.65-10.93% with a median of 2.64% for cycling females and 0.23-5.40% with a median of 1.70% for postmenopausal females. AFTC: 0.11-0.66 nmol/L with a median of 0.38 nmol/L for males, 0.006-0.055 nmol/ L with a median of 0.018 nmol/L for cycling females and 0.002-0.033 nmol/L with a median of 0.011 nmol/l for postmenopausal females. Bioavailable Testosterone: 2.8-15.5 nmol/L (25.7-70.5%) with a median of 8.8 nmol/ L (47.3%) for males, 0.13-1.30 nmol/L (14.8-57.4%) with a median of 0.43 nmol/L (33.2%) for cycling females and 0.049-0.762 nmol/L (15.3-59.0%) with a median of 0.26 nmol/L (31%) for postmenopausal females. Conclusions: As the FAI and calculated values for free testosterone and bioavailable testosterone become more accepted by the endocrinologist community, it is important to have reference intervals readily available. These reference intervals are useful as examples; however each laboratory should verify these intervals or establish their own intervals to assure proper representation of specific populations. Disclaimer: Product is currently not available in the US.
D-90 Comparison of a Reference Method and Three Immunoassay Methods for Estradiol (E2) D. Young, J. M. Mattke, A. Artus, S. Villeneuve, E. Romeu, W. Roudebush. Beckman Coulter, Inc., Chaska, MN
Objective: The objective of this study was to determine the correlation between the Access Estradiol assay and the International Federation of Clinical Chemistry (IFCC) ID-GCMS estradiol reference method. Two other automated immunoassays with ID-GCMS standardization were also selected for correlation. Relevance: Assay standardization is essential for superior patient care and ensuring high quality, reproducible results. The IFCC recognizes reference methods and/or reference standards for use to ensure commutability between methods. For estradiol, the IFCC recognized reference method is isotope dilution-gas chromatography mass spectrometry (ID-GCMS). In women, estradiol levels are used to monitor ovulatory status. Since estradiol levels reflect follicular maturation in women, they are a valuable tool in the assessment and treatment of sexual development, etiology of amenorrhea, causes of infertility and menopause. In men, abnormally high levels of estradiol are indicative of feminizing syndromes such as gynecomastia. Methodology: A set of 63 serum samples were collected for analysis across the range of the Access Estradiol assay for comparison and standardization to the IFCC recognized ID-GCMS method. Samples had Access Estradiol values ranging from 23 -4576 pg/mL. Additionally, a set of 247 serum samples was obtained from residual
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samples for comparison to the Access Estradiol assay and the Elecsys Estradiol II electrochemiluminescence assay (Roche Diagnostics) and the Architect Estradiol assay (Abbott Laboratories Diagnostics Division). Samples had Access Estradiol values ranging from 21-4572 pg/mL. Validation: The Access Estradiol assay and the ID-GCMS method demonstrated good agreement. The Deming Method Comparison yielded the following regression equation: Access = 1.02 (ID-GCMS) -6.18 pg/mL, R = 1.00 Correlations between the three immunoassay methods demonstrated good agreement, but there is bias of approximately 28% between the Architect and both the Elecsys and Access assays. The Deming Method Comparison yielded regression equations of: Access = 1.07 (Elecsys) - 41.49 pg/mL, R = 0.99 Access = 1.28 (Architect) - 38.03 pg/mL, R = 0.99 Elecsys = 1.29 (Architect) - 56.19 pg/mL, R = 0.99 Conclusions: This study effectively demonstrates that the results from the automated Access Estradiol method correlate well with the ID-GCMS and Elecsys methods. Interestingly, the Access Estradiol and Elecsys Estradiol II assays both demonstrate a similar bias when correlated with the Architect Estradiol assay. Laboratories transitioning between Access and Elecsys should expect similar patient values. Transitioning between Architect and either Access or Elecsys may translate to different patient values and reference intervals, thus requiring discussions with physicians.
D-92 Determination of intact parathyroid hormone (iPTH) reference intervals based on iPTH values from ambulatory patients M. M. Desouki, J. E. Madory, Y. Zhu. MUSC, Charleston, SC
Background: Serum iPTH reference intervals can be determined according to iPTH values in normal populations, but this approach requires recruitment of apparently healthy individuals. Since iPTH level is closely related to the levels of serum calcium (Ca) and vitamin D (25-OHD), and renal function, we may be able to define iPTH reference intervals based on its levels in ambulatory patients with normal serum Ca, 25OHD, and creatinine (Cr). The objective of this study is to determine if we need all or part of these three analytes to define iPTH reference intervals from ambulatory patients. Methods: We identified 2033 ambulatory patents who had PTH testing with ADVIA Centaur two-site sandwich immunochemiluminescent assay from our laboratory information system. From these individuals, we searched patients with normal levels of serum Ca (8.9-10.3 mg/dL), Cr (0.4-1.3 mg/dL), and 25-OHD (25-80 ng/mL). Since these laboratory results were obtained from ambulatory patients instead of normal control subjects, we employed Hoffmann statistical approach to define iPTH reference intervals. We used Microsoft Office Excel 2007 to generate Hoffman plot. The lowest and the highest iPTH levels in the linear portion of the S-shaped Hoffman plot are defined as reference intervals. Results: From 2033 subjects, we identified 1415 patients with normal Ca levels, 523 with normal Ca and Cr, 369 with normal Ca and 25-OHD, and 147 with normal Ca, Cr, and 25-OHD. iPTH reference intervals were determined as 10-110, 10-80, 10-75, and 10-70 pg/mL, respectively based on iPTH levels from ambulatory patients in the above four groups. Conclusions: iPTH reference intervals determined based on its levels in patients with normal Ca plus Cr, normal Ca plus 25-OHD, and normal Ca, Cr, and 25-OHD are very close to the manufacturer provided iPTH reference interval (14 - 72 pg/mL) obtained from 142 apparently healthy individuals with normal Ca levels. However, iPTH reference intervals determined from patients with normal Ca alone is higher. This study suggests that iPTH reference intervals can be determined from ambulatory patients with normal Ca plus normal Cr and/or 25-OHD levels. Serum calcium alone should not be used to determine iPTH reference intervals if the subjects are not from healthy populations.
D-91 Comparison of a newly developed ultrasensitive immunoassay with the ID-GCMS reference method estradiol
D. Young, J. M. Mattke, S. Bord, J. Fieschi. Beckman Coulter, Inc., Chaska, MN

Objective: To aid in the diagnosis of conditions where low concentrations of estradiol (E2) must be measured with precision, an ultrasensitive E2 immunoassay (usE2) was developed for the family of Access Immunoassay Systems from Beckman Coulter. Relevance: Estradiol-17 (estradiol, E2) is the most potent natural estrogen in humans and plays a major role during the sexual development and regulation of reproductive function. Most commercially available immunoassays accurately measure E2 concentrations during the ovulatory peak, luteal phase and pregnancy. However, due to assay sensitivity, most assays fail to accurately measure low concentrations of E2 which is necessary for several conditions such as precocious or delayed puberty, onset of menopause, investigation of the menstrual cycle (amenorrhea, infertility, Polycystic Ovarian Syndrome [PCOS]) or gynecomastia. In addition to the clinical need for an ultrasensitive estradiol assay, the International Federation of Clinical Chemistry (IFCC) recognizes the international reference method as isotope dilution-gas chromatography mass spectrometry (ID-GCMS) for estradiol assays. Methodology: The Access usE2 is a sequential, two-step competitive immunoassay with calibrators traceable to the ID-GCMS reference method. Assay performance, including limit of detection, imprecision and functional sensitivity were evaluated, in addition to a method comparison to the ID-GCMS reference method. Validation: Assay performance: The Access usE2 assay has a reportable range of <1 to 200 pg/mL, defined by the limit of detection and the highest calibrator. Within-run, between-run and total imprecision coefficients of variation (CV) were < 10% between 10 - 200 pg/mL. The functional sensitivity of the assay is <4 pg/mL. Time to first result is approximately 50 minutes. Comparison to ID-GC/MS: The Access usE2 calibrators were assigned values based on a reference curve established with 54 human serum samples values assigned by the ID-GCMS reference method. Correlation of this initial sample set achieved a Deming regression equation of Access = 0.99 ID-GCMS - 1.04, R = 0.98 (n=54; range 2.6 231pg/mL). The accuracy of the standardization was confirmed with a second set of 31 samples also tested by the ID-GCMS reference method. This comparison resulted in a Deming regression equation of Access = 0.98 ID-GCMS - 6.06, R = 0.98 (n = 31; range 6.0 - 181 pg/mL). Conclusions: This study effectively demonstrates that the results from the Access usE2 method correlate well to the ID-GCMS reference method. Analysis demonstrated a high level of precision and accuracy for low concentrations of estradiol which is preferable for use in patients with diminished estradiol levels, such as postmenopausal women. Disclaimer: Product is currently not available in the US.
D-93 Evaluation of the Beckman Coulter Access Testosterone Assay A. M. Dnistrian, R. Gonzalez-Espinosa, F. Celucia, A. Mathew, M. Fleisher. Memorial Sloan-Kettering Cancer Center, New York, NY
Introduction: The Beckman Coulter Access is recognized for the reliability of tumor marker assays for the management of prostate cancer patients (total PSA and free PSA). The availability of a sensitive and reliable testosterone assay on the same instrument as the tumor markers would be useful in monitoring prostate cancer patients during the course of androgen deprivation therapy, the basic intervention for hormone dependent prostate cancer. Methods: We evaluated the performance characteristics of the Access testosterone assay, a chemiluminescent immunoassay for the quantitative determination of total serum testosterone using the fully automated Access Immunoassay System. The sensitivity, accuracy, precision, linearity and reportable range for the Access testosterone assay were validated by standard statistical procedures for the Clinical Laboratory. A method correlation with the manual Siemens Coat-A-Count RIA procedure was performed with male specimens (N = 100) with testosterone concentrations ranging from 10 to 1205 ng/dL. Results: The Access testosterone assay exhibited an excellent analytical sensitivity (10 ng/dL) and a reportable range up to 1600 ng/dL. The within run imprecision was 1.8 - 3.2 % at three different levels of quality controls (140, 454 and 838 ng/dL, respectively), and the between run imprecision was 4.6 - 7.8 %. Regression analysis of correlation data for the automated procedure (y) with the manual radioimmunoassay method (x) yielded the equation y = 1.046x + 0.2 (R=0.9909) indicating the results were substantially equivalent. Discordant results between the two methods were observed for 7 of 27 specimens (26 %) with testosterone concentrations < 40 ng/dL signifying that accurate results at ultra low levels were not possible with these methods. Conclusion: The Beckman Coulter Access testosterone assay is a fast, reliable and reproducible method for the evaluation of male endocrine function. The test correlated well with a sensitive radioimmunoassay procedure but accurate measurements for patients with concentrations < 40 ng/dL would require an ultrasensitive method.
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D-94 The relevance of hormones ratio between cerebral spinal fluid (CSF)and serum for patients with pituitary tumors M. Purice1, M. Gheorghiu2, M. Coculescu2. 1"C.I.Parhon" National Institute of Endocrinology, Bucharest, Romania, 2"Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
It was previously shown by our group and others that pituitary tumors (PT) alter the permeability of blood-brain barrier (BBB) for the anterior pituitary hormones. Albumin CSF/serum ratio is generally considered an accurate index of the integrity of BBB (AR, albumin index).The aim of the study was to assess if evaluation of hormonal CSF/serum ratio (RC/S) has or not relevance for the damage of BBB and if it can help for the diagnostic of the tumor. Methods and patients: We evaluated CSF/serum ratio for GH, PRL, LH, FSH and TSH and albumin for 52 patients with pituitary adenomas (17-79 years, 25 M/27F, 16 before and 36 after pituitary surgery) comparative to a control group (n=10, 21-79 years, 6M /4 F, before undergoing abdominal or peripheral surgery). The study had the approval of the local Ethical Committee. Anterior pituitary hormones and albumin were simultaneously measuerd in serum and CSF by rapid fluoroimmunoassay (FIADELFIA Wallac)) and nephelometry (ARRAY, Beckman Coulter), respectively. FIA method for serum was prior validated for CSF and compared with a standard IRMA method. The report RC/S > 1 was considered as an index of hormonal secretion direct in CSF. An albumin index >0.007 was considered abnormal. Results: We found significant higher CSF/serum ratio comparative to control (p<0.001) for all pituitary tumor types but with normal concentrations LH,FSH and TSH in serum .For at least one pituitary hormone a RC/S > 1 was found in significantly more patients with tumors in contact with BBB- either before pituitary surgery (47%) or after surgery (56%)- compared with only 6% in patients with PT without contact with BBB before surgery (p = 0.001). Only patients with acromegaly and prolactinoma show significant high concentrations of GH and PRL, both in serum and CSF, comparative with control group (p<0.001) but with CSF/serum ratio <1. Albumin in CSF, in serum and the albumin index, were not statistically different between contact and non-contact tumors or in patients with hormonal CSF/serum ratio >1, compared to those with hormonal CSF/serum ratio (RC/S) <1. Conclusions: Pituitary hormones CSF/serum ratio can be a good index for BBB alteration but only for those hormones that are in low or normal serum concentrations (LH, FSH, or TSH). The alteration is correlated with a significant higher CSF/serum ratio mainly for tumors with suprasellar extension. CSF/serum albumin evaluation shows that there is no alteration of the CSF flow rate in patients with pituitary adenomas and with increased CSF/serum ratio for the anterior pituitary hormones, compared to controls.

carbimazole treatment for the hyperthyroidism, which uncovered the diagnosis of hypoparathyroidism. The hypoparathyroidism was managed with Calcichew and Alphacalcidol. The patient initially responded to treatment for both disorders but poor compliance of all therapies lead to a rebound pseudonormocalcemia. Most recently, in light of good compliance, the patient is clinically euthyroid with true normocalcemia. To our knowledge this is the 10th report of hyperthyroidism associated with DGS/ VCFS. As this syndrome is associated with other auto-immune conditions we investigated the possible underlying immunological mechanisms between hyperthyroidism and DGS/VCFS.
Changes in calcium concentration related to thyroid function Mar 07 Aug 07 Sept 07 Apr 08 Jan 09 Reference Interval
Treatment notes
Good Treatment compliance Treatment Non-compliance started for . Clinically started for Clinically to treatment thyrotoxicosis euthyroid. hypo- euthyroid 'Pseudo'PseudoTrue calcemia normocalcemia' normocalcemic' normocalcemia 8.04 (2.01) 6.24 (1.56 ) 8.24 (2.06 ) 9.56 (2.39 ) 9.16 (2.19) 8.8-10.4 mg/dL (2.2-2.6 mmol/L) 0.3-5.6 mIU/L 10-21 pmol/L
Adjusted calcium concentration Thyroid stimulating hormone Free thyroxine (FT4)
<0.03
<0.03
0.03
<0.03
<0.03
43
19.2
11.8
58.4
13.5
D-96 A Case of Biotin Interference in the Roche Elecsys 2010 Intact Parathyroid Hormone Assay D. L. Meany, S. M. Jan de Beur, M. J. Bill, L. J. Sokoll. Johns Hopkins Medical Institutions, Baltimore, MD
Objective: The objective of this study was to investigate biotin as an interfering substance in the Roche Elecsys 2010 intact PTH assay in a patient with renal impairment taking high doses of biotin. Background: A 64-year-old female with end-stage renal disease (ESRD) was evaluated for management of renal osteodystrophy. Adynamic bone disease (ABD) was suspected because of low normal serum intact parathyroid hormone (PTH), intermittently elevated serum calcium, and severe osteoporosis. However, her mildly elevated serum alkaline phosphatase (range: 149-196 U/L, reference range: 30-120 U/ L) was inconsistent with the low bone turnover observed in ABD. This discrepant clinical profile prompted investigation into the PTH assay used at our institution. Simultaneous samples were analyzed for intact PTH on our Roche Elecsys 2010 immunoassay analyzer and at a reference laboratory on the Siemens Immulite 2000 immunoassay analyzer. Discrepant values of 48 ng/L and 786 ng/L were obtained, respectively. Dilution studies confirmed the presence of a negative interference since PTH was higher in the diluted samples compared to the undiluted sample (undiluted = 48 ng/L; diluted 1:20 = 567 ng/L). After heterophilic blocking reagent had revealed no effects, we reviewed the patients medical history which suggested that biotin may be the cause of the interference since the Elecsys 2010 assay uses biotin-streptavidin mechanisms. Methods: We first determined the biotin concentration in the specimen to be 4.8 g/ L, approximately ten fold higher than the reference range upper limit (200-500 ng/L). Two different approaches were subsequently used to examine the interfering role of biotin: (i) studying the effect of added biotin and (ii) removing the effect of biotin, in this case using streptavidin-coated microparticles. The first approach was carried out by adding various amounts of free biotin (0- 160 g/L) into sera with normal and elevated intact PTH concentrations (33 and 487 ng/L, respectively). This second approach treated 50 L of the patients specimen with the streptavidin microparticles for 1 hour at room temperature with shaking. Results: The first approach failed to mimic the interference, whereas the second approach clearly identified biotin as the interference. In this experiment, the intact PTH concentration in the patients specimen increased from 32 ng/L pre-treatment to 419 ng/L post-treatment. The PTH recovery was >1,000% compared to recoveries of approximately 80% in 3 control specimens from patients not taking large doses of biotin. To further confirm the interfering role of biotin, serum intact PTH was measured in both laboratories after the patient stopped taking biotin for two weeks. The Elecsys 2010 result, 158 ng/L, was consistent with the result measured on the Immulite 2000 (223 ng/L) using the same specimen.
D-95 A case of pseudonormocalcemia and catch 22 A. Viljoen1, F. Kaplan1, S. Pereira1, P. J. Twomey2. 1East and North Hertfordshire NHS Trust, Stevenage, United Kingdom, 2Ipswich Hospital NHS Trust, Ipswich, United Kingdom
We present a case of dual pathology related to calcium homeostasis, assimilating to a normal calcium concentration. We coin the term, pseudonormocalcemia which has not been used before in the medical literature. Calcium homeostasis is a complex process which primarily involves: serum calcium and phosphate, 1,25-dihydroxyvitamin D and parathyroid hormone. Hypoparathyroidism is a relatively uncommon condition in which parathyroid hormone secretion is deficient or absent with subsequent hypocalcemia and hyperphosphatemia. It is most commonly seen following neck surgery. Hypoparathyroidism may also be due to developmental defects of the parathyroid glands as seen in the DiGeorge / Velocardiafacial syndrome (DGS/VCFS). Hyperthyroidism is associated with modesty raised calcium concentrations, with some reports of significant hypercalcemia. This association was noted more than a century ago. We present a case of an 18 year old female patient who initially presented with thyrotoxicosis and became acutely and severely hypocalcemic following treatment which yielded her to be clinically euthyroid. Subsequent investigations for the hypocalcemia revealed that the patient had hypoparathyroidism. The cause for this was shown to be DGS/VCFS, also known as CATCH22, and confirmed by FISH analysis revealing a deletion at the 22q11.2 location. As depicted by figure 1, the initial calcium concentrations at the time of diagnosis were near normal. The patient then became acutely hypocalcemic following
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Conclusions: Using the streptavidin microparticle treatment, we determined the interfering role of biotinylated molecules present in specimens from patients with renal impairment who take high doses of biotin with immunoassays that use biotin streptavidin interactions. An in vitro interference study using exogenous biotin failed to mimic the interference, which may be due to the discrepancy between exogenous biotin and endogenous biotinylated molecules.
determined by colorimetric assay, two-site chemiluminescence, or LC-MS/MS, respectively. fT concentration was determined either by ED or the mass action equation described by Vermeulen, et al. Association constants for testosterone binding to albumin and SHBG were previously published by Vermeulen, et al., Sodergard, et al. and Emadi-Konjin, et al. or individually derived for each patient group to yield the best fit with the ED data (cA and cB). fT concentrations measured by ED or calculated using these association constants were compared by weighted linear regression using Analyse-It. Statistical significance between groups was determined using JMP. Results: Individually derived association constants gave the best fit for their respective patient groups compared to previously published constants (A/cA: y=1.01x-0.23, r=0.941 and B/cB: y=1.00x-0.02, r=0.947). However, these constants yielded a poorer fit when applied to the other group (A/cB: y=1.29x-0.37, r=0.937 and B/cA: y=0.79x+0.03, r=0.952). Roughly 10-25% of each group had a poor fit (>15% CV) between calculated and ED fT results. SHBG levels in this subset were significantly higher than the subset with better fit (p<0.01). Albumin levels had minimal impact on fit, as calculated fT results were statistically similar whether albumin was measured or assumed to be 4.3 g/dL (p=0.94). Conclusion: Use of a mass action equation to calculate fT is a cheaper and faster alternative to ED. However, the choice of association constants has a significant impact on the fit between calculated and ED-measured fT. Constants that were individually derived to specific patient groups yielded a better fit for their respective groups than previously published constants, but yielded poorer fits when used on the other dataset. Poor fit is associated with higher SHBG levels and differences in fit between the two groups are likely due to their different gender makeup. This difference may be attributable to gender variability in the levels of other steroid hormones that bind SHBG. These results indicate that constants need to be verified in either large population sets or between numerous smaller datasets, and that different constants may be required for males and females to ensure goodness of fit.
D-97 Thyroid Autoimmunity and Trimester Specific Reference Intervals for Thyroid Function Tests in Pregnant Chinese Women S. Lu, Z. Xie, M. Wang, B. Zhu, Y. Sun. Zhejiang University, Hangzhou, China
Background: Proper maternal thyroid function is important during pregnancy. However, thyroid disorders are common in women of reproductive age, and maternal thyroid dysfunction is associated with a number of adverse outcomes. Because of physiological changes associated with pregnancy, interpretation of thyroid function tests (TFT) in pregnant women requires use of trimester specific reference intervals. In addition, ethnic and dietary differences are also known to influence thyroid function. For these reasons, reference intervals for TFT should ideally be established using local patient samples. The objective of our study was to establish trimester specific reference intervals for thyroid function tests in pregnant Chinese women. Methods and results: Serum samples were collected from 1409 pregnant women as part of routine antenatal care (n = 438, 400, and 571 for 1st, 2nd, and 3rd trimesters, respectively). All samples were tested for TPO-Ab, Tg-Ab, TSH, FT4, TT4, and TT3 using the Abbott ARCHITECT analyzer. In the study population, 18.7% of women were positive for TPO-Ab and/or Tg-Ab. Reference intervals (2.5th - 97.5th percentile) were calculated using antibody negative samples, and are reported in the table below. Trimester, Median (2.5th-97.5th percentile) First Second Third 1.219 1.314 1.619 (0.155-3.781) (0.339-3.512) (0.336-4.316) N=365 N=346 N=480 14.05 12.0850 10.66 (10.925-17.741) (9.288-15.240) (7.871-14.075) N=365 N=346 N=480 102.59 130.725 118.31 (73.148-164.422) (89.656-174.913) (83.493-170.143) N=365 N=346 N=480 3.82 3.765 3.65 (2.892-5.019) (2.914-4.603) (2.85-4.45) N=365 N=346 N=480 1.67 2.18 2.055 (1.202-2.570) (1.464-3.01) (1.37-2.889) N=365 N=346 N=480
D-99 Evaluation of the automated DxI direct Testosterone assay. D. Gruson, J. Ketelslegers, O. Alexopoulou, D. Maiter, C. Fille. Cliniques Universitaires St Luc, bruxelles, Belgium
Background: Radioimmunoassay (RIA) and chemiluminescence immunoassays are still the most widely used methods for measuring total testosterone (TT). RIA methods incorporating extraction procedure and chromatography offer several advantages, including removal of interfering proteins and separation of cross-reacting steroids, but are labor intensive and use a large serum aliquots to increase the sensitivity. Therefore, high-throughput and low volume direct automated assays employing serum or plasma without any extraction are often preferred nowadays but their performances and accuracy for measuring TT in male and female patients is still a matter of debate. The aim of this preliminary study was to assess the analytical performances of the DxI Testosterone assay and to compare it to our conventional RIA method. Methods: The linearity, precision and functional sensitivity were determined for the Beckman Coulter DxI Testosterone assay. Additionally, 100 patient plasma samples sent to the Cliniques Universitaires St-Luc Laboratory for testosterone testing were used to measure and compare the testosterone values obtained by the RIA method and the DxI Testosterone assay, respectively. Statistical analysis was performed on logtransformed data with the Medcalc software. Results: Within-run precision testing indicated maximum coefficients of variation (CV) for the DxI Testosterone assay of 27% at 0.35 nM, 1.8% at 7.4 nM and 2.2% at 15.7 nM. The between-run precision CVs for five levels of plasma pools were 24% at 0.43 nM, 9.9% at 1.0 nM, 8.7% at 1.5nM, 5.7% at 7.7nM and 4.7% at 16.0 nM. The functional sensitivity of the DxI Testosterone assay was estimated to be 0.65 nM. The average recoveries after dilution of two plasma samples titrated at 37.3 nM and 35.0 nM were 109% for a dilution factor of 2 - 97% for a dilution factor of 4 and 126% for a dilution factor of 8, reflecting a good linearity for this assay. The comparative analysis of testosterone results yielded the following regression equations: log[DxI] = 0.95 log[RIA] + 0.23 in males (r=0.953; p<0.001; n=74) and log[DxI]= 1.31 log[RIA] + 0.17 in females (r=0.757, p<0.001; n=26). Bland and Altman plots showed a systematic positive bias for the DxI for both males and females with mean differences of 4.1nM and 0.51 nM, respectively. Conclusions: The results of this preliminary study show that the DxI Testosterone assay appears to be a reliable method for the routine determination of TT without previous extraction. The accuracy and sensitivity of this assay are suitable for the determination of TT in both men and women. For both sex groups, the correlation between the DxI assay and our reference RIA was excellent but with a systematic positive bias for DxI. Additional studies should confirm the exact reference intervals using well-defined and characterized populations and the reliability of the DxI assay for its use in paediatric populations.
TSH (mIU/L)
FT4 (pmol/L)
TT4 (nmol/L)
FT3 (pmol/L)
TT3 (nmol/L)
Conclusions: Laboratory evidence of thyroid autoimmunity was relatively in the study population. Trimester specific reference intervals for TFT were established in pregnant Chinese women. These reference intervals were significantly different from those in non-pregnant women, and should prove useful as an aid in the interpretation of TFT during pregnancy.
D-98 Variability in Calculated Serum Free Testosterone Concentrations J. Hackbarth, J. Hoyne, S. K. Grebe, R. J. Singh. Mayo Clinic, Rochester, MN
Background: Free testosterone (fT) levels are important in the assessment of androgen status. However, equilibrium dialysis (ED) and immunoassay, two common methods of measuring fT, are either expensive and laborious or inaccurate, respectively. One alternative is to calculate fT levels using mass action equations based on serum albumin, sex hormone binding globulin (SHBG), and total testosterone (TT) levels. Several different formulas are commonly used. The goal of this study was to compare fT calculated from an equation with fT measured by ED to determine if variables including albumin, SHBG, association constants, and population makeup affect goodness of fit. Method: Serum concentrations of albumin, SHBG, TT and fT were measured for two separate groups of patients who underwent fT testing at Mayo Clinic Rochester: A (n=191, 93M/98F) and B (n=209, 171M/38F). Albumin, SHBG, and TT levels were
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D-100 Technical performance of the first fully automated assays for human soluble fms-like tyrosine kinase 1 and human placental growth factor E. Schneider, A. Gleixner, R. Haenel, W. Kraus, T. Ciesiolka, B. Denk, D. Gassner. Roche Diagnostics GmbH, Penzberg, Germany
Objective: An imbalance in circulating soluble fms-like tyrosine kinase 1 (sFlt-1), a splice variant of Flt1, and placental growth factor (PlGF) appears to be involved in the pathogenesis of preeclampsia (PE). Pregnant women who develop PE exhibit higher levels of sFlt-1 and decreased levels of PlGF. sFlt-1, an antagonist of PlGF (and VEGF), contributes to PE by absorbing free VEGF and PlGF in the maternal circulation and thereby preventing interaction with their respective membrane receptors. Automated immunoassays were established for sFlt-1 and PlGF measurements in human serum as an aid in diagnosis of PE in conjunction with other clinical findings. Materials and Methods: Elecsys sFlt-1 and PlGF are sandwich immunoassays for the quantitative determination of sFlt-1 and PlGF. Each assay applies a biotinylated mouse monoclonal capture and a monoclonal detection antibody labeled with a ruthenium complex. PlGF capture antibody is directed to an unique epitope on the PlGF molecule involved in receptor binding. sFlt-1 antibodies used do not interfere with the PlGF binding site of sflt-1. Basic assay characteristics were assessed. Molecular forms of sFlt-1 and PlGF in normal pregnant and PE samples were analyzed by Elecsys sFlt-1 and PlGF following gel filtration over Superdex 200. sFlt-1/PlGF ratios in normal pregnancies and pregnancies diagnosed with PE were determined. Results: Elecsys PlGF in serum detects biologically active PlGF, i.e. free PlGF as well as that part of sFlt-1-complexed PlGF which can still bind receptor. Elecsys sFlt1 detects total sFlt-1 (free multimeric and ligand-complexed multimeric sFlt-1) independent on PlGF binding. Destabilization of receptor ligand interaction by heat treatment released receptor masked PlGF from multimeric sFlt-1. Elecsys sFlt-1 covers a measuring range of 10 to 85000 pg/mL. The limit of detection (LOD) using CLSI protocol EP17A was found <10 pg/mL . The limit of quantitation (LoQ) was <15 pg/mL. Assay standardization against sVEGFR-1 ELISA (R&D Systems) demonstrated good agreement (P/B: y = 1.025*x-7.1, r = 0.967, n= 120, range < 2000 pg/mL). Total precision (CLSI protocol EP5-A2) resulted in CVs between 1.8 to 5.0% for concentrations from 59 to 72350 pg/mL. No high dose hook effect was found up to 200000 pg/mL. Elecsys PlGF covers a measuring range from 3 to 10000 pg/mL. LoD was <3 pg/mL. LOQ <10 pg/mL. Standardization against PlGF ELISA (R&D Systems) yielded comparable results (P/B: y = 1.013*x+0.58, r = 0.994, n= 119, range < 1000 pg/mL). Total precision CVs ranged from 1.0 to 2.6% for concentrations between 17.8 to 5730 pg/mL. No high dose hook effect was observed up to 100000 pg/mL. Both assays enable results within 18 minutes. In 75 normal pregnancies PlGF (sFlt-1) values ranged from 23 to 1818 pg/mL, mean 429 pg/mL (354 to 5282 pg/mL, mean 1883 pg/mL). The mean sFlt-1/PlGF ratio was 12.5 (range 1-114) in controls vs. 590 (range 145 -1440) in 21 pathological cases. Conclusion: Elecsys sFlt-1 and PlGF are the first, fast and automated immunoassays for convenient and reliable measurement of PE markers in maternal serum. Availability of these assays will offer new possibilities in the diagnosis of PE.

a more detailed study, we have now further investigated the ultrafiltration temperature effects on free thyroid hormone measurements by UF ID-LC-MS/MS, and compared its analytical performance with ED ID-LC-MS/MS at 37C. METHODS: An API-5000 triple-quadrupole mass spectrometer (AB-Sciex) coupled with electrospray ionization (ESI) source and Shimadzu HPLC system was used employing isotope dilution with 13C6-labeled internal standard (IS) for each analyte. 400 L of undiluted serum/plasma were placed in a Centrifree YM-30 ultrafiltration device (30,000 MW cut-off, Millipore), and centrifuged at 2900 rpm for 30 min at 25C. A duplicate ultrafiltration was repeated at 37C. 150 L of ultrafiltrates were further deproteinized by adding 450 L of methanol containing internal standards (IS). After centrifugation, 500 L of supernatant were diluted with 400 L of deionized water, and 650 L aliquot was injected onto a C-18 column. The tandem mass spectrometer was operated in negative ion mode, and thyroid hormones were quantified in multi-reaction monitoring (MRM) mode (649.7/126.7 for T3; 655.8/127 for T3-13C6; 775.7/127 for T4; 781.8/127 for T4-13C6). RESULTS and CONCLUSION: The results of ultrafiltration temperature effect demonstrated a linear least squares regression equation of y = 1.515x - 0.097 (n = 28; r = 0.974; Syx = 5.59; range 1.14-85.2 pg/mL) for FT4, and y = 1.495x + 0.038 (n = 24; r = 0.964; Syx = 1.414; range 0.55-23 pg/mL) for FT3. Both the serum concentrations of FT4 and FT3 show an expected temperature-dependency: by switching the ultrafiltration temperature from 25 to 37C, the results for both free T4 and free T3 increase by a factor of ~1.5. A comparison study between the ultrafiltration tandem mass spectrometric method and an equilibrium dialysis tandem mass spectrometric method was also performed at 37C, and results show good agreement, with a linear least squares regression equation of y = 0.428x + 2.712 (n = 37; r = 0.847; Syx = 0.803; range 5-21 pg/mL) for FT4.
D-102 Performance Characteristics of Six Intact Parathyroid Hormone Assays S. L. La'ulu1, W. L. Roberts2. 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 2Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT
The aim of this study was to evaluate the performance characteristics of six intact parathyroid hormone (PTH) assays: Beckman Coulter Access, Abbott ARCHITECT i2000SR, Siemens ADVIA Centaur, Roche Modular E170, Siemens IMMULITE 2000, and DiaSorin LIAISON. Imprecision studies were performed using three concentrations of commercially available quality control materials. Two runs of duplicate testing were conducted per day, for 5 days with a minimum of 2 hours between runs. For method comparison, 203 serum samples were tested by all methods with the E170 as the comparison method. A study comparing sample types and their stabilities was conducted where 20 individuals had two types of BD Vacutainer tubes drawn: red top and SST. An aliquot from each tube was tested after being subjected to the following conditions: tested immediately, frozen immediately and tested after thawing, after 24 hours at room temperature, and after 24 hours and 48 hours refrigerated. Total CVs ranged from 1.6 to 10.9%, 1.5 to 8.1%, and 1.1 to 5.5% for levels 1, 2, and 3, respectively. The E170 always had the lowest within laboratory CV and the LIAISON had the highest. Method comparison results by Passing-Bablok regression had slopes of 1.01-1.32 and correlation coefficients of 0.93-0.99. The results of the stability study are summarized (Table). Red Top Access ARCHITECT Centaur E170 LIAISON IMMULITE SST Access ARCHITECT Centaur E170 LIAISON IMMULITE Percent recovery compared to fresh Frozen 24h RT 24h 4C 48h 4C 87.9 82.4* 86.5* 92.4* 102.4 86.4* 91.3* 96.6 99.6 82.0* 87.4* 86.4* 101.2 92.7* 96.6 100.5 100.4 85.9* 94.0 97.1 81.0* 77.0* 84.6 86.0* Frozen 24h RT 24h 4C 48h 4C 95.2* 79.1* 89.3* 84.0* 99.9 80.8* 91.1* 89.0* 103.2* 76.7* 84.9* 80.5* 99.2 86.2* 97.3 92.4* 96.0* 82.3* 98.4 98.3 92.0* 116.5 95.7 86.3
D-101 Temperature Effects on Free Thyroid Hormone Measurements by Ultrafiltration and Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry T. Guo1, A. Xu1, S. J. Soldin1, S. J. Soldin2. 1NMS Labs, Willow Grove, PA, 2Georgetown University, Washington, DC
BACKGROUND: The free thyroid hormones, including free thyroxine (FT4) and free 3,5,3'-triiodothyronine (FT3), are the most physiologically active fractions of thyroid hormones. The two recommended laboratory tests to assess thyroid function are sensitive thyroid stimulating hormone (TSH, thyrotropin) and FT4 tests. Most clinical chemistry laboratories measure FT4 by various immunoassays (IAs), which have been criticized for their lack of specificity. Recently, isotope dilution (ID)-liquid chromatography tandem mass spectrometry (LC-MS/MS) assays have been reported to measure FT4 and FT3 in serum, following a physical separation by either equilibrium dialysis (ED) or ultrafiltration (UF) of serum. We previously developed the first UF and ID-LC-MS/MS assay to simultaneously measure FT4 and later FT3 in undiluted serum, with UF performed at 25C. A small comparative study had shown results were higher at 37C. The results at 25C were, however, identical to the then gold standard Nichols equilibrium dialysis method, which was performed at 37C. In
*Indicates a statistically significant difference (p<0.05) between fresh and other storage conditions. For red top tubes, one freeze-thaw cycle was not different from immediate analysis for all methods except the IMMULITE. Serum samples show statistical differences after
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24 hours refrigerated for three of the six methods. In summary, imprecision was acceptable for all methods. All methods correlated well to the E170 (r = 0.92 to 0.99). The IMMULITE exhibited the highest positive bias. The types of collection tube and sample storage conditions are more important for some methods than others.
with 10 mol/L aqueous formic acid) as the second dimension. Analysis was performed using Agilent 6410 QQQ with Hot Box Upgrade. Mass spectrometry conditions were as described above for the LC-MS/MS method with the addition of the MS/MS transitions for testosterone: 304 to 124 and 304 to 112. Results: LC-MS/MS method results: limit of quantification (LOQ) and upper limit of linearity (ULOL) were 0.33, and 92 ng/mL, respectively. Intraassay CVs were 15.9% (0.3 ng/mL) and 3.8% (2.3 ng/mL) and interassay CVs were 5.0% and 3.6% at concentrations of 0.3 and 2.3 ng/mL, respectively. The LC-MS/MS method comparison with chemiluminescent immunoassay (Siemens) resulted in a regression equation y= 0.59x + 2.02, r= 0.41, Sy/ x= 0.80 (n=31 samples). Out of 22 structurally similar steroids, none seemed to interfere with the analysis. The 2D-LC-MS/MS method results: LOQ and ULOL were 0.05 and 40 ng/mL, respectively. Total imprecision of the method, at concentrations 0.7-3.0 ng/ mL, was less than 15% for androstenedione and testosterone. The method comparison with a chemiluninescent immunoassay for androstenedione resulted 2D-LC-MS/ MSevaluated= 0.53*CEIA + 0.06, r=0.93, Sy/x= 0.17 (n=20 samples); and testosterone comparison with an LC-MS/MS resulted 2D-LC-MS/MSevaluated = 1.00*LC-MS/ MSreference + 0.04, r=0.98, Sy/x= 0.01 (n=20 samples). No interference was observed with 19 structurally similar steroids. Conclusion: LC-MS/MS method is free of cross reactivity and has acceptable sensitivity for the measurement of androstenedione in serum. 2D-LC-MS/MS showed acceptable performance using Agilent 6410 QQQ with Hot Box Upgrade and allowed the simultaneous measurement of androstenedione and testosterone.
D-103 Comparison of RIA for the measurement of 25-OH Vitamin D with automated Liaison CIA and with LC-MS/MS: A need for standardization. R. Kreller, M. K. Gupta, T. M. Daly. Cleveland Clinic Foundation, Cleveland, OH
With the recognition of high prevalence of Vitamin D insufficiency/deficiency in general population there has been growing interest in the screening for 25-OH Vitamin D levels. The increasing volume requires a simple reproducible method that will accurately estimate the total circulating 25-OHD independent of circulating 25OHD2 and D3. There is a growing confusion and concern about the variability among available Vitamin D assays i.e RIA vs. automated chemiluminescence immunoassay [CIA, Diasorin] and LC-MS/MS. To assess this variability we selected 148 samples in different range [between <7-150 ng/mL by RIA, Diasorin] and froze 4 aliquots and shipped one to a lab performing 25OHD analysis by CIA [Liaison, Diasorin] and another to a lab performing analysis by LC-MS/MS and compared their results with RIA. Both RIA and CIA utilize same antibody that reacts to both D3 and D2 forms equally. Data showed the median values [25-75 percentile range] of 23.65 (12-32.8 ng/mL) for RIA, 29 (10.2-32.8 ng/mL) for CIA and 32.0 [14.5-42.5]for LC-MS/MS. Linear regression analysis showed the following equations: CIA vs. RIA = 0.944 + 2.05 (r = 0.899); LC-MS/MS vs. RIA = 0.74 + 0.94 (r=0.91) LC-MS/MS vs CIA.= 0.75 + 1.42 (r=0.89). The paired T-test statistics showed significant differences in the mean levels between LC-MS/MS and RIA or CIA [P (one tailed) = <0.0001]. Our results show that although all three assays correlated well with each other the 25OHD levels were significantly higher by LC-MS/MS than immunoassays. Using a cutoff level of 30 ng/ mL[defined by RIA] for optimum levels, about 18 % of patients had insufficient levels by RIA and CIA had adequate levels by LC-MS-MS. This number is higher [28%] if we use 25 as an optimum cutoff value that is defined with this LC-MS/MS method. In conclusion, Vitamin D assays compared here show good correlation but differ in defining an individual as deficient vs. adequate. Our results indicate the need of standardization of these methods in terms of reference cutoff level.
D-105 The Establishment of Reference Intervals of Thyroid Peroxide Antibodies, Thyroglobulin Antibodies, and Thyroglobulin Assay by Modular E170 in Korean Population Compared with Immulite 2000 W. Lee, S. Yoon, O. H. Kwon, J. Kim. Yonsei University College of Medicine, Seoul, Republic of Korea
Measurement of thyroid peroxidase antibodies (TPOAb) is useful in diagnosing patients with autoimmune thyroid disease (AITD). Measurement of thyroglobulin antibodies (TgAb) is used to detect potential interferences with thyroglobulin (Tg) immunoassays and in limited situations for the diagnosis of AITD. The primary use of serum Tg measurements is as a tumor marker for patients carrying a diagnosis of differentiated thyroid cancer. We determined TPOAb, TgAb, and Tg by Roche Modular E170 analyzer. We determined the reference intervals for above items using 139 healthy subjects (Male/ Female, 79/60; mean age, 39.2; range, 25~49). We used the following exclusion criteria for reference subjects: blood glucose <50 mg/dL or >126 mg/dL; AST >36 U/ L (Male), AST >30 U/L (Female); ALT >50 U/L; Hb <11.0 g/dL; platelet <100,000/ mcL or >1,000,000/mcL; WBC <3,000/mcL or >15,000/mcL; and TSH <0.1 mIU/mL or >8 mIU/mL. Volunteers with known underlying diseases such as diabetes mellitus, hypertension, thyroid disease, gastric ulcer, cancer, and those taking medications were excluded. We also compared E170 with Immulite 2000 and calculated the kappa agreement ratio of each test to determine abnormal subjects. Nonparametric reference intervals (2.5 to 97.5 percentile) were 2.5 to 13.6 IU/mL (TPOAb), 5.0 to 124.2 IU/mL (TgAb), and 2.5 to 32.5 ng/mL (Tg). Passing-Bablok comparison of each test items (y) with Immulite 2000 (x) were as follows: y=0.637x0.872 (r=0.77, n=57) for TPOAb, y=5.913x-108.3 (r=0.84, n=123) for TgAb, y=1.281x-0.156 (r=0.94, n=130) for Tg. Kappa agreement ratios were 0.81 (TPOAb), 0.64 (TgAb), and 0.76 (Tg). We could establish reference intervals of TPOAb, TgAb, and Tg in Korean population. Although there were some bias of those test between Modular E170 and Immulite 2000, the concordance rate were relatively good.
D-104 LC-MS/MS methods for the measurement of androstenedione and testosterone in serum. E. Erdogan1, M. M. Kushnir2, B. Yue2, T. L. Blamires2, W. L. Roberts1, A. W. Meikle1, A. L. Rockwood1. 1Department of Pathology, University of Utah, Salt Lake City, UT, 2ARUP Laboratories, Salt Lake City, UT
Introduction: Androgens, a group of 19-Carbon steroids derived from cholesterol, have direct effects on diverse physiological and behavioral systems. Androstenedione and testosterone are frequently measured for detection of androgen-secreting tumors of ovarian and adrenal origin, evaluation of inborn errors of sex-steroid metabolism and disorders of puberty. Sensitivity, specificity and high throughput make LC-MS/MS methods advantageous over immunoassays for androgen analysis. Here we describe an LC-MS/MS method for the measurement of androstenedione and a two dimensional (2D)-LC-MS/MS method for the simultaneous measurement of androstenedione and testosterone using Agilent 6410 triple quadruple (QQQ) tandem mass spectrometer with Hot Box Upgrade (Santa Clara, CA). Methods: LC/MS-MS method: samples were derivatized using aqueous hydroxylamine and extracted using methyl tert-butyl ether. Chromatographic separation was performed on a 1200 series HPLC system (Agilent); 4.6 x 50 mm Rapid Resolution HT C18 column with 1.8-m particles (Agilent), the mobile phase (methanol with 10 mol/L aqueous formic acid). Analysis was performed using Agilent 6410 QQQ in the multiple-reaction monitoring (MRM) mode using mass transitions 317 to 124 and 317 to 112 for androstenedione and 307 to 124 and 307 to 112 for d3-testosterone (internal standard). 2D-LC-MS/MS method: the sample preparation was performed as described above using d3-testosterone and d7-androstenedione. A twodimensional chromatographic separation was performed using a Gemini Phenyl cartridge with mobile phase (gradient, methanol with 10 mol/L aqueous formic acid) as the first dimension and a Gemini C18 column with mobile phase (gradient, acetonitrile
D-106 Prolactin Reference Intervals With Polyethylene Glycol Precipitation on VITROS 5600 Integrated System S. SAW, C. Wang, S. Sethi. NATIONAL UNIVERSITY HOSPITAL, SINGAPORE, Singapore
Background: Recently we have seen publications providing reference intervals for monoprolactin samples treated with polyethylene glycol (PEG) precipitation of different immunoassay platforms. With the placement of the new VITROS 5600 Integrated System (Ortho Clinical Diagnostics, USA) in our laboratory we chose to determine the reference intervals on this platform. Methods: We assayed anonymised blood donor samples from healthy males (n=113)
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and healthy females (n=109) for prolactin on VITROS 5600. From these samples 12 male subjects and 21 female subjects were greater that the reference interval published by the manufacturer (male: 78-380mIU/L; female: 64-395mIU/L) and omitted. 250uL of patient serum was mixed with equal volumes of 25% PEG 6000 in PBS (137mmol/L sodium chloride, 10mmol/L sodium phosphate), pH 7.4. Samples were mixed well and incubated for 10 minutes, then centrifuged at 10000g for 30 minutes. The supernatant required a dilution 1:1 in normal saline prior to aspiration on VITROS 5600. Results: Post PEG treatment prolactin results for male: mean 137, minimum 29, maximum 224, median 139, SD 45; for female: mean 131, minimum 18, maximum 219, median 132, SD 47. The PEG treated means recovery was 53%, range 19-66% (male) and 50%, range 18-79% (female). These histograms showed a central tendency for both sexes in the range of 45-65%. Conclusions: We compared the results obtained to our current method of reporting and interpretation: Recovery >60%: sample contains mostly monomeric prolactin. Recovery 40-60%: greyzone - in addition to monomeric prolactin, sample also contains macroprolactin and/or oligomeric prolactin. Results need to be reported as such. Further assessment is necessary (e.g filtration chromatography). Recovery <40%: sample contains mostly macroprolactin results need to be correlated to clinical findings. This shows that our current reporting format is not likely to be transferable between instruments. It is known that there are differences in immunoassay antibody specificity for the prolactin isoforms. With this in mind it may then mandate manufacturers to include a reference interval for PEG treated samples to allow appropriate interpretation.

D-108 Glycated hemoglobin, insulin resistance and glucose tolerance in individuals at high risk of developing type 2 diabetes K. Santos-Rey, P. Fernndez-Riejos, J. Mateo, V. Snchez-Margalet, R. Goberna. HUVM, Sevilla, Spain
Objective To compare the glycated hemoglobin (HbA1c) levels with insulin resistance and glucose tolerance in individuals at high risk of developing type 2 diabetes. Research design and Methods A total of 685 patients with at least two risk factors for the developement of type 2 diabetes (obesity, dyslipidemia, hypertension, previous history of impaired fasting glucose or family history of diabetes) underwent an oral glucose tolerance test (OGTT) with 75 g glucose, as well as an HbA1c and a basal insulin measurement. Insulin resistance was assesed by Homeostasis Model Assessment (HOMA). Our laboratory follows the NGSP recommendations for HbA1c measurement, with a Level I Certification. Results From the 685 patients, 234 were euglycemic (1st group), 185 had impaired fasting glucose (2nd group), 116 presented impaired glucose tolerance (3rd group) and 150 met the diagnostic criteria for type 2 diabetes (4th group). First group presented a main HbA1c value of 5.02 % 0.36, and a HOMA value of 2.01 1.6; in the second group, HbA1c was 5.35 % 0.35, HOMA 3.14 1.7; third group, HbA1c = 5.66 % 0.33, HOMA = 4 2.4; fourth group, HbA1c = 6.13 % 0.61, HOMA = 5 2.6. Statistically significant differences were observed for HbA1c values in each group. Differences for HOMA values did not reach statistical significance. Conclusions HbA1c assay could be usefull to classify patients at high risk of developing diabetes.
D-107 Determination of Trimester-specific Reference Intervals for Thyroid Function Tests in a mixed South East Asian Population T. Loh1, S. Sethi1, S. Saw1, B. Saw1, S. Ong1, P. Wong1, M. Thevarajah2, N. W. Tee3, N. Sabir2, J. Sickan4. 1National University Health System, Singapore, Singapore, 2University of Malaya Medical Centre, Kuala Lumpur, Malaysia, 3KK Women and Children's Hospital, Singapore, Singapore, 4Abbott Diagnostics South Asia, Singapore, Singapore
The prevalence of thyroid autoimmunity and possibility of relative iodine deficiency during pregnancy appear to influence and determine the occurrence of thyroid underfunction. In addition, physiological changes in thyroid hormone status during the course of pregnancy complicate the assessment of maternal thyroid status. We undertook this study to establish a trimester-specific reference interval for a South East Asia population. Serum free-T4, free-T3, total T4, total T3, TSH and TPO-Ab were measured in 1024 pregnant women of mixed South East Asian ethnic origin using Abbott AxSYM analyzers. Thyroid autoantibodies were detectable in 11.7% (n = 120). We found significant difference in mean values between the 1st and 2nd, 1st and 3rd as well as 2nd and 3rd trimester using post-hoc ANOVA (p < 0.05) in our analysis of fT3, fT4, TSH and fT4:fT3 ratio. However, the mean values of fT3, fT4:TT4 ratio and fT4:fT3 ratio failed to show significance between the 2nd and 3rd trimester. No significant difference was found among the different trimesters for TT4 and TT3. fT4 (pmol/L) TSH (mIU/L) fT4 (pmol/L) TSH (mIU/L) fT4 (pmol/L) TSH (mIU/L) Mean 13.57 1.26 9.93 1.72 9.25 2.01 95% CI 12.97- 14.17 1.11 - 1.40 9.70 - 10.16 1.61 - 1.84 9.02 - 9.47 1.90 - 2.12
D-109 RELATIONSHIP BETWEEN GESTATIONAL MELLITUS AND PERIODONTAL DISEASES. DIABETES
P. Fernndez-Riejos1, G. Cisneros1, K. Santos1, J. Mateo1, R. Jaramillo2, R. Santos2, J. Rios2, P. Bullon2, V. Snchez-Margalet1, R. Goberna1. 1Department of Clinical Biochemistry, Virgen Macarena University Hospital, Seville, Spain, 2School of Medicine, Seville, Spain
Background: The association between periodontal diseases and diabetes mellitus has been recognized in the dental literature for several decades. The recent accumulating evidence of a relationship between periodontal infections and certain systemic diseases has renewed interest in this association. However, there are no comparable published studies evaluating this relationship in subjects with gestational diabetes mellitus (GDM).Objective: To investigate the relationship between periodontal diseases and GDM in pregnant women. Methods: We conducted a cross-sectional prospective pilot study to investigate the relationship between periodontal diseases and GDM in pregnant women from Seville, who came to University Hospital from November 2007 to November 2008. The subjects completed a questionnaire and underwent full-mouth periodontal examinations at enrollment (prior to 32 week gestational age). MAIN OUTCOME MEASURES: Plaque and bleeding scores, pocket probing depth and loss of attachment. Hemoglobin A1C level and C-reactive protein (CRP) were collected prospectively. Hemoglobin A1C reflects the mean blood glucose concentration over the preceding 1-3 months and CRP is a marker of systemic inflammation. Periodontitis was defined as the presence of at least four teeth with one or more sites with probing depth 4 mm and clinical attachment loss 3 mm. Known risk factors like smoking, alcohol, drug consumption, socio-economic status and the periodontal status were also recorded. Results: Data were collected from 165 subjects. A Mann-Whitney U test and a Students t-test were carried out in which N.D.S. were found. Conclusion:There is a good correlation between biochemical markers and the risk of periodontal diseases in women with GDM.
1st Trimester 2nd Trimester 3rd Trimester
Fine tuning of the management of subtle thyroid dysfunction during gestation requires a strict definition of the reference intervals of functional thyroid hormones, since both hypothyroidism and hyperthyroidism can lead to maternal and fetal complications. Data indicates significant differences among the mean values of the thyroid hormones across the three trimesters, supporting the utility of trimester-specific reference intervals.
D-110 Connective tissue growth factor: A marker for liver fibrogenesis? E. Kovalenko1, O. Gressner1, R. Weiskirchen1, A. Janetzko2, C. E. Geacintov3, A. M. Gressner1. 1RWTH University Hospital, Aachen, Germany, 2DRG Instruments GmbH, Mountainside, NJ, 3DRG International, Inc., Mountainside, NJ
CTGF, a member of the CCN protein family, is relevant in many physiological and pathological processes (1). Both, clinical and experimental studies have demonstrated
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that CTGF is overexpressed in fibrotic human liver and in experimental animal models of liver fibrogenesis (2). CTGF is detectable in various human fluids (serum, plasma, urine) and can potentially provide information about fibrotic remodelling processes (3). We established a novel method for the measurement of serum CTGF levels and tested if this CCN protein is a suitable marker for staging liver fibrosis. We examined 138 patients with chronic Hepatitis C virus infection (median age 47 years, range 17-85 years; 76 males and 62 non-pregnant females) and healthy controls (n= 80). CTGF levels were measured using a novel ELISA test that utilized capture and detection antibodies directed against the C-terminal and N-terminal part of CTGF. The CTGF level was determined to 15.410.0 g/L in healthy volunteers and 53.834.68 g/L in patients with chronic Hepatitis C. Moreover, the CTGF concentration was elevated in all patients, irrespective of the severity of hepatic fibrosis that was scored after Desmet Scheuer (F1 47.5 (median 45; range 11-171) g/ L, F2 48.2 (median 42; range 14-189) g/L, F3 52.9 (median 41; range 11-150) g/L, F4 81.5 (median 76; range 21-148) g/L). Therefore, we conclude that serum CTGF levels determined by the new DRG ELISA are usable for classification of diseases that are associated with fibrogenesis in general. References cited 1.Brigstock DR. Endor Rev 1999;20:189-206. 2.Paradis V, Dargere D, et al. Hepatology 1999;30:968-76. 3.Moussas E, Brigstock DR. Mol Genet Metabolism 2000;71:276-92.
D-112 Measurement of serum Testosterone: Waters Quattro Premier XE Micromass System versus ElecsysTestosterone II and ElecsysTestosterone F. Streit, N. von Ahsen, G. Brandhorst, M. Oellerich. University Medicine Goettingen, Goettingen, Germany
Immunoassays for quantification of testosterone can be affected by cross-reactivity with structurally related compounds, in particular with specimens in the low assay range (e.g. females) or from dialysis patients 1, 2. The purpose of the present study was to compare the performance of the new ElecsysTestosterone II assay (Roche Diagnostics) with the current ElecsysTestosterone (I) assay. The accuracies of the two immunoassays were evaluated against an in house lc-ms/ms-method, which in turn had been validated with a reference id-gc-ms-method (x): y=1.05*x-0,032 (r=0,998; n=38). Imprecision of the new Elecsys II assay was determined according to the CLSI-Protocol. Within run imprecision was 3.2% and 1.2% while total imprecision was 6.0% and 3.4% at testosterone concentrations of 0.22g/L and 13.00g/L respectively. The measuring range for the Elecsys II was from 0.02 g/L (LLOQ) to 15 g/L. A method comparison (Passing Bablock) with the specific lc-ms/ ms method is shown in the following Table. N 120 Male 120 151 Female 144 20 Dialysis male 20 20 Dialysis female 17 method Elecsys I Elecsys II Elecsys I Elecsys II Elecsys I Elecsys II Elecsys I Elecsys II conc. range (g/L) 0.063-12.55 0.04 - 12.86 0.02 - 5.09 0.02 - 4.15 1.61 - 7.71 1.07 - 5.76 0.08 - 1.4 0.07 - 0.61 slope intercept 1.055 1.056 1.336 1.074 1.431 1.196 5.098 1.098 0.043 0.016 -0.031 -0.035 -0.150 -0.332 -0.255 -0.029 r 0.976 0.982 0902 0.959 0.765 0.993 0.668 0.661 md95 0.817 0.702 0.388 0.206 1.011 0.19 0.073 0.211
D-111 Understanding Diabetes Mellitus in Singapore S. Ong1, X. Lin2, D. Poo2, S. Sethi1. 1National University Hospital, Singapore, Singapore, 2National University Singapore, Singapore, Singapore
Aim: To use data mining techniques in the understanding of diabetes mellitus in Singapore. Method: We extracted 65536 records in year 2003-2008 from the LIS database at National University Hospital of diabetes mellitus patients diagnosed on fasting plasma glucose 7.1mmol/L criterion. Each data set was anonymised with a unique number. Data field included patients demographic, fasting glucose, HbA1c and lipids(cholesterol, LDL-C, HDL-C, triglycerides). Descriptive statistics were done by calculating the demographic-based breakup for diabetes related tests for ethnic Chinese, Malay and Indian. Predictive modeling was done by regression analysis. Exploratory study was constructed using Microsoft Excel and SPSS 15.0 for cleaning and statistical analysis. Permission to use database was approved by our Domain Specific Review Board Ethic Committee Results: Descriptive modeling indicated that diabetics fasting glucose meansd was 9.73.3mmol/L (n=5186; male:female=1.1:1.0; age 16-86 years) with Chinese:Malay:Indian ethnicity of 65%:20%:15%. The ethnic Malay subjects had highest mean fasting glucose of 10.3mmol/L. Diabetic HbA1c was 8.11.8% (n=1307), total cholesterol was 5.21.1mmol/L (n=3271), LDL-C was 3.21.0mmol/ L (n=3273), HDL-C was 1.180.40mmol/L (n=3403) and triglycerides was 1.91.7mmol/L (n=3453). 60% of patients had HbA1c 7.0% and 40% has LDL-C 2.6mmol/L with 75% diabetics had both HbA1c and LDL-C in these ranges. Ethnic Chinese diabetics had the lowest mean HbA1c, total cholesterol and LDL-C and highest HDL-C. The Indian male had higher fasting glucose than female, Malay female has the highest HbA1c and females had higher LDL-C than males. In preliminary calculation of predictive modeling (n=869) of fasting glucose and LDLC, we used Tenure = (collection date - collection date of first visit)/age 30 * 24 * 3600. ANOVA analysis at p<0.05 change rates are: a) one year increase in age increased monthly change rate of fasting plasma glucose by 0.05 mmol/L, b) Fasting glucose of Chinese female had higher tendency to decrease over time compare to Chinese male, c) 1 mmol/L increase in baseline fasting plasma glucose resulted in monthly change rate of fasting glucose decrease by 0.79 mmol/L, d) 1 mmol/L increase in baseline LDL-C increases monthly change rate of fasting glucose by 0.46 mmol/L (R-square=0.268). Discussion: Based on criterion of fasting glucose7.1mmol/L, status of subjects duration of diabetes mellitus was not known. Further calculation of predictive modeling would include the contribution of factors such HbA1c, smoking, family history, body-mass-index measurements and blood pressure readings. Conclusion: Database mining tools appear to be able to define the theoretical foundation for the development of clinical decision support systems to assist in the management of diabetes mellitus.
The new Elecsys Testosterone II immunoassay showed good precision in the low assay range as well as markedly improved accuracy in female patients and hemodialysis patients as compared with the first generation assay. (1) Fitzgerald R, Herold DA. Clin. Chem. 1996, 42, 749-755. (2) Taieb J, Benattar C, Birr AS, Lindenbaum A. Clin. Chem. 2002, 48, 583-585.
D-113 Development of a Cortisol Assay using LOCI technology on the Dimension Vista Intelligent Lab System S. A. Lewisch, C. Clark, V. Desai, M. Drinan, L. Schiavoni, M. Sharma, Z. Teng. Siemens Healthcare Diagnositics, Newark, DE
Cortisol is a glucocorticoid hormone secreted in a diurnal pattern by the adrenal glands in response to adrenocorticotropic hormone (ACTH) which is released by the pituitary. Additionally, cortisol levels are increased due to stress. Measurements of cortisol aid the evaluation of adrenal insufficiency and excess. Low levels of cortisol imply hypocortisolism; high levels of cortisol indicate hypercortisolism. We describe the development and initial analytical performance of a fully automated, high sensitivity immunoassay for cortisol* employing LOCI technology on the Dimension Vista System (Siemens Healthcare Diagnostics, Newark, DE). LOCI technology is a homogenous, chemiluminescent immunoassay approach utilizing singlet oxygen transmission between sensibead and chemibead particles, incorporating two bead reagents and a biotinylated species. A generic bead reagent (sensibead) is coated with streptavidin and contains a photosensitive dye. A second bead reagent (chemibead) is coated with a cortisol analog. The chemibead contains a chemiluminescent dye as the signal generating component; it competes with analyte for limited amount of biotinylated antibody specific for cortisol. During the progress of the assay, the three reactants combine to form a bead-aggregated immunocomplex. Illumination of the complex by light at 680 nm generates singlet oxygen from sensibeads, which diffuses into chemibeads to trigger a chemiluminescent reaction that is measured at 612 nm. The resulting signal is inversely proportional to the concentration of analyte in the sample. The analytical range is 0.1 to 60 g/dL cortisol (spanning the interval from the limit of detection, mean plus 2 SDs of an analyte-free sample, to the upper calibration standard). The assay has a 16 minute turn around time and demonstrates excellent precision (testing was conducted over a ten day interval, using commercial quality control materials). Repeatability was 2.6 % CV and within lab precision was 4.8 % CV at 4.3 g/dL; at 23.0 g/dL repeatability was 2.0 % CV and within lab precision
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was 3.7 % CV; at 39.9 g/dL repeatability was 2.3 % CV and within lab precision was 4.1 % CV. Good agreement was observed between the comparative system, Siemens ADVIA Centaur, and the Dimension Vista system: Dimension Vista cortisol = (1.02 X Siemens ADVIA Centaur) + 0.9 g/dL (r = 0.97, n = 71). No significant interference (<10 % bias) was seen from lipemia (3000 mg/dL triglycerides), hemolysis (1000 mg/dL hemoglobin) or icterus (40 mg/dL conjugated bilirubin or 80 mg/dL unconjugated bilirubin). Minimal cross reactivity is observed. We conclude that use of LOCI technology provides excellent sensitivity, precision, turnaround time, and dynamic range suitable for measurement of cortisol. *product under development - not available for sale

molecular variants is not fully understood. Macroprolactin (macroPRL) has been listed as a diagnostic pitfall, and it is recommended to screen all patients for it in the routine investigation of hyperprolactinemia. Many studies have shown the association of macroPRL with certain autoimmune diseases particularly those with increased IgG type (e.g. SLE), hence our interest in individuals with increased thyroid autoantibodies (tAb+). We compared different PRL isoforms in healthy individuals with those having tAb+: anti-thyroglobulin greater than 105 IU/ml and/or antithyroid peroxidase greater than 30 IU/ml (both assayed by Abbott Axsym). PRL was assayed using Roche Modular analytics. Serum macroPRL was precipitated after treatment with equal volume of 25% polyethylene glycol (PEG) solution. MonoPRL was then determined in the supernatant; a recovery greater than 60% was negative for macroPRL. The method precision was 5.4% and 3.9% (between-run) for PRL & monoPRL respectively, and 0.94% (within-run). The analytical measuring range is 0.05-470 ng/ml. Serial dilutions on a sample with high PRL gave a linear response between 2.4-161.7 ng/ml and 1.1-69.3 ng/ml for PRL and monoPRL respectively. Healthy Males Females 34 26 3.6-19.7 2.8-16.9 60.0-95.6 0 0 0 4.1-23.0 3.8-19.5 75.5-99.0 0 0 0 tAb+ Males 11 6.1-24.6 5.2-20.2 82.0-100.0 1 2 0 Females 44 4.1-65.1 3.6-55.4 53.5-96.3 2 2 1
D-114 Commutability of Serum-based Reference and Proficiency Testing Materials for Total Testosterone with Selected Immunoassays J. C. Botelho, C. Shacklady, R. Razdan, A. Smith, H. W. Vesper. Centers for Disease Control and Prevention, Atlanta, GA
Testosterone is important in the diagnosis of androgen diseases and disorders such as androgen deficiency in men or polycystic ovary syndrome in women. Problems in the performance of testosterone tests have been described and the need to improve testosterone testing has been stated by many researchers and organizations. CDC is addressing this with its steroid hormone standardization project. One tool to help address current problems in testosterone testing is the use of reference materials to assess assay trueness and allow for traceable calibration. One material characteristic that describes the suitability of a reference material for use with certain assays is its commutability. Currently, no information about the commutability of reference materials (RM) and proficiency testing (PT) materials for testosterone testing exists. The aim of this study was the assessment of commutability of serum-based reference and PT materials. Serum-based RM from NIST (SRM 971) at two levels: 27.6 ng/dL (F) and 641 (M) ng/dL, dilution of these RM with assay specific diluents, and 6 materials intended for PT programs were assessed for commutability. 14 immunoassays from 8 manufacturers using 40 fresh-frozen patient samples (3.66-788 ng/dL) over 3 days (n=7) were used to make the evaluation. Measurement results obtained by the immunoassays were compared against those obtained by a mass spectrometry-based certified reference method. Initial assessment of commutability was made following CLSI protocol EP14-A2 Evaluation of Matrix Effects. Results are summarized in the table below. This initial assessment found the materials and dilutions being commutable for most immunoassays. For some assays SRM materials were commutable while PT materials were not commutable, which may lead to incorrect detection of bias in the PT study. Assay %CV Male %CV Female Material Percent Commutable 1 6.21 13.2 SRM-M Undiluted 71.4% 2 3.28 9.19 SRM-M Dilution 1 71.0% 3 3.00 7.52 SRM-M Dilution 2 92.9% SRM-M 4 11.5 24.3 92.9% Dilution 3 5 10.8 21.8 SRM-F Undiluted 92.9% SRM-F 6 10.9 29.5 92.9% Dilution 1 SRM-F 7 2.68 5.17 85.7% Dilution 2 8 2.00 5.19 PT 1 71.4% 9 1.67 3.75 PT 2 100% 10 2.38 10.4 PT 3 71.4% 11 2.07 8.36 PT 4 71.4% 12 1.27 6.21 PT 5 100% 13 2.81 11.8 PT 6 100% 14 6.67 7.71
Number (N) Prolactin (ng/ml) (ranges) Monomeric (ng/ml) (ranges) Recovery (%) (ranges) Increased PRL (N) Increased MonoPRL (N) Macroprolactin (N)
The ranges for PRL and monoPRL obtained for the healthy individuals in this study were used as cutoffs for normal. As described in the table, 3 out of 55 individuals with tAb+ had increased PRL. One additional male had increased monoPRL only. A recovery of 53.5%, highly suspicious for macroPRL, was found in one of the two females. In comparison with the control group, both PRL and monoPRL levels were higher in the group with tAb+, but this increase was not considerably associated with the presence of macroPRL.
D-116 The analysis of 25-hydroxyvitamin D in serum using semi-automated solid phase extraction and LC/MS/MS L. J. Calton1, B. J. Molloy1, B. G. Keevil2, D. P. Cooper1. 1Waters Corporation, Manchester, United Kingdom, 2University Hospital South Manchester (UHSM), Manchester, United Kingdom
Introduction: In recent years the demand for serum 25-hydroxyvitamin D (25OHD) analysis has increased significantly. In addition to the role vitamin D plays in bone metabolism, several clinical studies now link vitamin D deficiency with increased risk for certain cancers, multiple sclerosis and heart disease. 25OHD concentration is accepted as the clinical indicator for determining vitamin D status and is also important for monitoring supplementation therapy, which is available in two forms, vitamin D2 and vitamin D3. Many clinical laboratories have now adopted LC/MS/MS based methods for measuring 25OHD to enable the simultaneous and reliable measurement of 25OHD2 and 25OHD3. Some immunoassays may under report the total 25OHD level for patients on Vitamin D2 supplementation due to the crossreactivity of the antibody for 25OHD2 being less than 100%. The analysis of 25OHD by LC/MS/MS requires a number of sample pre-treatment steps to release 25OHD from Vitamin D binding protein and to minimise matrix effects. This method describes a semi-automated sample pre-treatment protocol in combination with UPLC/MS/MS for the simultaneous analysis of 25OHD2 and 25OHD3. Methodology: For the initial study, twenty samples were analysed using an established manual liquid/liquid solvent extraction LC/MS/MS method at UHSM. All serum samples were anonymised and stored at -20C. Calibrators were prepared in horse serum (Sigma) over the concentration range 2.5-100ng/mL and QC samples were prepared independently. Commercially available calibrator and QC material from Chromsystems were also analysed. Primary serum samples and calibrators were placed on a robotic liquid-handling system and identified by bar code to be tracked throughout the extraction procedure. Hexa-deuterated internal standard was added to the dispensed samples prior to protein precipitation. Following centrifugation (offline), the supernatant was transferred to a conditioned Oasis HLB Elution SPE plate and washed. The retained analytes were eluted by the liquid-handling system and the eluant was chromatographed using a Waters ACQUITY UPLC with an ACQUITY BEH Phenyl column (2.1x50mm) with a water/methanol/ammonium
D-115 Prolactin Isoforms in Healthy Subjects Versus Individuals with Increased Thyroid Autoantibodies R. T. Daher, R. A. Nassar, D. S. Sarriedine, N. K. Cortas. Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon
Hyperprolactinemia described in autoimmune diseases, supports an immunomodulatory role for prolactin (PRL) through activation and enhancement of the immune process by binding to PRL receptors. The biological significance of PRL
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acetate gradient. A Waters TQD mass spectrometer was used to quantify 25OHD2 and 25OHD3, monitoring two transitions for each analyte. Results: The assay was linear over the range 2.5-100ng/mL with a coefficient of determination (R2) of > 0.997. The inter- and intra-assay imprecision at three levels was less than 10% CV (n=5, over 5 days). The calculated 25OHD concentrations of the twenty patient samples were in good agreement with the LC/MS/MS measurements from UHSM, using Passing-Bablok analysis Waters=1.01(UHSM)1.45. Ion suppression studies were conducted and demonstrated minimal ion suppression for 25OHD3. Conclusion: A routine semi-automated solid phase extraction UPLC/MS/MS method for the analysis of 25OHD2 and 25OHD3 in serum has been developed. The assay has acceptable performance characteristics (linearity, sensitivity, precision and accuracy) and allows the analysis of up to 196 samples per working day. The semi-automated procedure significantly reduces the manual operation steps, eliminates solvent evaporation, reconstitution and operator variability to ensure more consistent and reproducible results.
D-118 LC-MS/MS Reference Intervals for Androgens in Serum of Children M. M. Kushnir1, T. Blamires1, W. L. Roberts2, B. Yue1, A. L. Rockwood2, A. M. Bunker1, E. Erdogan2, A. W. Meikle3. 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 2Department of Pathology, University of Utah, Salt Lake City, UT, 3Departments of Medicine and Pathology, University of Utah, Salt Lake City, UT
Dehydroepiandrosterone (DHEA) and androstenedione (A4) are androgen precursors of male and female sex hormones. Measurement of DHEA and A4 is useful in diagnosing sex hormones-related disorders because abnormal production of these steroids may cause pathological changes in masculine and feminine characteristics. Gender and age-specific reference intervals (RI) for androgens are required for interpreting results of diagnostic testing. Available RI of DHEA and A4 established with automated analyzers are reagent- and method-specific and not transferable between instruments of different manufacturers. As a general rule validated liquid chromatography tandem mass spectrometry (LC-MS/MS) methods provide accurate measurement of analytes, independent of the instrument type and vendor of the reagents and are transferrable between laboratories. We developed a high sensitivity/ high specificity LC-MS/MS method for DHEA and A4 and established age and Tanner Stage specific RI in children 7 - 17 years of age. The method utilizes solvent extraction and derivatization of the androgens to form oxime derivatives to enhance sensitivity. Instrumental analysis was performed using two-dimensional chromatographic separation and MRM acquisition on an API 4000 (Applied Biosystems/ MDS Sciex) LC-MS/MS. The assay was linear up to 9 ng/mL for DHEA and 40 ng/mL for A4. Total imprecision of the method at concentrations of 0.05, 0.3, 0.7, and 2.5 ng/mL was less than 9.3% and 11.2% for DHEA and A4, respectively. The lower limit of quantitation was 0.05 ng/mL for DHEA, 0.01 ng/mL for A4. Method comparison with another LC-MS/MS yielded linear regression equations of Y =0.89 * X - 0.04, r =1.00, Sy,x = 0.05; and Y =0.95 * X - 0.50, r =1.00, Sy,x = 0.27 for DHEA and A4, respectively. Nonparametric reference intervals for children are summarized in the table. The method requires small sample volume (0.2 mL) and is sufficiently sensitive to measure DHEA and A4 in all population groups. Gender and age Boys 7 to 9 Boys 10 to 11 Boys 12 to 13 Boys 14 to 15 Boys 16 to 17 Girls 7 to 9 Girls 10 to 11 Girls 12 to 13 Girls 14 to 15 Girls 16 to 17 N 206 140 143 141 136 206 148 142 143 138 Androstenedione, ng/mL 0.031-0.31 0.072-0.41 0.11-0.64 0.18-1.01 0.31-1.14 0.038-0.49 0.04-1.27 0.19-2.09 0.43-2.09 0.39-2.15 DHEA, ng/mL 0.092-2.46 0.3-3.81 0.58-4.11 0.87-6.64 1.21-7.63 0.12-2.70 0.55-3.92 0.81-6.34 1.23-7.63 1.46-9.51
D-117 ANALYTICAL AND CLINICAL EVALUATION OF THE AUTOMATED LIAISON INSULIN-LIKE GROWTH FACTOR (IGF-I) ASSAY H. Mekouar, D. Gruson, D. Maiter, M. Maes, J. Cumps, J. Ketelslegers. Clinique Universitaire Saint-luc, UCL, brussels, Belgium
Background. IGF-I assay is essential for management of Growth Hormone Deficiency (GHD) in children and adults as well as diagnosis and follow-up of acromegaly. The Liaison from Diasorin allows automation of IGF-I assay and is based upon a sandwich chemiluminescence immunoassay, after neutralization of IGFI binding proteins by excess of IGF-II. The performances of this automated assay are currently poorly documented. The aim of this study was to determine the analytical and clinical performances of the IGF-I Liaison assay. Methods. Precision, linearity and functional sensitivity were determined for the IGF-I Liaison assay using different serum pools covering a broad spectrum of IGF-I concentrations. IGF-I Liaison assay was also compared with 490 samples previously measured with the former Advantage Nichols Institute Diagnostics (NID). In addition, normal values over the life span were derived from the correlation with Advantage assay. Finally, to assess the concordance with our reference values, we determined IGF-I in patients samples with clinically established GHD (children and adults) or active acromegaly. Results. Intra-assay coefficient of variation (CV) obtained with 20 replicates of serum pools: were 6.4% at 19ng/mL, 6.5% at 57ng/mL, 2.0% at 251ng/mL and 4.0% at 575ng/mL. Inter-assay variation were assessed with four levels IGF-I serum pools (9, 20, 21 and 31 ng/mL) run as singles (n = 20) for 2 weeks with 4 calibrations, CVs obtained were 33.2%, 15.3%, 12.7% and 8.1% respectively, consistent with a functional sensitivity of 15 ng/mL . After serial dilutions of serum pools, the linearity was supported. After log transformation of the data, the Liaison IGF-I assay correlated closely with the Advantage IGF-I assay: Liaison = 0.315 + 0.875 x Advantage (r = 0.99; n = 490; range: 15 - 1246 ng/mL). Passing and Bablock regression analysis (distribution of Studentized residuals) showed no significant deviation of linearity. Estimated reference values for Liaison were derived using the regression equation, from 1656 normal samples previously published. Liaison baseline values (0 - 5 years) rose from 71 (30 - 170) ng/mL to a peak of 372 (211-655) ng/mL (13 - 16 years); values declined to 240 (130 - 440) ng/mL (20-25 years), and further decreased to 145 (93 - 225) ng/mL at 60 - 70 years (geometrical means; 95% CF). To evaluate the clinical concordance of our reference values, IGF-I was tested in 11 GHD children (3 - 11 years), before treatment, IGF-1 was < 15 ng/mL (n = 4) or low (17 to 32 ng/mL); eleven adult patients with GHD were also evaluated; 4 subjects with childhood onset GHD (19 - 48 years) had IGF-I levels of <15 - 28 ng/mL; in 7 patients with adulthood GHD (47 - 84), IGF-I ranged from <15 ng/mL to 34 ng/mL. In 14 active acromegal patient, IGF-I range was: 530ng/mL to 903 ng/mL. Conclusion. Liaison IGF-I assay offers analytical performances suitable for routine determination of IGF-I and excellent correlation with the used reference assay. After establishment of the assay reference values, we also demonstrated the reliability of this assay through different patients samples.
D-119 Performance evaluation of an automated immunoassay for the determination of Cortisol on the Olympus AU3000i Immunoassay System M. Evans, A. O'Sullivan, S. Hennessey, J. Hanley, S. Gaston. Olympus Life Science Research Europa (GmbH), Co.Clare, Ireland
Cortisol, also known as hydrocortisone, is the major glucocorticoid produced and secreted by the adrenal cortex and has a number of functions including the regulation of carbohydrate, lipid and protein metabolism together with immunosuppressive and anti-inflammatory activity. Cortisol circulates either bound to transport proteins (90%) or as free hormone. Only the free, unbound cortisol fraction can interact with its receptor and is thus physiologically active. In individuals with normal renal function, about 1% of the plasma cortisol is excreted unchanged into the urine and levels can be related to the concentration of biologically active plasma free cortisol. Plasma cortisol levels normally follow a diurnal rhythm, with maximum concentrations (around 750nmol/L) usually reached early in the morning and followed by a gradual decrease during the day. The Olympus Cortisol assay is a one step paramagnetic particle enzyme immunoassay. It is based on the competitive principle and is used to quantitate cortisol in serum, plasma and urine. Reported here are results from the development and evaluation of the automated assay for cortisol on the Olympus AU3000i Immunoassay System. This assay is
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referenced to ID-GC-MS with a limit of detection and limit of quantification of 5nmol/L and 8nmol/L, respectively. Assay imprecision was characterized over 5 days according to CLSI protocol EP15-A. Total coefficients of variation (CV) for low (<230nmol/L), medium (600-950nmol/L) and high (>1500nmol/L) human serum and urine pools were determined to be 6.5%, 4.0% and 3.2 % respectively for serum and less than 7% for all urine pools tested. A method comparison was performed against the Roche Cobas e411Cortisol assay using serum samples over a concentration range of 10-2000nmol/L, producing a slope of 1.05, an intercept of -1.09nmol/L and a correlation coefficient of 0.95. Dilution linearity was demonstrated over the entire range of the assay from 103000nmol/L. No significant interference was detected from bilirubin, hemoglobin triglycerides, Intralipid, protein and urea. Reagents were optimized to negate RF and HAMA interference. No significant cross-reactivity was observed with any of the compounds and drugs normally associated with automated cortisol immunoassay; these include all other major steroid hormones and compounds known to cause interference in cortisol assays, e.g. cortisone, 11-deoxycortisol, dexamethasone, methylprednisolone, prednisolone and progesterone. Based on our evaluation, we conclude that the Olympus Cortisol assay is a sensitive, precise, and specific method that meets the requirements of measuring cortisol in human serum, plasma and urine.

menopause. AMH is produced in the primordial follicle. In conjunction with FSH and Age, it is the most used marker for estimating ovarian reserve. Unilab of Dade is a private laboratory dedicated to reproductive medicine. Procedures used for patient diagnosis are first validated and then verified from time to time for linearity, normal range, specimen requirement and additional parameters that may be deemed necessary by our Director or clinicians. This study evaluated 900 orders received for AMH levels during 2008. The following conditions were analyzed: 1. Specimen Stability 2. Validity of the calculated normal range 3. AMH levels vs. Age 4. AMH levels vs. FSH 5. AMH level and incidence of pregnancy Method: Subjects were screened for AMH levels using the Diagnostics Systems Laboratories (DSL) ACTIVE MIS/AMH ELISA procedure. This is an enzymatically amplified two-site immunoassay. All orders received were used for the calculated range. Patients that were followed in treatment towards pregnancy were used in the physiological range. Three age groups were evaluated: 18-30 years, 31-36 years and 37-50 years. These samples were evaluated for Normal Range, Age, FSH and Pregnancy correlation. Results: 1. Sample Stability according to the insert range is 24 hours at 2-8C. Stability was expanded to one week at 2-8C with less than <1% decay. 2. The normal estimated range was first calculated for each age group by determining a Mean and 2 Standard Deviations for results obtained between <0.05-5.0 ng/mL. 3. Physiological range was obtained by determining the AMH level detected for each group with patients resulting in an elevated HCG: 4. Additional relationships: A. AMH vs. AGE B. AMH vs. FSH C. None detectable levels of AMH Conclusions: 1. Normal levels of AMH correlate with age and FSH. 2. Age Group 18-30 years: AMH levels of 0.5-5.0 ng/mL correlate well with FSH values of 0.0-8.0 miu/ml 3. Age Group 31-36 years: Incidence of higher FSH values begins to increase with lower AMG counterparts. 4. Age group 37-50 years: Shift of FSH to higher levels with large percent in abnormal range. AMG values continue to decrease with a high percent of NonDetectable noted. 5. AMH Non-Detectable may be inducible towards pregnancy as occurred in two cases of this study. 6. An AMH normal level is not sufficient to determine the successful rate of pregnancy.
D-120 Performance evaluation of an automated immunoassay for the determination of estradiol on the Olympus AU3000i Immunoassay System P. O'Dea, P. Curley, B. de Grimaudet de Rochebout, A. O'Sullivan, S. Gaston. Olympus Life Science Research Europa (GmbH), Co.Clare, Ireland
Quantitation of estradiol (E2) in human serum and plasma has been utilized as a means for monitoring ovulatory status and is used extensively in in-vitro fertilization monitoring. Distinguishing low levels of estradiol in pre- and postmenopausal women also enables risk assessment of common fertility related diseases in older women. Abnormally high levels of estradiol in males are indicative of gynecomastia and other feminizing syndromes. The Olympus Estradiol assay is a paramagnetic particle, chemiluminescent immunoassay for the quantitative determination of estradiol levels in human serum and plasma using the Olympus AU3000i Immunoassay System. Reported herein are results from the development and evaluation of the automated assay for estradiol on the Olympus AU3000i Immunoassay System. This assay is referenced to ID-GC-MS assigned sera. Limit of detection and functional sensitivity (at a CV of 20%) were determined to be 15pmol/L and 70pmol/ L respectively. Assay imprecision was characterized according to CLSI protocol EP15-A. Within run coefficients of variation (CV) for low (<184pmol/L), medium (184pmol/L -1835pmol/L), and high (>1835pmol/L), human serum pools were determined to be 4%, 3%, and 1% respectively. Total precision was determined to be 5%, 3%, and 2% for the same serum pools. Method comparisons performed against the Roche Cobas e 411 Estradiol assay using 126 serum samples over a concentration range of 50pmol/L to 6000pmol/L yielded a slope of 0.9874, y-intercept of -69.2pmol/L and correlation coefficient of 0.984. No significant interference was detected from bilirubin, hemolysate, triglycerides and Intralipid. Reagents were optimized to negate RF and HAMA interference. No significant cross-reactivity was observed with estrone, estriol and other potentially cross-reactive analogues. Based on our evaluation, we conclude that the Olympus Estradiol assay is an exceptionally sensitive, precise, and specific method that meets the requirements of measuring estradiol in human serum and plasma.
D-122 Increased leptin expression in placenta from pathological pregnancies A. Perez-Perez1, Y. Gambino2, J. Maymo2, J. L. Dueas1, R. Goberna1, C. Varone2, V. Sanchez-Margalet1. 1Virgen Macarena University Hospital, Seville, Spain, 2University of Buenos Aires, Buenos Aires, Argentina
Objectives: Leptin is a regulatory hormone in many cellular systems, including trophoblastic cells, where it is produced and may function as a trophic factor. In the present work we aimed to study the expression level of leptin in pathological pregnancy (gestational diabetes and pre-eclampsia) compared with control placenta from normal pregnancies. Materials and Methods: We have studied trophoblastic samples from placenta obtained from donors after full-term delivery. 8 control placenta from normal pregnancies, 6 placenta from gestational diabetes, and 5 placenta from pre-eclampsia were studied. Leptin expression level was determined by real time PCR and the protein level by immunoblot. Students t test was employed to assess statistical differences. Results: We have found that every placenta from pathological pregnancy have increased expression level of leptin, compared with control placenta from normal pregnancy. Both placenta from gestational diabetes and preeclampsia expressed in average twice as much leptin levels than control placenta, significant differences (p<0.001) between placenta from pathological pregnancy and control placenta were found. Conclusions: Pathological pregnancy (gestational diabetes and pre-eclampsia) produces an increase in the leptin expression in trophoblast, and leptin may be a useful marker in the diagnosis and follow-up of these pathological pregnancies.
D-121 Correlation of Factors affecting the AMH Normal Range for Women in the Reproductive Years G. A. Agramonte1, J. M. Cortes2, K. Gelman3, N. Goodman4, C. Ferguson1, A. Menocal1. 1Unilab, Fort Lauderdale, FL, 2Unilab of Dade, Fort Lauderdale, FL, 3Infertility and Reproductive Medicine of South Broward, Pembroke Pines, FL, 4Neil Goodman, MD, Miami, FL
Background: AMH has important functions for both male and female reproductive organ development. AMH/HIS begins circulating after puberty, when menstrual cycling begins and slowly decreases throughout life until it becomes undetectable at
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D-123 Free Cortisol Measurement in Human Serum by Combined Equilibrium Dialysis and LC-MS/MS J. Twentyman, K. Cradic, S. Grebe, R. Singh. Mayo Clinic, Rochester, MN
Background: Free serum cortisol is perceived to be superior to total serum cortisol for assessment of adrenal function in severely ill patients. Historically, free serum cortisol has been calculated from the concentrations of total serum cortisol and cortisol binding globulin (CBG). Questions have been raised as to the validity of these methods.1 Equilibrium dialysis combined with LC-MS/MS offers an effective means of measuring free cortisol directly. We offer a method for directly quantifying free cortisol in serum using equilibrium dialysis and LC-MS/MS. Methods: Serum samples were dialyzed in phosphate dialysis buffer, pH 7.4, in a single-use Pierce rapid equilibrium dialysis (RED) plate. A pipetting station was used to transfer all components of the assay which greatly improved speed and precision of the assay. 250 L of serum was added to the sample chamber and 500 L of dialysis buffer was added to the buffer chamber of the RED plate. The plate was sealed and incubated for 16-18 hours at 37C on an orbital shaker. After equilibrium was reached, 150 L of the dialysate was removed and transferred directly into a 96 deepwell plate. Deuterated cortisol (d3-cortisol) was added as an internal standard. The resulting dialysate mixtures underwent on-line high-throughput liquid chromatography (HTLC) followed by conventional liquid chromatography on an XDB-C18 analytical column. Cortisol and d3-cortisol were analyzed using a heated nebulizer ion source (APCI) and tandem mass spectrometry. Results: Intra-assay imprecision (CV) was found to be 3.4-8.9% (n=20) for three different levels (0.29-6.84 g/dL). Linearity of the dialysate (4 patient dialysates) ranged from 78-107% with an average of 95% for samples ranging from 0.238-7.99 g/dL. Free cortisol results by equilibrium dialysis showed significant systematic bias to calculated free cortisol, with a slope of 0.3637 and intercept of 0.3639 (n=5, one sample removed as it exceeded the highest calibrator for our method). Conclusions: The dialysis method provides a simple and reproducible means of directly quantitating free cortisol. There is a systematic difference when compared to calculated free cortisol. Assuming that equilibrium dialysis is closer to the true in vivo free cortisol levels, this suggests that calculated free cortisol measurements result in inaccurate estimation of free cortisol levels. 1. Vogeser et.al. Clin Chem Lab Med. 2007;45(4):521-5.
calibrators for the Centaur assay with assigned estradiol concentrations 97.9 pg/mL and 726 pg/mL gave values of 67.3 pg/mL and 599 pg/mL when analyzed on the VITROS 3600. Analysis of 18 samples by LC-MS yielded regression data as follows: VITROS = 0.99 LCMS - 6.4, r = 0.9168, and Centaur = 1.06 LCMS + 1.3, r = 0.9450. Conclusion: Based on this limited evaluation, the new VITROS 3600 Systems estradiol assay is a sensitive and precise assay that readily meets the routine needs of the clinical laboratory. The assay demonstrated excellent precision at low estradiol concentrations. The VITROS 3600 assay correlates well with the ADVIA Centaur assay, and it appears that a significant amount of the difference between the assay results can be attributed to assignment of calibrator values. The VITROS 3600 assay gave an acceptable correlation with a LC-MS estradiol assay performed at a reference laboratory.
D-125 Performance characteristics of the Tosoh G8 for the determination of Hemoglobin A1c. R. Khoury, A. H. Gandhi, B. P. Salmon, A. D. Patel, P. Gudaitis, D. Gudaitis, S. S. Gibbs. Aculabs, Inc., East Brunswick, NJ
Background: Diabetes is a chronic illness marked by high levels of blood glucose due to either an improper insulin production, insulin action, or both; more than 10 million or 20 % of people age 60 and older in the Unites States have diabetes. The first step in diabetes care is the glycemic control to prevent and reduce the risk of the complications. Techniques to assess the effectiveness of a management plan on glycemic control include: Self-Monitoring of Blood Glucose and the measurement of hemoglobin A1c. The objective of this study is to validate the Tosoh G8 and compare the results to the Tosoh G7 and Roche Integra 800 results. Methodology: The Tosoh G8 Glycohemoglobin analyzer uses non-porous ion exchange HPLC and microcomputer technology. There is no interference from Schiff base and no pretreatment is required. The assay is NGSP certified, and there is a minor difference between G7 and G8 which helped reducing the assay time to 1.6 minutes from 2.2 minute for G7, although the assay time is shorter but high resolution was maintained. We evaluated the assay accuracy, linearity, precision, reportable range, and samples correlation with Tosoh G7 and Roche Integra 800. Results: The assay %CV was 0.9 % for a mean A1c of 5.9%, the assay linearity was confirmed with an average recovery of 100.9%. Reportable range was 3.0 to 18.1%; the following is the summary of the correlation: Instrument G8 vs. G7 G8 vs. Integra 800 Correlation Coefficient 0.9993 0.9945 Slope 1.03 1.00 Intercept -0.19 -0.14
D-124 Performance Evaluation of the VITROS 3600 Immunodiagnostic System Estradiol Assay D. Giacherio, E. VasBinder, K. Forbing. University of Michigan Hospital, Ann Arbor, MI
Background: Estradiol measurement is a key component in the assessment of reproductive function in women, including infertility, oligo-amenorrhea, and menopausal status. It is also commonly used by in vitro fertilization programs for monitoring ovulation. Immunoassays for estradiol have historically had accuracy and reproducibility issues at low estradiol concentrations. This study evaluated the performance of the estradiol immunoassay on the new Ortho Clinical Diagnostics' VITROS 3600 Immunodiagnostic System. Methods: The VITROS 3600 estradiol assay is a competitive immunoassay technique. Estradiol in the sample competes with a horseradish peroxidase labeled estradiol conjugate for a limited number of biotinylated anti-estradiol antibody binding sites. The antibody complex is captured on streptavidin coated well. Following washing to remove unbound material, the HRP catalyzes the oxidation of a luminogenic substrate producing light. The VITROS 3600 estradiol assay was compared to the Siemens ADVIA Centaur estradiol assay which is routinely used in our clinical laboratory. A subset of patients also had estradiol determinations by an LC/MS methodology. Results: To assess day-to-day assay imprecision, BioRad LiquichekTM Immunoassay Plus control material was analyzed for 10 consecutive days. The CVs for Levels 1, 2, and 3 were 7.25 %, 7.20 %, and 6.70 % respectively on the VITROS 3600 System, and 28.7 %, 6.03%, and 7.14 % respectively on the ADVIA Centaur. Three serum pools with low estradiol concentrations were analyzed on the Vitros 3600 System with 5 replicate determinations of each. These gave the following mean and CV data: Pool 1 = 9.66 pg/mL and 9.9 %, Pool 2 = 21.62 pg/mL and 6.3 %, Pool 3 = 33.75 pg/mL and 2.9 %. A method comparison with the Centaur estradiol assay using 177 serum samples with Centaur values ranging from 12 to 1074 pg/mL yielded a regression equation of VITROS = 0.59 Centaur + 2.3, r = 0.9536. The two
Conclusion: the Tosoh G8 offers the gold standard technology for the measurement of A1c which eliminate interferences, it is precise, fully automated, user friendly, offers up to 290 samples loader; in addition it offers the fastest throughput of the 3 instruments tested.
D-126 Relationship between Extremely Elevated Serum Human Chorionic Gonadotropin (hCG) and the Thyroid Hormones Thyroid Stimulating Hormone (TSH) and Free Thyroxine (fT4) C. M. Lockwood1, D. G. Grenache2, A. M. Gronowski1. 1Washington University School of Medicine, St. Louis, MO, 2University of Utah Health Sciences Center, Salt Lake City, UT
Background: During pregnancy, when hCG concentrations are highest, there is a transient suppression of serum thyroid stimulating hormone (TSH). Numerous studies have established that hCG can stimulate release of the thyroid hormones, which inhibit the pituitary release of TSH through negative inhibition. In normal pregnancy, TSH suppression is a transient phenomenon and TSH concentrations generally remain within non-pregnant reference intervals; however, in some patients TSH is lowered below the non-pregnant reference interval. It has been reported that suppressed TSH concentrations are usually only observed in cases when the hCG concentration exceeds 50,000 IU/L. In a pilot study, we reported that if there is an hCG concentration above which all serum TSH or free thyroxine (fT4) concentrations can be expected to be abnormal, it is >330,000 IU/L. However, the pilot study was limited because it contained very few specimens with hCG concentrations >200,000 IU/L (n=18). Objectives: To determine: 1) if there is an hCG concentration, above which, TSH
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concentrations consistently fall outside the normal reference interval; 2) how quickly thyroid hormone concentrations change in response to changes in hCG concentrations; and 3) the clinical symptoms in patients with such extremely elevated hCG concentrations. Methods: Over a twenty-six month study period, 15,597 physician-ordered hCG tests were performed. 69 specimens from 63 women with hCG concentrations >200,000 IU/L were identified with sufficient sample remaining to measure TSH and fT4 concentrations. Medical records were reviewed for clinical symptoms and pregnancy diagnosis. Approval for this study was provided by each institutions IRB. Results: 37% (23/63) of subjects had hyperemesis gravidarum, 19% (12/63) had gestational trophoblastic disease (GTD), and 10% (6/63) had a twin pregnancy (5 of which also had hyperemesis gravidarum). TSH was suppressed below the reference interval in 74% of the study specimens and 100% of specimens with an hCG concentration >400,000 IU/L. fT4 concentrations were elevated in 32% of specimens and in 80% of specimens with an hCG concentration >400,000 IU/L. Interestingly, only 6% (4/63) of subjects had documented signs of hyperthyroidism. During the study period, multiple hCG concentrations >200,000 IU/L were collected in 8% (5/ 63) of serially monitored subjects, which allowed an assessment of how rapidly the thyroid hormones changed relative to hCG concentration. In all cases, as hCG varied, fT4 and TSH also changed in a predictable manner. Conclusions: These data confirm the relationship between very high hCG concentrations and the thyroid hormones TSH and fT4. TSH is consistently suppressed at an hCG concentration >400,000 IU/L, a threshold which is substantially higher than previously published estimates. This study also illustrates that varying hCG concentrations can rapidly influence serum fT4 and TSH. Finally, despite the presence of biochemical hyperthyroidism in 32% of study subjects, very few (6%) exhibited overt hyperthyroid symptoms.

of 20-5,000 pg/mL. The significant postive bias shown in certain patient cohorts by the Roche immunoassays compared to LC-MS/MS, suggests interference by various non-1-84 PTH fragments in these immunoassays. The LC-MS/MS method of quantification presented here is not plagued by interference by non-1-84 PTH fragments and therefore has clear advantages over ICMA and IRMA immunoassays currently used to quantify 1-84 PTH.
D-128 Plasma Concentrations of Adiponectin Hormone in Type 1 and Type 2 Diabetes S. L. Barnes, E. P. Womack, M. Bon Homme, W. Tucker, R. Valdes, S. A. Jortani. University of Louisville, Louisville, KY
Background: It has been previously shown that patients with metabolic syndrome and type 2 diabetes have significantly lower plasma concentrations of adiponectin compared with non-diabetic controls. However, it is not clear as to whether non-obese patients such as type 1 diabetics would also have reduced plasma concentrations of adiponectin. This hormone is released by the adipose tissue to slow the production of glucose by the liver and to initiate the oxidation of fatty acids and glucose in the muscle. Ironically, obese individuals have reduced expression of adiponectin and its receptors. Furthermore, patients with reduced concentrations of adiponectin have difficultly controlling insulin and obesity. Hypothesis: Since plasma concentration of adiponectin is inversely proportional to the degree of obesity, we hypothesized that type 2 diabetics generally known to be obese would have lower expression of adiponectin than do type 1 diabetics. Methods: Plasma specimens from patients >18 years of age with blood glucose values greater than 200 mg/dL (n = 59) and those with blood glucose values less than 120 mg/dL (n = 60) were collected. Plasma concentrations of adiponectin were measured using a singleplex adiponectin kit on the Luminex 100 analyzer. Plasma concentrations of C-peptides were also measured. Various parameters such as clinical diagnosis of type 1 versus type 2 diabetes, height, weight and gender were recorded and body mass indices (BMI) were calculated. Results: As expected, patients with glucose >200 mg/dL had significantly higher Cpeptide concentrations compared with patients with normal glucose values (p < 0.05). Clinically diagnosed type 2 diabetics had significantly lower plasma adiponectin concentrations compared with clinically diagnosed type 1 diabetics (10.26 5.85 vs. 16.47 9.86 g/mL, p < 0.05). In this cohort, we also noted that adiponectin in the leaner patients (i.e. normal BMI) who had elevated glucose (>200 mg/dL) did not differ from those with elevated BMIs regardless of their diabetes status. In type 1 diabetics, plasma concentration of adiponectin was not reduced and was comparable to non-diabetic controls. Conclusions: We conclude that plasma concentrations of adiponectin are lowered in patients diagnosed with type 2 diabetes. The possibility exists that this phenomenon is related to the degree of obesity in such patients.
D-127 Sensitive and Specific Quantification of 1-84 PTH in Serum and Plasma by Immunocapture-in situ digestion LC-MS/MS V. Kumar, D. R. Barnidge, J. M. Twentyman, K. W. Cradic, S. K. Grebe, R. J. Singh. Mayo Clinic College of Medicine, Rochester, MN
Background: Specific 1-84 PTH assays have been reported to be reflective of the bioactivity of the PTH hormone. Most existing immunoassays reflect the sum of 1-84 PTH and other cross-reacting PTH fragments (predominantly 7-84 PTH) that accumulate in patients with Chronic Kidney Disease (CKD). In addition, regardless of the degree of crossreactivity, consistent calibration over time remains an issue. A physico-chemical reference methodology may help address these problems. Methods: We have developed an accurate and robust method for measuring 1-84 PTH in serum and plasma. 1-84 PTH and 15N-PTH (internal standard) were recovered from serum and plasma by immunocapture using anti-44 - 84 PTH antibody coupled to quarter -inch polystyrene beads. Briefly, after washing the beads with PBS, the bound PTH molecules were digested in 50mM Ammonium Bicarbonate buffer, pH 8.0, containing 1g trypsin to generate the unique proteotypic N-terminal tryptic peptide 1-13 PTH (SVSEIQLMHNLGK) that was analyzed using LC-MS/MS method. The tryptic peptides were resolved by high performance liquid chromatography carried out on a C18 column with a gradient flow rate of 0.25mL/min either with acetonitrile or methanol organic solvent. The PTH results obtained from 111 patient samples by immunoassays were compared with their corresponding LC-MS/MS values. The patient samples were subdivided into three groups: i) 29 patients in whom th PTH levels were between 150 and 299 pg/mL, the desired range for PTH in patients with stage 5 CKD according to KDOQI guidelines, ii) 54 CKD patients undergoing dialysis at Mayo clinic (PTH: 20 - 3,638pg/mL), and (iii) 28 patients undergoing parathyroidectomy (IOPTH) at Mayo clinic (PTH: 10 - 1,951pg/mL). Results: Standard curves were made in charcoal stripped human serum using recombinant 1-84 PTH in concentration of 20-5,000 pg/mL (r2= 0.996). The intraassay CVs for replicate extracts of three levels of pooled controls-sera (27.2, 83.1 and 269.3 pg/mL) were 14%, 9.1% and 12.8%, respectively. The LC-MS/MS method showed poor correlation (R2 = 0.68, slope = 0.925) with the Roche Cobas immunoassay in group 1 patients. Furthermore, 10 of 29 immunoassay values showed a significant (>2SD) positive bias compared to the LC-MS/MS method. Comparison in Dialysis patients showed a good correlation (R2 = 0.93, slope = 0.87) and only 3 of 54 samples showed a significant (>2SD) positive bias compared to LC-MS/MS method. Finally, comparison for IOPTH patients showed a poor correlation (R2 = 0.59, slope = 0.57), but only 1/28 samples showed a significant (>2SD) positive bias compared to LC-MS/MS method. Conclusions: The LC-MS/MS method we have developed quantifies specifically the unique proteotypic N-terminal tryptic fragment 1-13 derived from immunocaptured 184 PTH. This novel PTH method is robust and reproducible over an analytical range
D-129 Use of Serial Patient Differences of HPLC HbA1c to Determine Long Term Instrument Performance C. L. Wiley1, D. V. Tran2, T. N. Higgins3, L. Vandergouwe4, G. S. Cembrowski2. 1Marshfield Clinic, Marshfield, WI, 2University of Alberta, Edmonton, AB, Canada, 3DynaLIFE Dx, Edmonton, AB, Canada, 4Chinook Health Region, Lethbridge, AB, Canada
Background: The usefulness of measuring HbA1c to assess glycemic control in diabetic patients is well established. The quality of the HbA1c assay is inversely proportional to the variation of the assay. Often estimates of assay imprecision are based on the repeated analysis of quality control specimens. These estimates are dependent on the matrix of the control specimen which may differ from actual patient specimens. We have developed a unique approach to determining the imprecision of HbA1c methods. Methods: HbA1c measurements of outpatient blood sample pairs drawn between 1 and 30 days were made on two Tosoh high performance liquid chromatography assays: Tosoh 2.2+ (total pairs, n=598) and Tosoh G7 (total pairs, n=626). The data collections period was two years. The standard deviation of duplicates was calculated for three day time intervals up to 30 days (1 to 3 days, 4 to 6 days, etc.). These intraindividual variations were then plotted. Extrapolation to zero time yields a combination of biologic and analytic variation (Figure). Since HbA1c is slow-forming and its biological variation is low, we assumed that the majority of this variation at time zero is due to analytic imprecision. Results: At the mean HbA1cs of 7.51 and 6.96% for populations tested on the Tosoh
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2.2+, and the Tosoh G7, respectively, the total analytic imprecisions (coefficient of variation, CV) are 1.66% and 1.14%, respectively.
Conclusion: Using our approach, the total analytic variation is attributed to the variation in multiple reagent lots, multiple calibrators and multiple calibrations during the study period as well as long and short-term environmental differences including variations in ambient temperature. Our analysis takes into consideration the variation observed in specimens that are separated over periods as long as 30 days. The variation of the patient duplicates is thus a measure of the average variation over this extended period.
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specifically with its pre-mRNA and suppresses the formation of mature mRNA during splicing in vitro. Human ribosomal protein S17 possesses high affinity to fragment of its own pre-mRNA, containing the first intron. Weak binding of the pre-mRNA fragment to other ribosomal proteins was an evidence for the specific nature of the rpS17 binding to its own pre-mRNA. Results of the present work together with the data obtained earlier for human ribosomal proteins S13 and S26 make it possible to draw a conclusion on existence of the pathway of feedback auto regulation general for all eukaryotes which provides the exact control of ribosomal proteins biosynthesis in the cell in the course of splicing of their pre-mRNAs. Probably, transgressions in this regulation result in uncontrolled protein synthesis typical for tumor cells.

Poster Session: 2:00 pm - 4:30 pm Proteins/Enzymes
D-130 Establishment of the biomarker signature of age-related skeletal muscle weakness K. Ohlendieck, P. Donoghue, K. O'Connell, J. Gannon. National University of Ireland, Maynooth, County Kildare, Ireland
Frailty and fragility among sedentary elderly patients is related to an impaired structure and function of contractile fibres. Proteomic research into the mechanisms that underlie age-related skeletal muscle wasting investigates the scientific basis of evidence for designing new diagnostic and therapeutic strategies for treating sarcopenia of old age. Once new treatment options such as cell therapy or pharmacological intervention have been developed, it will be essential to also establish more reliable diagnostic markers. With respect to preventive medical approaches to sarcopenia, the identification of a reliable set of disease-specific biomarkers would be crucial for the proper diagnosis of age-dependent muscle degeneration long before the onset of serious clinical symptoms. To identify novel sarcopenia markers, we have conducted a mass spectrometry-based proteomic profiling exercise to investigate muscle specimens from young, adult and senescent rat and human fibres. We are currently comparing the soluble protein complement from muscle versus distinct subcellular fractions representing microsomal membranes, mitochondria and the contractile apparatus. Using fluorescently tagged adult versus old muscle proteomes and high-resolution two-dimensional gel electrophoresis (1st dimension: pH 3 to pH 10; 2nd dimension: 10 kDa to 220 kDa), the differential expression pattern of several thousand muscle proteins could be determined. For statistical purposes, 4 biological repeats and 3 technical repeats were performed per analysis using differential fluorescent labeling with the dyes Cy5, Cy2 and Cy3. Both, peptide mass fingerprinting and peptide fragmentation technology was used for the identification of tryptic digests of proteins that exhibited a marked change in abundance. Immunoblotting surveys and confocal microscopy was used to confirm these findings. Major age-related alterations in the muscle proteome included proteins involved in fibre contraction (myosin heavy chains, myosin light chains, tropomyosin, troponins, actin), excitation-contraction coupling (dihydropyridine receptor), ion homeostasis (SERCAtype calcium pumps, sarcalumenin, sodium-calcium exchanger, plasma membrane calcium pump), metabolite transportation (fatty acid binding protein, myoglobin), cellular stress response (small heat shock prteins), and metabolism (glycolytic enzymes, mitochondrial enzymes). Based on these proteomic findings, we are currently screening different sub-types of muscle fibers to establish a comprehensive biomarker signature of sarcopenia. Hopefully muscle proteomics will lead to a better molecular understanding of age-related muscle wasting and thereby help in the future design of superior diagnostic procedures to evaluate muscle biopsies.
D-132 LIVER PROTEIN PROFILING OF MICE WITH HIGH FAT DIETINDUCED NONALCOHOLIC FATTY LIVER DISEASE I. Kirpich, M. Bon-Homme, I. Deaciuc, C. McClain. University of Louisville, Louisville, KY
Introduction. Non-alcoholic fatty liver disease (NAFLD) is present in up to one-third of the general population and in the majority of patients with metabolic risk factors such as obesity and diabetes. NAFLD refers to a wide spectrum of liver damage, including simple steatosis, steatohepatitis, fibrosis and cirrhosis. Insulin resistance and oxidative stress play an important role in NAFLD development and progression to non-alcoholic steatohepatitis (NASH). In spite of a large number of clinical and experimental studies, the intimate molecular mechanism of NAFLD and progression to the more complicated form, NASH, remains to be elucidated. The goal of the present study was to achieve a more detailed understanding of the molecular changes in response to high fat dietinduced liver steatosis through the identification of differentially expressed liver proteins. Materials and Methods. C57/BL6 mice were fed high fat diet (HFD, 60% lard) or control diet (CD) for eight weeks. Liver injury was evaluated by histopathology and plasma alanine aminotransferase (ALT) activity; hepatic and plasma triglyceride (TAG) and free fatty acid (FFA) were measured. Hepatic proteome was analyzed by twodimensional difference gel electrophoresis with mass spectrometry. Results. At the end of the experiment, HFD-fed mice developed hepatic steatosis (fat accumulation in the shape of macrovacuoles) and showed increased plasma ALT activity (58.3 + 7.4 vs 26.2 + 1.8 U/L, p > 0.001). Hepatic TAG (148.0 + 6.8 vs 42.0 + 2.9 mg/g of liver, p > 0.05) and FFA (11.9 + 0.8 vs 8.5 + 0.29 moles/g of liver, p > 0.001) were significantly higher compare to their CD-fed cohorts, as well as plasma TAG (80.4 + 4.4 vs 56.4 + 4.3 mg/dL, p > 0.05) and plasma FFA (0.37 + 0.02 vs 0.29 + 0.02 mEq/L, p > 0.05). Proteomic analysis identified 9 proteins whose expression levels changed by greater than 1.5 fold with significant changes (p > 0.05) in the HFD group compared to the CD group. Of these, five proteins were up-regulated and seven were down-regulated. The identified proteins can be divided into six different categories according to their functional properties: (i) proteins involved in cholesterol and triglyceride metabolism, such as apolipoprotein E and 3-hydroxy-3-methylglutaryl-Coenzyme A synthase, both up-regulated; (ii) enzymes involved in carbohydrate metabolism, such as ketohexokinase, up-regulated; (iii) proteins involved in stress response and xenobiotic metabolism, such as selenium-binding protein 2, glutathione S-transferases mu1 and pi, all down-regulated; (iv) proteins involved in methionine metabolism, such as methionine adenosyltransferase I alpha, up-regulated; (v) molecular chaperons, such as chaperonin subunit 5 epsilon, down-regulated; (vi) intracellular inhibitor of the lysosomal cysteine proteinases, such as stefin A, up-regulated. Conclusion. In summary, the current study has identified a set of proteins linking HFDinduced metabolic shift to the development of hepatic steatosis.
D-131 Human ribosomal protein S17 inhibits splicing of its own pre-mRNA K. A. Cheremisina, A. A. Malygin, G. G. Karpova. Chemical biology and fundamental medicine, Novosibirsk, Russian Federation
The computer and statistical analysis of the number of EST sequences corresponding to human ribosomal protein genes showed that large variations in the expression of these genes take place in different normal adult human tissues. Moreover, a number of studies have detected changes in the expression of ribosomal protein genes associated with the oncogenic state, including increase of amount of specific ribosomal protein mRNAs in tumors. Since eukaryotic ribosomal protein gene expression is coordinately regulated at the transcription and translations steps, it is possible to assume that differences in quantities of ribosomal protein mRNAs in the cell are concerned with regulation of their genes expression at the pre-mRNA splicing stage. Our investigation was aimed at studying regulation of splicing of human ribosomal protein S17 (rpS17) pre-mRNA. Interestingly, overexpression of this protein was observed in tumorous lymph tissues. The cDNA of rpS17 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in E. coli cells and highly purified protein was isolated. A method for refolding the protein from inclusion bodies was optimized. The secondary structure content of the refolded protein was analyzed by CD spectroscopy. In the present work, we showed that rpS17 binds
D-133 Multicenter evaluation OF a NEW Roche CRP (LATEX) ASSAY ON HITACHI and COBAS SYSTEMS R. Roeddiger1, F. Ceriotti2, A. Bezzu2, C. Mller3, F. Enzmann3, G. Sarfati4, M. Castro-Castro5, M. Widmann1. 1Roche Diagnostics GmbH, Mannheim, Germany, 2Diagnostica e Ricerca San Raffaele S.p.A., Milano, Italy, 3Virchow Klinikum, Berlin, Germany, 4Hpital Cochin, Paris, France, 5Hospital Universitario de Bellvitge, L'Hospitalet de Llobregat, Catalonia, Spain
Background/Objective: The present abstract describes the analytical performance of a new microparticle-enhanced turbidimetric assay for C-reactive Protein (CRP Gen. 3, Roche Diagnostics GmbH, Mannheim, Germany) with an extended primary
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measuring range for serum and plasma on RD/Hitachi and cobas c systems. Methods: The new method uses a reagent which was prepared by coupling two monoclonal antibodies independently to latex particles of two different sizes. The two microparticle reagents are mixed in an appropriate ratio. On both systems, the sample is first mixed with reaction buffer and thereafter with latex reagent. The immunoreaction is monitored at 800/570 (sub/main) nm after the addition of the latex reagent. The CRP concentration in patient samples is determined automatically from a six-point calibration curve. Results: The primary measuring range, which was defined by linearity testing, was confirmed from 0.3 to 350 mg/L (recovery rate: 0.3 - 5 mg/L 0.5 mg/L; 5 - 350 mg/ L 100 10 %), with an extended measuring range of up to 700 mg/L achieved by an additional dilution step that is automatically performed by the analyzer. Limit of Quantitation (CV 10 %) was found within a range of 0.44 - 0.61 mg/L. Within-lab precision (%CV) ranged from 1.4 - 4.9 for concentrations between 1.4 - 350 mg/L. Statistical Passing/Bablok analysis of method comparison against Tina-quant [a] CRP and Tina-quant [a] CRP HS yielded correlation coefficients > 0.98, slopes between 0.91 - 1.05 (Median 0.98) and intercepts ranged from - 0.38 to - 0.05 mg/L (Median - 0.15 mg/L). 10 calibrations at each site were performed to verify the calibration reproducibility. CV ranged from 0.4 to 1.4 % for standards 2 to 6. Conclusions: The new, sensitive CRP assay provides highly precise and accurate measurements with a broad primary measuring range from 0.3 to 350 mg/L. Disclaimer: The Roche CRP Gen. 3 assay is not cleared for use in the U.S.
Proteins/Enzymes
D-135 Re-Optimisation of Angiotensin Converting Enzyme Activity on Dimension RXL MAX using reagent from Trinity Biotech plc O. A. Joshi, I. Halsall. Cambridge University Hospital NHS Trust, Cambridge, United Kingdom
Introduction: Kinetic measurement of Angiotensin Converting Enzyme (ACE; EC 3.4.15.1) was performed on Dimension RXL Max (Siemens Healthcare Diagnostics Inc) using kit from Trinity Biotech plc (Catalogue No. 305-10). Concerns following poor performance (positive bias) in external quality control scheme (Biorad Immunoassay Programme 2) and imprecision at low activity, prompted re-optimisation of the instrument method parameters. Method: Concentrated substrate (N-[3-(2-furylacryloyl)-L-phenylalanylglycylglycine (FAPGG), was loaded on the analyser and automatically diluted at the time of analysis. Dimension RXL Max method kinetic programme was utilised to optimise the following method parameters: Reaction times, Reagent and sample mix rate and limit of detection. Calibration stability was also determined. Patient samples were analysed in duplicate to determine within batch imprecision. Total imprecision was assessed using quality control material. Results: See Results Camparison Table Result Comparison Table Before Reoptimisation 5:00-10:00 minutes Moderate 5 U/L Daily After Reoptimisation 07:30-11:30 minutes Moderate 10 U/L 3 months
D-134 Cystatin C estimated Glomerular Filtration Rate (eGFR) in the Elderly. R. Bck1, P. Thyln2, I. Klarin2, U. Bergman3, I. Odar-Cederlf4, A. Helldn4. 1Clinical Chemistry, Karolinska University Hospital Huddinge, Stockholm, Sweden, 2Dept. of Geriatrics, Karolinska University Hospital Huddinge, Stockholm, Sweden, 3Clinical Pharmacology, Karolinska Univeristy Hospital Huddinge, Stockholm, Sweden, 4Clinical Pharmacology, Karolinska University Hospital Huddinge, Stockholm, Sweden
Introduction: Elderly patients have decreased kidney function caused by ageing and diseases. Information on renal function is important for clinical assessment of the patient and for drug selection and dosing. Determination of plasma creatinine is easily available but may present unreliable results in elderly patients, due to influence of muscular mass. Plasma cystatin C has been considered a better marker of renal function than creatinine and allows calculation of estimated glomerular filtration rate (eGFR).Its reliability in very old patients (>75 years) has not been studied. Patients and methods: Ninety-seven inpatients, 64 women and 33 men, aged >75 years were examined. P-cystatin C was analysed with Dade-Behrings assay on a BNProspec nefelometer. The routine equation for adults (>20years) used at Karolinska University Laboratory was used for calculation of cystatin C estimated GFR (eGFR) and compared with GFR determined by iohexol clearance. We calculated the percent of estimates falling within 30% and 50% above or below the measured GFR (i.e. iohexol clearance), which is considered a measure of accuracy. Results and discussion: When we used the routine equation for cystatin C eGFR, we found that only 62% of the estimates were within 30% deviation from iohexol clearance (gold standard). We therefore derived a new equation based on the correlation between cystatin C and iohexol clearance in these elderly patients: y=60.173*CystC-1.0721 With this new equation 84% of the estimates fell within 30% deviation from the gold standard. GFR below 30 mL/minute is frequently recommended as a cut off point. When we calculated eGFR with our routine equation for adults 18% had a eGFR below 30 mL/min/1.73 sq.m. body surface area (BSA), compared to 31% when we used our new equation. Our data suggest that cystatin C based eGFR may overestimate GFR in old patients unless the routine equation is adapted to this group of patients. It should be taken into consideration that analysis of Cystatin C is not standardized between laboratories so algoritms cannot be used generalized without adjustment for the methods used. Conclusion: eGFR based on Cystatin C adds information on renal function, but the equation needs to be adjusted for elderly patients.
Parameter Reaction times Mix rate Limit of detection Calibration stability Total imprecision: Mean (%CV, n) Total imprecision: Mean (%CV, n) Total imprecision: Mean (%CV, n)
Mean: 17 U/L (30.5, 5) Mean:15 U/L, (8.1, 73) Mean: 45 U/L, (14.8, 58) Mean:44 U/L, (3.3, 28) Mean: 64 U/L, (6.8, 15) Mean: 64 U/L,(1.7, 19)
Discussion: Following re-optimisation, performance in the external quality control scheme and precision at low activity has improved. Method kinetics has demonstrated that previously recommended measurements at 5 and 10 minutes leads to imprecision due to, sample specific, varied rate of decrease in absorbance. If the readings are taken after 7 minutes then precision improves, particularly at low activities. Conclusion:This method allows the users of Dimension RXL Max (Siemens Diagnostics Inc) to add ACE activity measurement to their routine chemistry repertoire, improving turn around time whilst reducing sample handling. Singleton analysis and improved on-board reagent stability has reduced reagent cost per test.
D-136 A Multicenter Prospective Evaluation of the Access Soluble Transferrin Receptor (sTfR) Assay and the sTfR / log Ferritin Index (sTfR Index) for Differential Diagnosis of Iron Deficiency Anemia and Anemia of Chronic Disease B. S. Skikne1, K. Punnonen2, P. H. Caldron3, M. T. Bennett4, M. Ervasti2, G. H. Gasior5, J. S. Chamberlin5, L. A. Sullivan5, K. R. Bray5, P. C. Southwick5. 1University of Kansas Medical Center, Kansas City, KS, 2Kuopio University Hospital, Kuopio, Finland, 3Arizona Arthritis and Rheumatology Associates, Paradise Valley, AZ, 4Medical Associates Research Group, San Diego, CA, 5Beckman Coulter, Inc., San Diego, CA
Background: Iron deficiency anemia (IDA) and anemia of chronic disease (ACD) are the most prevalent forms of anemia. Standard tests of iron status used in differential diagnosis are affected by inflammation, making clinical interpretation difficult. In contrast, soluble transferrin receptor (sTfR) is a valuable indicator of iron deficiency and is unaffected by inflammation. Objectives and Methods: Our study assessed the clinical utility of the new automated Access sTfR assay (Beckman Coulter) and sTfR/log ferritin index (sTfR Index) in a large prospective multicenter clinical trial consisting of patients with common disorders associated with anemia, and included a sufficient number of subjects to determine definitive cutoffs. Four sites consecutively enrolled 145 patients. Results: Subjects with IDA or ACD+IDA showed significantly higher sTfR and sTfR Index values than subjects with ACD (p<0.0001). The following cutoffs were derived
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Proteins/Enzymes
using ROC curves: sTfR 21 nmol/L (1.55 mg/L), sTfR Index 14 (using nmol/L in calculations) or 1.03 (using mg/L in calculations). The figure below illustrates the distribution of sTfR Index values by diagnostic group, with the double y-axis showing values in either nmol/L or mg/L in the Index calculation; note the clear separation of ACD subjects from those with ACD+IDA or IDA. The sTfR Index is superior to sTfR (AUC 0.87 vs 0.74, p<0.0001). Use of all three parameters in combination increased sensitivity from 41% (ferritin alone) to 92% (ferritin, sTfR, sTfR Index).

averaging 6 runs performed over 3 days. Using a Deming regression analysis, 271 values ranging from 9.16 to 87.94 nmol/L were compared using the Access sTfR assay and the R&D Systems Inc. Quantikine sTfR ELISA. The slope was 0.90 (0.87-0.93 [95% CI]), the intercept was 0.55 (-0.59-1.68 [95% CI]) and the correlation coefficient was 0.96 (0.95-0.97 [95% CI]). The sTfR assay exhibited within-run imprecision of 1.74 %CV and total imprecision of 5.4 %CV using 4 controls (9.9194.28 nmol/L) analyzed 2 times per day over 20 days for a total of 80 individual results. Imprecision of the sTfR / log of ferritin index was 6 % when analyzing the variability of duplicate CV% on 334 samples. Using a Deming regression analysis, 263 values ranging from 10.02-40.33 nmol/L were compared in the Access sTfR assay using matched serum (SST) and plasma (Li-heparin PST) samples. The slope was 0.95 (0.92-0.97 [95% CI]), the intercept was 0.16 (-0.37- 0.68 [95% CI]) and the correlation coefficient was 0.98 (0.97- 0.98 [95% CI]). Conclusions: The Access sTfR assay and sTfR Index demonstrated acceptable assay performance on the Access automated analyzer sufficient to support accurate detection of IDA and the differentiation between IDA and ACD in the presence of chronic inflammatory diseases and infections.
D-138 The establishment of the reference range of the ratio between the urine amylase and urine creatinine Y. L. Hou, H. Jiang, Y. P. Tian. Chinese PLA General Hospital, BeiJing, China
Objective: To establish the reference ratio of urinous amylase and urinous creatinine in local laboratory. Methods: The samples were derived from 11 acute pancreatitis in-patient (acute pancreatitis group), 326 urine specimens of normal local adults (man 169,woman 157) (control group) and 126 urine specimens of 21 nomal adults (6 specimens in each person are separately taken before and after three meals in a day). Analytical instrument: Roche Modular P was used in the test. Result: The reference of ratio of urinous amylase and urinous creatinine of 326 urine specimens of nomal local adults is 9.36~19.48. The reference of urinous amylase of 11 acute pancreatitis in-patient is 918.9868.2 (97.7-2784.9), the reference ratio of urinous amylase and urinous creatinine of 11 acute pancreatitis in-patient is 58.6026.70 (33.12-129.95), The reference of ratio of urinous amylase and urinous creatinine of 21 urine speciments of nomal adults are 12.2912.91, 12.571.98, 12.191.94, 12.241.84, 12.191.74 and 12.261.80 separately. The reference of urinous amylase of 326 urine speciments of nomal local adults is 128.498.4 (7.8626.4), The reference of urinous amylase and urinous creatinine of 326 urine speciments of nomal local adults is 14.425.06 (4.11-29.26). Conclusion:1. When the reference value of urinous amylase of in acute pancreatitis in-patient may be normal, the ratio of urinous amylase and urinous creatinine may far varid from the normal level. 2. The ratio of urinous amylase and urinous creatinine may be stable while the urinous amylase and the urinous creatinine of the nomal adults alter greatly. 3. Using the ratio between the urine amylase and urine creatinine is better than urine amylase singly on acute pancreatitis diagnosis. Discussion:There was no significant difference between each reference value of ratio of urinous amylase/creatinine in nomal adults (P>0.05). It is showed that individual ratio of urinous amylase/creatinine may change little in a day and the ratio from random urine could reflect the normal level of whole day. The urinous amylase in random urine of acute pancreatitis often increased greatly, but it was not sometime. And it could be easily detected with the help of the ratio of urinous amylase/ creatinine, which was more sensitive than the urinous amylase in acute pancreatitis. With the ratio, it is easy to differentiate the acute pancreatitis from other kinds of acute abdominal pain in clinic.
Conclusions: The Access sTfR assay and sTfR Index improve detection of IDA, particularly in situations where routine markers provide equivocal results. Our findings demonstrate a significant advantage in the simultaneous determination of ferritin, sTfR and the sTfR Index in anemic patients requiring a differential diagnosis of IDA and ACD. Obtaining a ferritin level alone may delay diagnosis or recognition of the presence of combined IDA and ACD.
D-137 ANALYTICAL PERFORMANCE OF THE NEW ACCESS SOLUBLE TRANSFERRIN RECEPTOR (sTfR) ASSAY AND THE sTfR / LOG FERRITIN INDEX (sTfR INDEX) ON THE ACCESS IMMUNOASSAY SYSTEM J. S. Chamberlin, G. H. Gasior, L. A. Sullivan, P. C. Southwick, K. R. Bray. Beckman Coulter, Inc., San Diego, CA
Background: Anemia of chronic disease (ACD) and iron deficiency anemia (IDA), the most common forms of anemia, are difficult to differentiate by standard tests which are affected by chronic inflammatory diseases or infections. sTfR is unaffected by inflammation and therefore provides an advantage over standard measures. The sTfR assay may also be used in conjunction with an Access ferritin measurement to provide a calculated sTfR / log ferritin Index. sTfR and the sTfR Index are intended as aids in the diagnosis of IDA and for the differential diagnosis of IDA and ACD. Objectives and Methods: This study was performed at Specialty Laboratories to determine assay performance of the new sTfR assay and Index on the Access 2 Immunoassay System, a random access automated analyzer. The sTfR assay is a twosite immunoenzymatic (sandwich") immunoassay. A serum or heparinized plasma sample is added to a reaction vessel along with paramagnetic particles coated with anti-sTfR antibody and an anti-sTfR alkaline phosphatase conjugate. After incubation in a reaction vessel, materials bound to the solid phase are held in a magnetic field while unbound materials are washed away. The chemiluminescent substrate LumiPhosTM 530, is added to the vessel and light generated by the reaction is measured with a luminometer. The light production is directly proportional to the concentration of sTfR antigen in the sample. The amount of analyte in the sample is determined from a stored, multi-point calibration curve. The time to first result is 35 minutes. The Index is determined by dividing sTfR by the log of ferritin. Results: The analytical range of the sTfR assay is 3-150 nmol/L. The following results were obtained in our studies. The analytical sensitivity was 0.03 nmol/L when
D-139 Comparison of the mass and activity of serum isoenzyme of creatine phosphokinase(CK-MB) in children R. Zheng, H. Jiang, Y. P. Tian. Chinese PLA General Hospital, BeiJing, China
Background: CK-MB is specific enzyme in myocardial cells and is becoming an important biochemistry marker to diagnose myocarditis and myocardial injury in hospitals. Nowadays, there are two main methods, one is the enzyme inhibition immunoassay, and another is the electrochemiluminescence immunoassay, but the reference intervals are mainly defined for adults, which may not always be
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appropriate for children. Objective: In order to know the children's CK-MB value by comparing the mass and activity of CK-MB in children with two methods. Materials and methods: 165 children were divided into 5 groups from 1 to 15 years old and 19 adults were in group 6 as control. The activity and mass of CK-MB were detected by the method of enzyme inhibition immunoassay and electrochemiluminescence immunoassay, with the reference interval of 0-16U/L and 0-6.5ng/ml. Results: CK-MB activity and mass analysis were linear correlated (P<0.01). CK-MB activity mean value from group 1 to group 6 were 23.903U/L, 20.872U/L, 15.310U/L, 13.530U/L, 11.688U/L, 8.916U/L; and their 5th-95th percentile confidence intervals were 22.05~25.76U/L, 17.84~23.90U/L, 11.75~18.87U/L, 11.03~16.04U/L, 10.66~12.72U/L, 6.51~11.32U/L. CK-MB mass mean value from group 1 to group 6 were 4.899ng/ml, 4.999ng/ml, 3.010ng/ml, 2.862ng/ml, 2.725ng/ml, 2.926ng/ml; and their 5th-95th percentile confidence intervals were 4.57~5.23ng/ml, 3.61~6.39ng/ml, 2.46~3.56ng/ml, 2.56~3.17ng/ml, 2.43~3.02ng/ml, 2.51~3.34ng/ml. It showed a common tendency that the age of children were younger, the value of CK-MB activity and mass were higher. And CK-MB activity test results in group 1 and group 2 age below 5 years old were beyond its reference interval, meanwhile, all CK-MB mass test results and other groups of CK-MB activity test results were in their reference intervals. Thus it is found that there were no significant correlation with CK-MB activity and CK-MB mass analytical methods between group 1 and group 2(P>0.05). Conclusion: Children CK-MB activity and CK-MB mass value are higher than adults. It is advisable to define young children's CK-MB reference value based on their physiological feature. At present, CK-MB mass analysis is better for clinical diagnosis of young children.
Proteins/Enzymes
the day of presentation (day 0), eight days after presentation (day 8), and twenty-eight days after presentation (day 28) for analysis. On day 28, the patients were clinically examined for post-herpetic neuralgia, with nine patients exhibiting PHN and fourteen patients without PHN (six patients that were immunocompromised were excluded from this study and one patient was lost to follow-up, n = 23). All of the whole blood samples of the nine patients with PHN and the fourteen without PHN on day 28 were then divided into PHN-positive and PHN-negative groups and pooled by time of collection to yield four sample groups: PHN-positive, day 0; PHN-negative, day 0; PHN-positive, day 8; PHN-negative, day 8. The pooled serum of the patients with PHN were compared to those without PHN using 2-dimensional gel electrophoresis followed by Progenesis Discovery v.2005 software analysis. All protein spots with a 2 fold change between the PHN-positive and PHN-negative groups on either days 0 or 8, were considered significant and eligible for mass spectrometric identification. Of these protein spots, spot 636 was the most significant, exhibiting a greater than 3 fold increase on both day 0 and day 8 in the group that ultimately developed PHN when compared to the group that did not develop PHN. Mass spectrometry followed by tandem mass spectrometry were utlilized to identify spot 636 as peroxiredoxin, a thioredoxin peroxidase. Western blot analysis of the pooled whole blood samples with a peroxiredoxin-specific antibody followed by band density analysis verified and quantified the significant upregulation of peroxiredoxin in the patients with PHN versus the patients without PHN on day 0. This is the first description of increased serum peroxiredoxin in association with pain and the first association peroxiredoxin levels as a predictor of pain The peroxiredoxin family of proteins function as reducers of reactive oxygen species and are associated with oxidative stress. In vivo, peroxiredoxin levels have been shown to correlate with increases in reactive oxygen species. Furthermore, reactive oxygen species have been shown to play a vital role in both neuralgic and inflammatory nociception. Our future research into the novel association of peroxiredoxin and nociception may prove beneficial in understanding the pathophysiology of nociception, the role of the peroxiredoxin family in nociceptive signaling, and may ultimately lead to the recognition of the peroxiredoxin family as sensitive, objective biomarkers of reactive oxygen species-associated pain.
D-140 Rapid Identification of Amyloid Proteins from Formalin-Fixed Paraffin-Embedded Sections Using Laser Microdissection and Triple Quad MS/MS J. D. Gamez. Mayo Clinic, Rochester, MN
Amyloidosis is a condition in which one of 25 known proteins form non-soluble fibrils in various organs and tissues throughout the body. Amyloid deposits are identified by staining sections of formalin fixed paraffin embedded (FFPE) tissue biopsies with Congo Red. When amyloidosis is confirmed immunohistochemistry is performed in order to determine the subtype. In a number of cases the results are ambiguous, and a subtype is not defined. In amyloidosis definitive identification of the amyloid protein is essential for proper patient treatment. We have developed a new method that determines amyloid subtypes using laser microdissection, isotopically labeled synthetic peptides (acting as internal standards), and triple quad mass spectrometry. We have tested 40 samples with this method consisting of 15 transthyretin, 5 serum amyloid-associated protein 10 immunoglobulin light chain lambda, and 10 immunoglobulin light chain kappa. These samples incorporate a wide variety of tissue samples: (heart, lung, GI, kidney, and bone marrow). These samples were previously characterized using the LTQ orbitrap MS/MS method and immunohistochemistry. In this study we used these results to gauge the level of sensitivity with the triple quad mass spectrometer. Laser microdissection was used to collect amyloid plaques from a 10 micron thick FFPE section exhibiting positive Congo Red staining. Proteins were extracted, digested with trypsin, spiked with isotopically labeled peptides to the 4 amyloid subtypes, and MS/MS performed. Analyst software was used to interpret the data. A majority of the time we were able to identify the 4 amyloid subtypes using this method. In clinical testing the ambiguous samples would be reflexed to testing on the LTQ orbitrap mass spectrometer, the current clinical testing method. With this new Triple Quad method we are able to identify amyloid proteins in a high through put manner with accuracy and sensitivity.
D-142 The transferrin soluble receptor (sTfR): an application protocol for the Roche/Hitachi Modular P800 Chemistry System M. E. Mendes, P. Romano, V. R. Morsoleto Batista, P. A. Rezende Ebner, M. N. Burattini, N. M. Sumita. Central Laboratory Division & Laboratories of Medical Investigation (LIM-03) of Hospital das Clnicas da Faculdade de Medicina da Universidade de So Paulo (HCFMUSP), So Paulo, Brazil
Introduction: Some studies in hematology therapy suggest that blood concentration of transferring soluble receptor (sTfR) should be monitored to maximize efficacy control to reposition of iron. The transferrin receptor is an essential component of cellular uptake of iron, and it binds to serum transferrin. Recently, 2 different types of transferrin receptors have been recognized: transferrin receptor (TfR or transferrin receptor 1) and transferrin receptor 2. Most cells possess an ubiquitous system controlling the biosynthesis of TfR at the posttranscriptional level to avoid excess iron influx into the cells through TfR. During the process of recycling of transferrin receptors, some are shed and appear as soluble or serum transferrin receptors. Measurement of serum transferrin receptor is a new marker of iron metabolism that reflects body iron storage and total erythropoiesis. It has been shown that serum transferrin receptor and ferritin ratio has significant predictive value for differentiating iron deficiency anemia from non-iron deficiency anemia, such as anemia of chronic disorders. Objective: The aim of this study was to evaluate the performance of sTfR imunoturbidimetric Tina-Quant assay from Roche ((Roche Diagnostics, Mannheim, Germany). Materials and Methods: The reagent contains antibody anti-sTfR linked to a latex reagent in a bovine serum albumin (BSA) matrix. The complex antigen-antibody agglutination is measured by turbidimetric method. The assay has been optimized to measure total sTfR from 0.50 to 40.0 mg/L. The assay was performed on Roche/ Hitachi Modular P800 (Roche Diagnostics, Mannheim, Germany), using low and high control samples. The detection limit was defined evaluating a sample of serum without sTfR for 20 times. The recovery test was assessed using a saline solution adding a sample containing sTfR. Two samples with different concentrations were obtained: 1.05 mg/L and 3.60 mg/L. Results: The assay was found to be linear up to 20.7 mg/L. The detection limit was 0.01 mg/L in 20 dosages. The functional sensitivity was 0.22 0.014 g/mL. The
D-141 The Correlation of Peroxiredoxin Levels and Pain in Patients with Varicella Zoster Reactivation: A Novel Biomarker for Postherpatic Neuralgia M. P. Thompson, M. Kuro-o, J. Pastor, D. A. Payne. UT-Southwestern, Dallas, TX
Postherpetic neuralgia (PHN) affects approximately 30% of shingles patients. The aim of this study was to identify predictive biomarkers of shingles-associated PHN. Thirty patients presenting with newly diagnosed shingles had their blood collected on
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Proteins/Enzymes
within-run precision ranged from 3.44 0.07% to control sample with a concentration of 2.11 mg/L and 0.70.06% for concentration of 7.2 mg/L. The between-run precision ranged from 4.250.09% to control sample with 2.11 mg/L and 4.660,39% for concentration of 7.20 mg/L. The recovery test ranged from 92.8% to 2.11 mg/L and 109.3% to 7.20 mg/L. Conclusion: The Tina-Quant sTfR assay is a simple fast and convenient alternative to evaluate sTfR in blood serum sample. This parameter is useful in the diagnosis of iron deficiency, especially for patients with concurrent chronic disease, where routine tests of iron status are compromised by the inflammatory condition.

found that high molecular mass forms of albumin were aggregation and intermolecular link of albumin and other protein by disulfide bond. We undertook the present study to clarify the characterization of the low molecular mass forms of albumin and its possible relation to oxidative damage. H2O2-treated and -untreated human serum albumin (HSA), representative urine sample with GN and healthy urine sample were analyzed by non-reducing SDS PAGE, Western Blot using anti albumin antibody. These samples were also incubated with D-biotin-hydrazide (BHZ) to detect protein carbonyl using Western Blot based method [2]. As a result, among other proteins, the low molecular mass form of albumin (approximately 60 kD) was clearly carbonylated in GN subject (band no. 2 in the figure) similarly to the H2O2-treated HSA as a reference. The results from in-gel digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed that the peptide including N-terminal of the sequence was preserved in the 60 kD of albumin, and the detected peptides pattern was well-correlated with un-carbonylated albumin (band no.1 in the figure). These findings suggest that the electrophoretically low molecular mass form of albumin is not a simple fragment and strongly relevant for the mobility shift to the cathode due to oxidation, and carbonylated urinary albumin provides a new aspect for further understanding of posttranslational modification. [1] Nakayama A et al. Molecular heterogeneity of urinary albumin in glomerulonephritis: Comparison of cardiovascular disease with albuminuria. Clin Chim Acta 2009 in press. [2] Oh-Ishi M et al. Proteomic method detects oxdatively induced protein carbonyls in muscles of a diabetes model Otsuka Long-Evans Tokushima Fatty (OLETOF) rat. Free Radic Biol Med. 2003;34(1):11-22.
D-143 Meagerment of Beta-2 microglobulin Intermediate in serum by Capillary Electrophoresis Y. Uji1, M. Miura2, Y. Motomiya3, Y. Ando4, C. Yoshida1, I. Kitajima1. 1Clinical Laboratory Center,Toyama University Hospital, Toyama, Japan, 2Department of Clinical Chemistry,Hokuriku University, Kanazwa, Japan, 3Suiyukai Clinic, Kashihara, Japan, 4Department of Laboratory Medicine,Kumamoto University Graduate School, Kumamoto, Japan
Background: The beta-2 microglobulin (B2 m) conformer has been proven to be an amyloidogenic intermediate using capillary electrophoresis(CE) in several studies [Biol Chem 2002,277,11184-9 (1)and Electrophoresis 2002,23-918-25(2)] . However, this technique has never been used to determine the presence of the B2 m conformer in clinical specimens, such as hemodialysis(HD) patients serum. We established measurement of amyloidgenic intermediate B2m(I-B2m) and native B2m(N-B2m) in serum by CE. Methods and Materials: All experiments performed on a P/ACE 2100 CE system (Beckman Coulter Instruments, Inc., Fullerton, Calif., USA). Fused-silica capillaries(50 micro meter of ID, 57 cm of length) were also purchased from Beckman Coulter Instruments. Before the CE analysis, the serum samples were centrifuged for 30 min at 2,500 g using a Sartorius Ultrafilter Centrisart I (Sartorius AG, Goettingen Germany) to exclude proteins of more than 20,000 kDa in the sample. Using a pressure injection of 0.5 psi (5 s) applied to the capillary inlet for the sample (approximately 12.5 nL) in each cycle, capillaries were pretreated with 1 M NaOH for 1 min, followed by 3 min of washing with water and 5 min with 100 m M phosphate buffer solution. A positive polarity of 15 kV was applied at the inlets from the anode side to the cathode side, and through all experiments the currents varied from 89 to 90 micro A. The capillary was maintained at 24 C, and the wavelength was fixed at 200 nm. All serum samples were obtained at Suiyukai Clinic (Nara,Japan) after receiving informed consent for this study. The purified B2m from human urine was purchased from Sigma(St. Louis, MO.,USA) for a standard B2m. Polyclonal antibody of B2m was purchased from Sanyo Kasei (Kyoto,Japan). Results: Standard B2m and all serum samples including healthy subjects showed two peaks, major and minor, with migration time of 9.8 and 10.2 min, respectively. We judged the major peak to be the N-B2m and the minor peak to be the I-B2m using affinity column of B2m polyclonal antibody and according to (1) and (2). Linearity up to 100mg/L for N-B2m and 50 mg/L for I-B2m. Detection limits was 0.38 mg/L. The within-assay and between assay precision (CVs) were less than 6.5% (N=10). Recovery studies were performed 96-104% was achieved. The concentrations of NB2 m and I-B2 m were: 29.4 6.8 (17.5-48.6) and 2.7 81.4 (0.8-6.2) mg/L in HD patients (N=31) ; 4.4 1.9 (2.2-6.4) and 0.6 0.6 (0.1-1.6) mg/L in non-dialysis chronic renal failure patients (N=5), and 1.2 8 0.1 (1.0-1.3) and 0.2 0.1 (0.1-0.3) mg/L in healthy subjects (N=5). In addition, dialysis related amyloidosis (DRA) patients (N=13) of I-B2m concentration (N=13) were significantly lower than nonDRA patients (N=13, p<0.004). Conclusion: The proposed method suggested that can be new tool for diagnosis of DRA.
D-145 Evaluation of a new Cystatin C assay on the ABBOTT ARCHITECT cSystems and AEROSET Clinical Chemistry Systems F. Rota1, R. Dioli1, L. De Angelis1, R. Lucini1, J. Herzog2, H. Troonen2. 1Sentinel CH., Milan, Italy, 2Abbott GmbH & Co. KG, Diagnostics, Delkenheim, Germany
Objective: A new Cystatin C assay was evaluated on the ABBOTT ARCHITECT cSystems and AEROSET Clinical Chemistry Systems. The scope of the study was to verify analytical performance and agreement with NACBs guidelines for Emerging Biomarkers of Cardiovascular Disease and Stroke (allowed total analytical error 15%). Materials/instruments: New Cystatin C reagent, manufactured by SENTINEL CH., is a latex enhanced immunoturbidimetric (PETIA) assay based on microparticles coated with anti-hu CysC (rabbit). The assay was optimized to enhance data transferability and comparability with the nephelometric (PENIA) Cystatin C. The Abbott ARCHITECT c8000 and AEROSET Systems are random-access clinical chemistry analyzers. ARCHITECT c8000 can be integrated with ARCHITECT i2000SR, an immunoassay module, to form the ci8200. Study Design: Assay performance was investigated using modified CLSI protocols. Acceptance criteria were set at 5.0% CV for total imprecision and at 5.0% for accuracy throughout the study (allowed Total analytical error 13.2%). LOQ was defined as the analyte concentration at which imprecision was less than 20% CV. Analytical measurement range was defined through linearity. Method comparison consisted in comparing ARCHITECT and/or AEROSET results for patient serum samples with those generated by PENIA on the BNA II system (n=61, 0.58 to 6.91
D-144 Oxidized urinary albumin directly effects on the electrophoretic mobility A. Nakayama1, M. Sakatsume2, T. Kasama1, T. Kawara1, F. Gejyo2, M. Isobe1, K. Shiba3, K. Sato1. 1Tokyo Medical and Dental Univeresity, Tokyo, Japan, 2Niigata University, Niigata, Japan, 3Bunkyo Gakuin University, Tokyo, Japan
In patients with glomerulonephritis (GN), thirteen different mobility of urinary albumin bands were identified in the molecular mass range of 55 to 172 kD [1]. We
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mg/L). Interference due to endogenous substances and drugs was investigated following CLSI EP7-A2. Results: Performances Assay range Precision (10-days) Units: mg/L LOD LOQ Comparison Slope Intercept R Calibration stability ARCHITECT c8000 2.1% at 0.6 2.0% at 0.8 1.9% at 1.1 0.03 mg/L 0.04 mg/L 0.932 0.110 0.998 ARCHITECT c16000 0.05 to 8.0 mg/L 1.5% at 0.8 1.1% at 1.7 1.2% at 4.1 0.05 mg/L NA vs. Penia on BNA 0.929 0.126 0.998 45 days AEROSET 4.3% at 0.6 2.9% at 0.9 1.9% at 1.1 0.04 mg/L 0.06 mg/L 0.918 0.123 0.998
Proteins/Enzymes
D-147 Evaluation and Method Comparison of Three ELISAs for Quantifying S-100B in Serum J. A. Erickson1, D. G. Grenache2. 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 2University of Utah Health Sciences Center, Department of Pathology, Salt Lake City, UT
Background: S-100B is a calcium binding protein with a molecular weight of approximately 22 kD found in astroglial cells, Schwann cells and melanocytes. Several studies have demonstrated that serum concentrations of S-100B can be used to monitor ongoing brain damage and predict outcome in patients with acquired brain injury. Other studies have demonstrated that S-100B is a useful tumor marker for monitoring the treatment of malignant melanoma. Objectives: The purpose of this study was to evaluate the performance characteristics and perform method comparison studies for three commercially available S-100B ELISAs. Assays included in the study were the Sangtec 100 ELISA (DiaSorin, Stillwater, MN), CanAg S100 EIA (Fujirebio Diagnostics, Malvern, PA) and YK150 Human S-100B ELISA (Yanaihara Institute, Inc, Shizuoka, Japan). Methods: Residual serum samples sent to ARUP Laboratories for routine testing were utilized in this study. S-100B protein was measured according to each kit manufacturers testing protocol. Performance characteristics and split-sample comparison studies were determined for all assays with the exception of between-run precision, sample stability and reference interval determination for the YK150 assay. Reference intervals (97.5th percentile) were established by non-parametric analysis of serum S-100B results generated from 150 healthy adult volunteers (75 males, 75 females). This study was approved by the University of Utahs Institutional Review Board. Results: The results are given in the table below.
Sangtec Upper limit of AMR Limit of Detection Linearity 5000 ng/L 15 ng/L CanAg 3500 ng/L 12 ng/L YK150 6300 ng/L 39 ng/L YK150 YK150 CanAg vs. vs. vs. Sangtec Sangtec CanAg (N = 39) (N = 40) (N = 39) NA NA NA NA NA NA NA NA NA NA NA NA
Interfering substances (max allowable bias: 0.1 mg/L): Bilirubin (conjugated 66 mg/ dL, unconjugated 60 mg/dL), Hemoglobin (1000 mg/dL), Intralipid (1000 mg/dL), and RF (500 IU/L) did not interfere. 19 drugs and 9 drug combinations were tested and found to give a bias < 3%. Conclusion: Performance of the new Cystatin C assay fully meets NACBs guidelines for Emerging Biomarkers of Cardiovascular Disease and Stroke on both ABBOTT systems.
D-146 A New Method for Cystatin C Evaluated on the ADVIA Chemistry Systems from Siemens Healthcare Diagnostics P. Datta, M. Losapio, J. Dai. Siemens Healthcare Diagnostics, Tarrytown, NY
Objective: Quantitative determination of cystatin C in human serum or plasma is useful in the diagnosis and treatment of renal insufficiency. Serum concentrations of cystatin C are almost totally dependent on the glomerular filtration rate (GFR). A reduction in GFR causes a rise in cystatin C concentration. Cystatin C has not been shown to be affected by factors such as muscle mass and nutrition, factors which have been demonstrated to affect creatinine values. In addition, a rise in serum creatinine does not become evident until the GFR has fallen approximately by 50%. Recently, Siemens released a new cystatin C assay (CYSC) to measure cystatin C on the automated, random access ADVIA 1650, ADVIA 1800, ADVIA 2400, and ADVIA 1200 Chemistry Systems. Our evaluation of this method included precision, linearity, correlation, and interference performance studies. Methods: All ADVIA Chemistry Systems use the same CYSC reagent packs, calibrators, and commercial controls. In this assay, a specimen containing human cystatin C is diluted and then reacted with antibody (rabbit) coupled to latex microparticles. The increased turbidity is measured at 571 nm. By constructing a sixlevel standard curve (water is used as reagent blank) from the absorbances of standards, the analyte concentration of the sample is determined. Results: The within-run and total CVs (40 replicates per sample) of the new method with a two-level commercial control and three serum pools (~0.6, 3.0, 0.6, 2.0, and 5.0 mg/L cystatin C) on all ADVIA Chemistry Systems were <1.8% and <3.1%, respectively. The analytical range/linearity of the method (all ADVIA systems) was from 0.1 mg/L to the cystatin C concentration in the highest level of calibrator (7.78.3 mg/L). The new method (CYSC) on the ADVIA 1650/1800 (y) correlated well with the BN II System (Siemens) Cystatin C method (x): y = 1.00x + 0.02 (r = 0.99, n = 55, range: 0.13-7.75 mg/L). The ADVIA 2400 and 1200 CYSC methods, in turn, agreed with the ADVIA 1650/1800 CYSC method: ADVIA 2400 CYSC = 0.99 (ADVIA 1650/1800 CYSC) + 0.05 (r = 0.99, n = 102, range: 0.18-7.57 mg/L); and ADVIA 1200 CYSC = 1.03 (ADVIA 1650/1800 CYSC) - 0.02 (r = 0.99, n = 104, range: 0.18-7.57 mg/L). The new method showed <10% interference with bilirubin (conjugated or free) up to 60 mg/dL, hemoglobin up to 1000 mg/dL, lipids (INTRALIPID, Fresenius Kabi AB Corporation) up to 1000 mg/dL, and rheumatoid factors up to 1200 IU/mL. The method has a minimum of 60 days on-system and calibration stability on all ADVIA Chemistry Systems. No prozone was observed with the method on any platform up to the highest cystatin C concentration tested in a sample (56 mg/L). Conclusion: We conclude that the new Cystatin C method, when used on any ADVIA Chemistry System, can measure serum or plasma cystatin C concentrations precisely and accurately over a broad range in routine laboratory use.
Precision
Analyte Stability Reference Interval
Linear 1.123xRegression 1.014x+65.1 1.038x+31.1 105.4 2 0.999 0.999 0.997 R 10.8% (335 ng/ 5.7% 6.3% L) (222 ng/L) (47 ng/L) Within4.7% 1.6% 2.9% Run ng/ ng/L) (680 ng/L) (704 (CV, Mean) (1182 L) 2.1% 1.9% 4.6% (3217 ng/L) (1837 ng/L) (2677 ng/L) 11.3% 6.2% ng/L) (80 ng/L) Between- (228 7.6% 4.1% Run ND (1162 ng/L) (509 ng/L) (CV, Mean) 5.4% 3.5% (2912 ng/L) (1604 ng/L) Ambient 24 h 24 h ND 4 C 7d 7d ND 141 ng/L NA NA 96 ng/L NA NA ND NA NA
NA
NA
NA
NA
NA
NA
NA NA NA
NA NA NA
NA NA NA
Deming Comparison Regression of Methods R2 NA = Not Applicable ND = Not Done
1.376x0.339x+24.1 0.226x140.0 13.1 0.932 0.690 0.860
Conclusions: The performance characteristics of the Sangtec and CanAg S-100B ELISAs are comparable and both assays have lower limits of detection, better linearity and are more precise than the YK150 assay. Although agreement is poor among all assay combinations, the Sangtec and CanAg ELISAs demonstrate better correlation versus comparison with the YK150 ELISA.
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Proteins/Enzymes
D-148 Determination of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in human plasma: analytical and clinical validation M. Plebani1, C. Artusi1, M. Ivanova1, G. Boffa2, M. Zaninotto1. 1Department of Laboratory Medicine, University-Hospital, Padova, Italy, 2Department of Cardiology, University-Hospital, Padova, Italy
Background: Due to the growing number of reports in the literature on ADMA as a possible marker of endothelial dysfunction, its use in the clinical practic is of increasing interest. In contrast there are a less studies concerning the role of SDMA, the structure isomer of ADMA, as an endogeneous marker of renal function. Objectives: The purposes of this study were: 1) Determination of reference values of ADMA and SDMA plasma concentrations after validation of a chromatographic HPLC method; 2) Evaluation of plasma concentrations of ADMA (p-ADMA) in patients with various disorders characterized by endothelial dysfunction; 3) Estimation of the relationship between plasma concentrations of SDMA (p-SDMA) and estimated GFR (eGFR) as well as plasmatic creatinine in patients with chronic kidney disease (CKD). Materials and methods: Blood samples were taken from 140 blood subjects (107 males, 33 females) aged from 18 to 65 years for determination of reference values. We selected 4 groups of patients with a high risk of cardiovascular complications for evaluation of p-ADMA and p-SDMA. A total of 168 patients were analyzed: 66 with chronic heart failure, 43 with type II diabetes (30 patients of this group were complicated with renal insufficiency), 42 with CKD, 17 with pulmonary arterial hypertension. Sample cleanup was performed by solid-phase extraction on polymeric cation-exchange columns. After derivatization with orthophthaldialdehyde reagent, analytes were separated by isocratic reversed-phase HPLC with fluorescence detection. Results: Calibration curve is linear throughout the selected ranges (0.510.0 mol/L); the absolute and relative recoveries (96-106%) were assessed by six determinations at 3 concentrations; the within- and between- day imprecision (CV< 5%) were evaluated by performing al least 5 replicate analyses of quality control samples at 3 different concentrations; the accuracy (-3.57/+6.00%, bias -0.03/+0.06 mol/L) was obtained by measurement of recovery because certified reference standards in the matrix is not available. Reference values (2.5-97.5 percentiles) for pADMA and p-SDMA are 0.34-0,63 mol/L and 0.26-0.57 mol/L respectively. ADMA levels were significantly elevated in all groups compared with controls (p<0.001). In contrast there isnt a statistically significant difference of p-ADMA between the group of patients with type II diabetes and controls. This can be explained by the fact that these patients are under tight glycemic control. SDMA levels were significantly elevated (p<0.0001) both in the patients with CKD and in patients with type II diabetes complicated with renal insufficiency. We found that SDMA levels correlated highly with both eGFR (R=0.740) and plasmatic creatinine (R=0.700). Conclusions: Our study, showing elevated p-ADMA levels in all groups of patients studied, suffering from diseases characterized by endothelial disfunction, suggests the peculiar usefulness of this measurement in the biochemical evaluation of endothelial function. Furthermore, the preliminary results suggest that SDMA could be a reliable marker of renal function, but further studies are in progress in order to investigate whether SDMA is indeed a stable and clinical reliable marker of renal function, especially in pediatric populations in which the use of eGFR is not recommended.

labeled antibody are added for 6 hours at 4C. Following a further wash the plate is counted in a luminometer, with the measured relative light units (RLUs) being proportional to the 14-3-3 concentration in the sample. Until 11/20/2007 NSE was measured with an in-house ICMA. Since then, the samples are analyzed on the BRAHMS Kryptor Compact using time resolved amplified cryptate emission technology. For data reduction purposes of this study, the results obtained after 11/20/2007 were recalculated using the regression equation derived from a method comparsion between the old ICMA and the Kryptor NSE assay. Results: The 14-3-3 standard curve ranges from 2 to 250ng/mL with a net increase of over 150,000 RLUs. Assay imprecision was <20% at concentrations from 3.8 to 40.6ng/mL. Linearity of serial dilution of 4 CSF samples with elevated 14-3-3 levels showed a mean recovery of 110%, range 93-132%. For clinical validation, CSF samples submitted from 2002-2008 (N=949) for NSE testing and then frozen, were thawed and analyzed for 14-3-3 protein in 2008. Thirteen samples came from autopsy-proven CJD cases and 936 came from patients with other neurological disorders. Using ROC analysis, the optimal cutoffs were determined to be 43ng/mL for NSE (76.9% sensitivity; 94.0% specificity) and 9.6ng/ mL for 14-3-3 (76.9% sensitivity; 96.7% specificity). In another group of samples from 33 clinically highly probable CJD cases without histological confirmation NSE was elevated in 28 (84.8%) cases, while14-3-3 was elevated in 24 (72.7%). The specificity of the 9.6 ng/mL cut-off for 14-3-3 was further validated in 235 CSF samples submitted for red and white blood cell counting (CJD was not suspected). Of these, 222 (94.5%) had 14-3-3 values 9.6ng/mL. Some of the elevated results (9.834.8ng/mL) came from patients with disorders known to elevate CSF 14-3-3, such as Guillain-Barre syndrome and viral encephalitis. Since erythrocytes contain NSE and 14-3-3, interference by blood-contamination was assessed. The 14-3-3 concentrations in 82 visibly blood-tinged CSF samples were 0281ng/mL, mean 47.8ng/mL, with 74 samples (90.2%) showing levels >9.6ng/mL. Conclusions: The ICMA performs well with adequate precision and linearity, but is affected by hemolysis. When measured along with NSE it provides additional diagnostic specificity in patients in whom CJD is being considered.
D-150 EVALUATION OF NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN AS A MARKER OF OUTCOME IN PATIENTS FOLLOWING CARDIAC SURGERY H. Turner, J. Reeve, J. McNeilly, W. Mutch, R. Peake, D. Rae, P. Gibson, G. Hillis, B. Cuthbertson, B. Croal. Aberdeen Royal Infirmary, Aberdeen, United Kingdom
Background: Serum neutrophil gelatinase-associated lipocalin (NGAL) has been suggested as an early marker of acute kidney injury in patients following cardiac surgery. We have evaluated NGAL along with, creatinine, cystatin C and their derived estimated glomerular filtration rates (eGFR-Cr; eGFR-CC respectively), at predicting mortality in patients following cardiac surgery. Methods: Serum NGAL (ng/mL, ELISA, AntibodyShop) was measured in early post-operative samples (6hr) in patients undergoing cardiac surgery (n=284). Preoperative levels of cystatin C (mg/L, Siemens Prospec) and creatinine (umol/L, Siemens ADVIA 2400) were measured prior to surgery and their respective eGFRs calculated. Creatinine continued to be measured until discharge. Patients were followed up for one year post surgery. Results: Early post-operative NGAL levels were not predictive of mortality (p=0.645) at 1 year. However, there were significant differences in the pre-operative cystatin C (1.13 v 0.94; p<0.001), eGFR-CC (72.6 v 99.8; p<0.001) and eGFR-Cr (65.9 v 82.4; p<0.001) levels in patients that subsequently died. Quartiles of NGAL were not useful in predicting survival (p=0.52), however cystatin C quartiles did show significant differences (p=0.001). Interestingly, post-operative renal function, as reflected by the highest measured creatinine or the lowest eGFR-CR, was related to the quartile status of early post-operative measurement of NGAL (p<0.001; p<0.001 respectively). In a linear regression model, quartile status for NGAL was predictive of the subsequent highest creatinine level measured during the 10 days following surgery (p<0.001). However, this was not predictive of the maximum rise in creatinine from baseline during this period (p=0.062)
D-149 Development and Validation of an Immunochemiluminometric Assay for 14-3-3 Protein C. M. Preissner, A. J. Aksamit, J. E. Parisi, S. K. Grebe. Mayo Foundation, Rochester, MN
Background: Elevation of 14-3-3 protein in cerebrospinal fluid (CSF) is one of the World Health Organization criteria supporting an antemortem diagnosis of sporadic Creutzfeldt-Jakob Disease (CJD). However, 14-3-3 testing is performed essentially exclusively by Western blot, relying on subjective visual interpretation. Elevated neuron specific enolase (NSE) levels in spinal fluid have also been found in CJD patients. Objective: To develop and validate an immunochemiluminometric (ICMA) microtiter plate assay for 14-3-3 protein. Assay Methodology: Two mouse monoclonal antibodies are used in the 14-3-3 assay. White high-binding 96-well plates are coated overnight with 0.5g antibody per well. The wells are then washed and blocked with Superblock for 1 hour at room temperature. Duplicate 200L aliquots of standards, controls and patient samples are added and incubated overnight at 4C. After washing, 200L of acridinium ester-
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TRAUMATIC BRAIN INJURY (TBI): S100B PROTEIN VERSUS GLASGOW SCALES M. C. GONZALEZ MAO, E. ALVAREZ GARCIA, A. REPARAZ ANDRADE, C. VARA PEREZ, P. POSADA GONZALEZ, A. ANDRADE OLIVIE. COMPLEJO HOSPITALARIO UNIVERSITARIO VIGO, Vigo pontevedra, Spain
BACKGROUND: Being able to predict the presence and severity of Nervous Central System (NCS) injury is vital at Neurological Intensive Care Units (NICUS). The availability of a marker before clinical symptoms allows modifying treatment criteria. Until now we have lacked a really specific marker to assess the severity of traumatic processes and to early detect adverse neurological effects. OBJECTIVES: To study S100B predictive capacity as a brain injury marker comparing this protein levels with patients clinical assessments using Glasgow Coma Scale (GCS), and S100B usefulness as a predictor of adverse neurological effects with patients neurological assessments at hospital discharge measured with Glasgow Outcome Scale (GOS). METHODOLOGY: 137 patients with TBI (110 males and 27 females), admitted to hospital ICU during the last year, with an average age of 42.69 (15-84) were selected. Inclusion criteria: arrival at hospital in the 6 hours following the injury, TBI as a main diagnosis, previous neurological assessment. Clinical variables assessed: GCS, GOS. Analytical criteria: S100B levels in blood in the first 6, 24, 48 and 72 hours after the injury. Negative urine drug screening. S100B concentration was determined with a quantitative automated luminometric inmunoassay (LIASON Sangtec 100 ).The detection limit is 0.02 g/L. Inter-assay variation (10.7% at 0.11 g/L; 2.2% at 2.6; 3.2% at 18.4). We used SPSS 16.0 . p<0,05 is considered statistically significant. Pearsons correlation among S100B levels and GSC and GOS scales is calculated. RESULTS: r Pearson p n Mild TBI S100B 6h -0.214 0.204 37 -0.085 0.626 35 -0.32** 0.011 63 S100B 24h -0.156 0.395 32 -0.44** 0.012 33 -0.37** 0.006 55 -0.41** 0.000 121 -0.258 0.154 32 -0.62** 0.00 33 -0.47** 0.000 55 -0.53** 0.000 120 S100B 48h -0.044 0.826 28 -0.43** 0.021 29 -0.21** 0.015 47 -0.25 ** 0.009 104 -0.279 0.150 28 -0.54** 0.003 29 -0.36** 0.012 47 -0.38 0.000 104 S100B 72h -0.221 0.323 22 -0. 36** 0 .034 23 -0.44** 0.010 34
Conclusion: NGAL measured during the early post-operative period would appear to be a potentially useful predictor of subsequent deterioration in renal function, however it does not appear to be related to subsequent mortality.
D-151 S100B PROTEIN AS A SERUM MARKER OF SURVIVAL AFTER TRAUMATIC BRAIN INJURY (TBI) M. C. GONZALEZ MAO, A. REPARAZ ANDRADE, E. ALVAREZ GARCIA, P. POSADA GONZALEZ, C. VARA PEREZ, A. ANDRADE OLIVIE. COMPLEJO HOSPITALARIO VIGO, Vigo -pontevedra, Spain
BACKGROUND: In the last years, there has been an increasing need of a new peripheral biochemical marker specific for the activity of damaged brain tissue that allows assessing the severity of traumatic processes. The early identification of severe patients after TBI is essential to assist them and crucial to establish prognosis. OBJECTIVE: To establish the relationship between S100B protein levels and the survival of patients with TBI. METHODOLOGY: N (137) patients with TBI (110 males and 27 females), admitted to hospital Intensive Care Unit (ICU) during the last year and with an average age of 42.7 years (15-84), were selected. Inclusion criteria: arrival at hospital in the 6 hours following the injury, TBI as the main diagnosis, previous neurological assessment. Clinical variables assessed: survival/exitus. Analytical criteria: S100B levels in blood in the first 6, 24, 48 and 72 hours after the injury. Negative urine drug screening at admission. S100B concentration was determined with a quantitative automated luminometric inmunoassay (LIASON Sangtec 100 Diasorin). Detection limit 0.02 g/ L Inter-assay variation (10.7% at 0.11 g/L; 2.2% at 2.6; 3.2% at 18.4) We used SPSS 16.0 statistical package (SPSS, Chicago, IL, USA). p<0,05 was considered statistically significant. Odds ratio (OR) and 95% confidence ratio are calculated. RESULTS: S100B values (g/L) in TBI (n= 137) S100B S100B S100B S100B 6h 24h 48h 72h Mean 2.18 1.34 1.17 0.60 (Confidence ratio 95%) (1.6-2.7) (1.0-1.7) (0.6-1.8) (0.4-0.8) Median 1.11 0.68 0.45 0.38 Range (0.04-26) (0.05-14) (0.04-28) (0.07-5.5) S100B marker as mortality predictor Mean S100B 3.6 / 1.7 2.9 /0.7 3.5 / 0.6 1.3 / 0.5 Exitus (Y/N) p 0.002 0.000 0.04 0.05(n.s) N 36/89 31/89 21/83 14/65 Exitus (Y/N) Predictive capacity regarding: Survival/lethality. Logistic regression Odds Ratio 1.2 3.1 2.7 OR (1.1-1.4) (1.8-5.2) (1.4-5.2) p 0.01 <0.001 0.002 CONCLUSIONS: S100B protein levels after 6, 24 and 48 hours are a good survival predictive marker after TBI. Significantly higher S100B levels are observed in those patients with fatal outcome. Each increase in one unit of the S100B level at 6, 24 and 48 hours of the injury rises the risk of death 1.2, 3.1 and 2.7 times respectively. The value with the highest predictive significance is taken 24 hours after TBI.
Moderate TBI GCS Severe TBI
Total TBI -0.294 0.078 37 -0.25 0.152 35 -0.31** 0.013 63
Mild TBI
Moderate TBI GOS Severe TBI
-0.50** 0.017 22 -0.63** 0.001 23 -0.40** 0.018 34
Total TBI
CONCLUSIONS: S100B measured in the initial 24, 48 and 72 hours correlates with GCS scale as a marker of brain injury severity and with GOS scale as a predictor of adverse neurological effects in moderate and severe TBI. S100B 6h only correlates in severe TBI. No correlation is observed in mild TBI, except with GOS scale at 72 hours.
D-153 Development of a Cystatin C assay using monoclonal antibodies for automated clinical chemistry analyzers S. Nakayama, Y. Nakamura, H. Takahashi, H. Yago, K. Ushizawa. Tsukuba Research Institute, Ryugasaki, Japan
Estimation of the glomerular filtration rate (GFR) is essential for the evaluation of patients with chronic kidney disease (CKD). Cystatin C has been suggested as a new
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marker of the GFR. We have developed a specific and sensitive reagent for the measurement of Cystatin C levels using various automated clinical chemistry analyzers. The assay is based on latex-enhanced immunoturbidimetry with a combination of antihuman Cystatin C mouse monoclonal antibodies. The Cystatin C concentration of a sample is determined by measuring the change in absorbance that results from agglutination of latex particles. For standard analysis ueing the Hitachi 917 auto analyzer, 2.4 L of human serum or plasma was mixed with 120 L of the first buffer solution and incubated for 5 min. at 37C. Then 120 L of the second reagent, which contains the monoclonal antibody-coated latex particles, was added and the absorbance was monitored at 570 nm /800 nm (main/sub wavelengths) for 5 min. The detection limit for Cystatin C was 0.05 mg/L. The measurable range was defined 0.5 mg/L to 10 mg/L. The within-run CV (n=10) at 0.5, 1, 2, 4, and 10 mg/L was 0.8%, 0.5%, 0.4%, 0.5%, and 0.9%, respectively. The between-run CV (n=10) at 0.6, 2.1, and 6.8 mg/L was 1.3%, 0.5%, and 1.7%, respectively. Interference studies showed no effect of bilirubin, hemoglobin, and rheumatoid factor (RF) at concentrations of 20 mg/dL, 500 mg/dL, and 500 IU/dL, respectively. Comparison with the N-Latex Cystain C kit using a BN prospec (Dade-Behring) yielded a correlation coefficient of 0.99 and an equation of Y (present method) = 0.965 X (nephelometric method) + 0.117 (n = 55). We conclude that this ready-to-use assay is an accurate, precise, and simple method for measuring Cystatin C concentrations in serum or plasma samples from patients with CKD.

with Type 2 diabetes, obesity, essential hypertension, and coronary heart disease. Thus diagnosis of insulin resistance is very important and immunoreactive insulin (IRI) has been used as a marker of insulin resistance.We have developed a method based on latex-enhanced immunoturbidimetry for measurement of the serum and plasma IRI concentrations. The IRI concentration of a sample is determined by measuring the change in absorbance that results from agglutination of latex particles. Our assay has two reagents and shows excellent performance. For standard analysis using a Hitachi 917 autoanalyzer, 10 L of human serum or plasma sample was mixed with 150 L of the first reagent and incubated for 5 min. at 37C. Then 50 L of the second reagent, which contains antibody-coated latex particles, was added and the absorbance was monitored at 570 nm /800 nm (main/sub wavelengths) for 5 min. The detection limit for IRI was 1 U/mL. The measurable range was defined as 1 U/mL to 200 U/mL. The within-run CV (n=10) at 4.2, 51.4, and 206.2 U/mL was 2.8%, 0.9%, and 1.2%, respectively. Comparison with data obtained using LUMIPULSE INSULIN N (FUJIREBIO) yielded a correlation coefficient of 0.96 and a regression formula of Y (present method) = 0.989 X (nephelometric method) + 0.263 (n = 142). We conclude that this ready-to-use assay is an accurate, precise, and simple method for measurement of IRI in serum or plasma samples from patients with insulin resistance.
D-156 D-154 Improved enzymatic cycling method for determination of ethanol concentration in Biological sample. T. Wang, Y. Tang, P. He. Yujiang Medical College for Nationalities, Baiso,Guangxi, China
INTRODUCTION: Gas chromatography is considered to be the reference method for ethanol determination. However, enzymatic ethanol assays have been selected as routine screening method. The most frequently used enzymatic assay utilizes the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase with concurrent reduction of nicotinamide adenine dinucleotide (NAD) to NADH while monitoring the increase in absorbance at 340 nm. Previously, several authors reported that increased concentrations of lactate and lactate dehydrogenase (LDH) can cause false-positive results in haemolytic sample, because of the NADH produced by LDH. The another disadvantage of the NAD-ADH system is insensitive in low alcohol sample. In this paper, we present an enzymatic cycling method to overcome the weakness of the NAD-ADH system, which includes two steps, in first step, ethanol is converted to acetaldehyde by ADH, and the potential lactate is depleted. In second step, acetaldehyde formed is subsequently oxided to acetic acid by aldehyde dehydrogenase, while thio nicotinamide adenine dinucleotide(thio-NAD) is reduced to reduced thio nicotinamide adenine dinucleotide(thio-NADH), in the presence of NADH, acetic acid is reversely catalyzed to acetaldehyde,. Thio NADH signal is amplified during the cycling reaction and can be monitored at 405nm. METHODS: The method involves use of thio-NAD(+), NADH , alcohol dehydrogenase(EC 1.1.1.1) and aldehyde dehydrogenase (EC. 1.2.1.5) , and measurement of the increase in absorbance at 405 nm of thio-NADH at 37 degrees C. RESULTS: The calibration curve for ethanol was linear (r=0.99) between 0.5 and 120 mmol/l. Analytical recoveries of exogenous ethanol added to serum and urine were 100105% and 98-103%, respectively. Within-run and between-run coefficient of variation (CV) were 2.8%(1 mmol/l), 1.6%(30 mmol/l) and 0.8%(80mmol/l) in blood sample, and 3.4%(1 mmol/l), 2.5%(30 mmol/l) and 1.2%(80mmol/l) in urine sample, respectively. There were no significant difference(p>0.05) in the results of the congenerous samples with or without lactate and LDH added(lactate 3.0 mmol/l, LDH 400 U/l). CONCLUSIONS: An improved enzymatic cycling assay for ethanol determination was successfully developed, which was more sensitive and able to eliminate the interference of lactate and LDH. This method is especially suit for the low ethanol sample and haemolytic sample.
Simultaneous measurement of interleukins, chemokines and growth factors in a single sample with an Evidence biochip array. P. Lowry, L. Farry, J. Porter, F. M. McPhillips, V. Toner, R. I. McConnell, S. P. Fitzgerald. Randox Laboratories, Crumlin, United Kingdom
Background: Cytokines are investigated in multiple fields of research as they play central roles in many biological processes. Cytokines are often pleiotropic and normally function as part of a complex network. Evidence Biochip Array Technology enables simultaneous measurement of multiple analytes with a single sample and this is beneficial as a patient profile is generated in real time. This multi-analyte profiling approach provides more information on the cytokine network than single-analyte determinations and leads to reduced consumption of sample/reagents and better costeffectiveness of the tests. Relevance: We report multiplexed immunoassays on biochip array for simultaneous quantitation of interleukins, chemokines and growth factors per sample using a single analytical device. The biochip array enables determination of eotaxin, IL-1Ra, IL12p40, IGF-I, IP-10, PDGF-AA, PDGF-BB, RANTES. Methodology: Simultaneous sandwich chemiluminescent immunoassays are employed. Antibodies for all the analytes are bound to a solid substrate (the biochip) in discrete test regions defining arrays. Assay specific reagents and sample are applied to the biochip and incubated under controlled conditions. Signal detection, data processing and storage were carried out using the semi-automated bench top analyser Evidence Investigator. Results: Initial evaluation of the biochip array shows that the immunoassays are target specific. All analytes are measured in picogram quantities with sensitivity ranging from 5pg/ml for PDGF-BB (calibration range 0 to 3,000pg/ml) to 140pg/ ml for PDGF-AA(calibration range 0 to 2,000pg/ml). For all the immunoassays, the intra-assay precision expressed as %CV is typically less than 12%. Conclusion: Biochip array technology is applicable to the real time measurement of interleukins, chemokines and growth factors. The use of a multi-analytical approach can provide a better understanding of the inter-relationship of cytokines in disease states with a view to aiding earlier diagnosis and potentially identifying new routes for treatment
D-157 Diagnostic Values of NMDA Receptor Peptide for Acute Ischemic Stroke S. A. Dambinova. Emory University, Atlanta, GA
D-155 Development of a novel IRI immunoturbidimetry assay for clinical chemistry analyzers J. Kondou, M. Yamamoto, H. Yago, K. Ushizawa. Tsukuba Research Institute, Ryugasaki, Japan
Insulin resistance is defined as a clinical state in which a normal or elevated insulin level produces an attenuated biologic response. Insulin resistance is observed patients
Introduction. NR2 peptide, an N-methyl-D-aspartate (NMDA) receptor fragment, has been proposed as a biomarker for TIA and ischemic stroke providing real-time evidence of neurotoxicity. Early microembolic/thrombotic processes activate NMDA receptor cleavage by thrombin-activated serine proteases, resulting in peptide fragments entering the bloodstream. Ability of the NR2 peptide to signal acute cerebrovascular events and diagnostic utility of this biomarker in different clinical settings were studied. Methods. An NR2 peptide assay based on magnetic-particle (MP) ELISA was developed and assay performance characteristics were established. Plasma specimens were collected at 3 clinical settings, where feasibility studies of the NR2 peptide assay
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for assessment of transient ischemic attack (TIA) and ischemic acute stroke were performed. Of 232 patients recruited between January 2006 and June 2008 according to local Institutional Review Board approvals, 141 had TIA/acute stroke. To define tissue-based evidence of injury all patients had DWI/MRI. Blood samples were drawn 1, 3, 6, 12, and 24 h after TIA/acute stroke onset. Plasma samples were also drawn from apparently healthy persons (n=154). Results. The reference interval calculated from the samples (central 94th percentile) was found to be 0.1-0.5 ng/mL for both genders. Positive likelihood ratio of the NR2 peptide assay to detect cortical ischemic events varied depending on time from onset: Clinical setting Surgery Neurology Clinic Emergency Department Positive Cut off, Hours from Sensitivity Specificity likelihood g/L onset % % ratio 0.5 1-6 88 95 18.5 0.9 2-12 99 90 9.7 1.5 12-24 83 84 5.2
Proteins/Enzymes
analytical goals. The method correlation data support the equivalence of the ARCHITECT c4000, ARCHITECT c8000, ARCHITECT c16000 and AEROSET Systems. The common reagent and commodity requirements across the four systems allow the laboratory the flexibility to use either instrument depending on the user needs. The ability to integrate the c4000 with an i1000SR immunoassay module provides the capacity to analyze most routine clinical requests on one system. * = In-development
D-159 Evaluation of Diazyme Total Bile Acids Assay on the Olympus AU680 Chemistry-Immuno Analyzer. M. Lin, C. Hoke, R. Dlott, T. Lorey. TPMG Regional Laboratories, Berkeley, CA
Total bile acids are metabolized in the liver, and hence, serve as a marker for normal liver function. Serum total bile acids are increased in patients with acute hepatitis, chronic hepatitis, liver sclerosis, and liver cancer. Total bile acid testing is also a sensitive test for obstetric cholestasis in pregnancy, where the flow through the bile ducts is reduced. The Diazyme Total Bile Acid reagents are in a stable liquid formulation that allows for ease of use. In the presence of Thio-NAD, the enzyme 3--hydroxysteroid dehydrogenase (3--HSD) converts bile acids to 3-keto steroids and Thio-NADH. The reaction is reversible and 3--HSD can convert 3-keto steroids and Thio-NADH to bile acids and Thio-NAD. In the presence of excess NADH, the enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring specific change of absorbance at 405 nm. Demings linear regression was used to compare the Diazyme Total Bile Acid assay to the LC/MS/MS method with the following results (n = 40): r = 0.9852, y = 1.04x 1.53, Syx = 2.00, average bias = -1.15 umol/L, range 0.8-57.4 umol/L. Precision Within-run (N=20) Mean (umol/L) SD 11.3 0.4 109.8 0.6 Total Precision (N=20) Mean (umol/L) SD 11.0 0.5 31.5 1.1 109.0 5.7 %CV 3.9 0.5 %CV 4.7 3.6 5.3
A significant correlation of NR2 peptide to stroke volume from 2 to 250 cc was observed. In patients with white matter strokes and lacunar lesions, NR2 peptide showed low sensitivity (about 63%), while specificity remained significant (95%). Conclusions. NR2 peptide is brain-borne microvascular biomarker that is released into the bloodstream as tissue-based evidence of cerebrovascular injury. The likelihood ratios, when used in conjunction with clinical findings, have the potential to decrease time to referral to a stroke specialist for patients suspected of having a cerebral ischemic event.
D-158 Evaluation of Enzyme Assays on the Abbott ARCHITECT c4000 Clinical Chemistry System S. Shaw, C. Kasal, J. Lucio, P. Bathory, J. Salazar, D. Bozimowski. Abbott Laboratories, Irving, TX
Objective: Verification studies established performance of enzyme assays on a clinical chemistry analyzer under evaluation, the Abbott ARCHITECT c4000* Clinical Chemistry System. Methodology: The ARCHITECT c4000 Clinical Chemistry System uses the reagent configuration, calibration processes, reaction modes, photometric technology and Integrated Chip Technology (ICT) that are available on the ARCHITECT c8000 System and the ARCHITECT c16000 System and the Abbott AEROSET System. The ARCHITECT c4000 is a fully automated analyzer that can be run as a standalone instrument or integrated with the ARCHITECT i1000SR (the immunoassay module) to form the ARCHITECT ci4100. The ARCHITECT ci4100 is the newest member of the ARCHITECT family of instruments. Results: Assay performance (Precision, Sensitivity, Linearity and Method Comparison was characterized using CLSI-derived protocols. Precision (5 days) was determined using a minimum of two levels of controls. LOQ was demonstrated to meet or exceed values previously determined for the ARCHITECT cSystem. The analytical range represents the observed linear low and high levels verifying the Linearity claim. Method Comparison was evaluated by assaying serum patient samples across the entire range of the assay, comparing the ARCHITECT c4000 to the ARCHITECT c16000. Bias was determined at the medical decision level(s). Sigma Metrics ([TEa - bias] / CV) were calculated and ranged from 6.2 (Acid Phosphatase) to 22.7 (Amylase).
Assay Acid Phosphatase Activated Alanine Aminotransferase Activated Aspartate Aminotransferase Alanine Aminotransferase Alkaline Phosphatase Amylase Aspartate Aminotransferase Creatine Kinase GGT Lactate Dehydrogenase Lipase Control Imprecision (Total %CV) Level 1 Level 2 1.52 1.33 2.92 1.14 2.22 2.13 0.62 0.98 1.33 0.86 1.45 2.60 1.52 0.30 1.02 0.61 0.35 1.50 0.91 0.71 1.14 1.78 Limit of Quantitation (Sensitivity) (U/L) 0.4565 4.1825 3.5490 2.6579 5.0424 2.2977 2.4844 4.9207 2.2674 0.7363 3.8589 Analytical Range (U/L) 0 - 88 6 - 5066 6 - 5450 6 - 4200 4 - 4712 3 - 6720 3 - 4531 6 - 4419 4 - 9396 3 - 5057 3 - 1256 Method Comparison (vs. c16000) Slope R % Bias 1.05 0.9994 -1 0.99 0.9997 3 0.99 1.0000 1 1.00 1.0000 0 1.00 1.0000 -2 1.00 1.0000 1 0.98 1.0000 -2 1.01 1.01 1.03 0.99 1.0000 1.0000 0.9999 0.9999 2 2 2 1
Precision was determined following CLSI Protocol EP5. The analytical measurement range (1 umol/L to 180 umol/L) and reference range from 66 males and 68 females (< 10 umol/L, mean 3.2 umol/L) were determined. The Diazyme Total Bile Acid test was easily implemented on the open channel Olympus AU680 analyzer. The assay demonstrated good performance and can be conveniently integrated into a high volume laboratory setting.
D-160 Method Comparison of Quantitative Serum Free Light Chain Immunoassays Performed on Nephelometric Versus Turbidimetric Platforms S. LeSourd, K. Sweat, M. Johnston, J. Marshall, J. Bornhorst. University of Arkansas for Medical Sciences, Little Rock, AR
Background: The serum free light chain assay, Freelite (The Binding Site, Inc),is a quantitative serum-based immunoassay for free circulating light chains, a surrogate marker for many plasma cell dyscrasias. The immune complexes formed using Freelite antibody reagents assay can be quantified by both nephelometry (light scatter) and turbidimetry (light absorption). The objectives of this study were to compare performance characteristics and analytical results of serum free light chain testing on a turbidimeter (The Binding Site SPAPLUS) versus the nephelometer (Dade Berhing BNII). Freelite reagents have been validated for both platforms and should provide comparable results. Methods: Intra-assay (ten repeats of control and pooled patient samples) and interassay (control samples run seven days three time daily) imprecision were analyzed for kappa and lambda free light chains on a nephelometer and a turbidimeter using abnormal and normal control samples provided by the manufacturer and a high abnormal patient pool provided by UAMS. Patient and control serum sample results were compared between both platforms using Freelite kit reagents and standards.
Conclusion: The ARCHITECT c4000 Clinical Chemistry System demonstrated acceptable performance characteristics that achieved or exceeded all pre-established
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Deming regression analyses were performed for method comparison. Results: The precision for kappa and lambda reagents on the SPAPLUS was determined for both intra-assay (CV range 1.31 - 3.22%) and inter-assay (CV range 3.4 - 8.3%) analysis. Historically, precision of the BNII for the assay has been comparable (average CV range - 2.75 - 6.94%). Normal serum range for free kappa concentrations is 0.33-1.94 mg/dL and for free lambda concentrations is 0.571-2.63 mg/dL. Free kappa results from normal and myeloma subjects ranged from 0.036 to 982.0 mg/dL on the SPAPLUS and 0.030 to 1110.0 mg/dL on the BNII (n=129); analyses between instruments for kappa reagents demonstrated a correlation coefficient of 0.9774 P<0.0001. The Deming regression equation for the kappa method comparison is SPAPLUS= 0.9790BNII + -1.3286. Free lambda results from normal and myeloma subjects ranged from 0.060 to 1153.0 mg/dL on the SPAPLUS and 0.040 to 1010.0 mg/dL on the BNII (n=129); analyses between instruments for lambda reagents demonstrated a correlation coefficient of 0.9933 P<0.0001. The Deming regression equation for the lambda method comparison is SPAPLUS= 1.1289BNII + -5.3222. General agreement between methods for measured free kappa, free lambda and Free Kappa/Lambda ratios values were observed in the normal patient samples. Conclusion: Method comparison of the serum free light assay, Freelite, by nephelometry and turbidimetry demonstrated that either platform produce comparable and precise results. Both platforms represent viable options for clinical serum light chain analysis.

D-162 A New Enzymatic Method to Measure Adenosine Deaminase in Human Body Fluids P. P. Chou, M. Lara, K. Sisco, N. Sherman. Quest Diagnostics Nichols Institute, Chantilly, VA
Introduction: Adenosine deaminase (ADA) is an enzyme catalyzing the deamination reaction from adenosine to inosine. This enzyme is widely distributed in human tissues, especially high in T lymphocytes. Elevated serum ADA activity has been observed in patients with acute hepatitis, alcoholic hepatic fibrosis, chronic active hepatitis, liver cirrhosis, viral hepatitis, and hepatoma. Increased ADA activity is also observed in patients with tuberculous effusions. Method: The ADA assay is based on the enzymatic deamination of adenosine to inosine, which is converted to hypoxanthine by purine nucleoside phosphorylase. Hypoxanthine is then converted to uric acid and hydrogen peroxide by xanthine oxidase. Hydrogen peroxide is further reacted with N-Ethyl-N-(2-hydroxy-3sulfopropyl)-3-methylaniline and 4-aminoantipyrine in the presence of peroxidase to generate quinone dye which is monitored kinetically. Results Pleural Peritoneal CSF Fluid Fluid Intra-assay Mean, U/L CV, % Mean, U/L CV, % Mean, U/L CV, % Level 1 0.35 20.20 3.55 1.99 4.23 2.73 Level 2 25.63 1.42 22.97 0.75 27.72 0.15 Level 3 39.92 0.23 134.67 1.01 46.35 0.20 Inter-assay Mean, U/L CV, % Mean, U/L CV, % Mean, U/L CV, % Level 1 0.35 15.65 3.55 2.35 4.23 15.50 Level 2 25.63 1.41 22.97 1.09 27.72 1.88 Level 3 39.92 0.96 134.67 0.99 46.35 0.38 Recovery, % 95.2-101.2 103.1-114.6 94.6-97.5 Limit of Qn, U/L 0.35 2.70 0.70 Ref Range, U/L <7.0 <9.2 <7.6 Conclusions: We have developed and validated the ADA assay for human cerebral spinal, pleural and peritoneal fluids.
D-161 Method Comparison for the Gentian Immunoturbidimetric Cystatin C Assay for Beckman Coulters Synchron System Versus a Siemens Nephelometric Cystatin C Assay on the Siemens BNII System S. LeSourd, K. Sweat, R. C. Faught, K. Sanderson, J. Marshall, J. Bornhorst. University of Arkansas for Medical Sciences, Little Rock, AR
Background: Cystatin C is a non-glycosylated peptide produced by all nucleated cells and is present in all body fluids. It is removed from circulation by glomerular filtration and is often considered a good indicator of glomerular filtration rate (GFR). Serum cystatin C concentrations are relatively unaffected by age, gender, muscle mass or inflammatory disease. Recently an user defined reagent immunoturbidimetric assay for Cystatin C by Gentian for the Beckman Coulter automated Synchron and UniCel systems was released for human serum or plasma. A method comparison was performed on the Beckman Coulter platform versus a nephlometric cystatin C assay on the Siemens BN II System. Precision studies and a patient linearity were also performed to validate the immunoassay on the Beckman Coulter UNICEL DXC 800 System. Methods: Sixty five serum specimen Cystatin C results were compared between the Gentian assay on the Beckman Coulter UNICEL DxC 800 System and the Siemens Cystatin C assay on the Siemens BNII System. Data from two calibrations for each assay were included in this data set. Samples were obtained from patients with low or normal estimated glomerular filtration rates (eGFR). Imprecision was analyzed on the Beckman Coulter DxC using low and high patient samples and 2 different levels of control material. Linearity was confirmed by diluting a high patient sample. The stated package insert analytical measurement range is 0.4-8.0 mg/L for the Cystatin C assay by Gentian and 0.2-6.8 mg/L for the Siemens Cystatin C assay. Results: The method comparison was performed on 65 patient serum samples ranging from 0.51 to 6.53 mg/L for the BNII and 0.7 to 7.99 mg/L for the DXC. The Deming regression equation for the method comparison is DxC=1.127(BNII assay) + 0.137 and with a correlation coefficient (r-value) of 0.9970. Intra-assay precision analysis for the Beckman Coulter DxC system had a CV range of 0.6-2.8%. Inter-assay precision studies are ongoing. Cystatin C precision studies performed on the Siemens BN II instrument showed comparable imprecision with an intra-assay CV range of 1.5-3.5%. Linearity was demonstrated on the Beckman Coulter system using the Gentian cystatin C assay over a measured range of 0.52 to 7.97 mg/L using a high patient serum sample (slope was 0.96 0.4 and the intercept was 0.00 0.19.) Conclusion: While the these two assays showed comparable precision, the Gentian Beckman Coulter cystatin C immunoassay as tested on the UNICEL DxC 800 system demonstated a ~13% positive bias when compared to the Siemens BNII system Dade-Behring nephelometric cystatin C assay. The stated package insert reference interval for the Siemens BNII assay is 0.53-0.95mg/L, and the stated reference interval Gentian Cystatin C assay is 0.62-1.15mg/L, roughly reflecting the observed positive method comparison bias. The Gentian cystatin C turbidimetric assay appears to be a potential alternative to the Siemens nephlometric cystatin C assay.
D-163 Characterization of Abnormal Lactate Dehydrogenase Isoenzyme with the Semi-Automated Hydrasys Electrophoresis System W. Lee, Y. Park, S. Yoon, J. H. Lee, O. H. Kwon. Yonsei University College of Medicine, Seoul, Republic of Korea
Macroenzymes are complexes of serum enzymes with a plasma protein, having higher molecular weight with prolonged half-life. Their presence can cause an elevation in the serum enzyme levels, possibly leading to misdiagnosis. Herein we present a 30-year-old man found with persistent elevation of LDH level measuring 885-1083 IU/L in a routine health check-up. Other laboratory findings including complete blood counts, routine chemistries, liver function tests and serologic tests for hepatitis virus were not remarkable. Radiologic studies including abdominal ultrasonography also showed no significant finding. The electrophoresis of LDH isoenzymes was performed with the Hydragel Iso-LDH kit used in conjunction with the semi-automated Hydrasys gel electrophoresis system, and the electrophoretic separation showed abnormal migration patterns of LDH2 and 3 isoenzymes with densely stained LDH3. The immunoelctrophoresis of LDH isoenzyme with antihuman IgG, IgA, IgM, kappa and lambda light chain antibodies was performed to identify the antibodies bound to LDH. The patient's LDH was presented to be bound to IgA and kappa light chain. This modified immunoelectrophoresis with semi-automated system could be useful to characterize these kinds of abnormal LDH isoenzyme pattern.
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D-164 Comparison of Cystatin C to various markers in chronic kidney disease patients Y. Minakawa1, Y. Meguro1, Y. Sato1, T. Asaumi2. 1Denka Seiken Co., Ltd., Tokyo, Japan, 2Tokyo Medical University Hachioji Medical Center, Tokyo, Japan
Objective: We compared clinical utility of serum Cystatin C in estimating GFR to various serum and urine markers that have been traditionally used for kidney function monitoring and estimation. We also address potential discordance in GFR estimation by the use of different methods for Cystatin C determination with the lack of the standardization. Methods: Serum Cystatin C, serum and urine beta2-microglobulin, serum and urine alpha1-microglobulin, creatinine and microalbumin were determined in 100 subjects. Each biochemical marker was evaluated for correlation with creatinine clearance estimation of GFR. The correlation with creatinine clearance was also evaluated in subgroups of subjects from each stage of chronic kindly disease. Statistical significance of each biochemical marker among different stages of chronic kindly disease was also evaluated. A latex-enhanced TIA (Denka Seiken, Japan) was used for serum Cystatin C measurement. Results: Serum 1/Cystatin C showed the best correlation with creatinine clearance among the various biochemical markers tested (r = 0.821). The following equation was derived to estimate GFR by serum Cystatin C: eGFR (mL/min) = 61.58 x Cystatin C (mg/L) -1.3691. Even when the subjects were subdivided into 5 groups according to the GFR levels, serum Cystatin C showed the good correlation with creatinine clearance in all the subgroups. We previously reported that there is a substantial difference in test results among Cystatin C assays from different manufactures (the slope of correlation curves varies from 0.88 - 1.11). The current study indicates in conjunction with such previous findings that there can be 53 - 73 mL/min discordance in estimating GFR by the use of different Cystatin C assays.Conclusions: Cystatin C was demonstrated as the most sensitive and specific biochemical marker estimating GFR in this study. Once an international reference preparation becomes available and a common equation for eGFR is established, Cystatin C will be further valuable tool for diagnosing and monitoring kidney status.
Proteins/Enzymes
this methodology could be adapted to other candidates, thus providing a bridge between the discovery and validation platforms for biomarkers. Furthermore, with this methodology, there is the potential to multiplex commonly co-requested analytes in routine clinical chemistry laboratories.
D-166 Liquid stable immunoturbidimetric assay for the measurement of cystatin C on RX series analysers. P. Armstrong, E. Donnelly, P. McGivern, J. Campbell, S. P. Fitzgerald. Randox Laboratories, Crumlin, United Kingdom
Background: Cystatin C is a small cysteine proteinase inhibitor that is steadily produced by all nucleated cells. The small molecular weight of cystatin C allows it to be freely filtered by the glomerular membrane and therefore cystatin C levels in the blood are indicative of a normal or impaired glomerular filtration rate(GFR). The GFR gives a good indication of renal function. Due to the strong link between kidney dysfunction and later cardiovascular problems, cystatin C has also been proposed for the early detection of coronary heart disease, stroke and death in the elderly. Cystatin C is broken down in renal tubular cells and is not secreted nor excreted by the kidneys. Therefore, levels of cystatin C in serum/plasma are almost entirely dependant on GFR. The use of methods enabling accurate determination of this compound is advantageous for the estimation of renal function in clinical and therapeutic applications. Relevance: We report the development of a liquid stable immunoturbidimetric assay for the measurement of cystatin C with an analytical range that allows determinations in human serum/plasma samples without additional dilutions. The assay is applied to the fully automated RX series analysers. This is of value as an accurate, stable and convenient tool for determination of cystatin C using these automated systems. Methodology: The principle of the assay is immunoturbidimetric. A latex agglutination complex (read at 570nm) is found between cystatin C and latex particles. The assay is applicable to the fully automated RX series analysers, RX Daytona and RX imola. These systems require 2.1l of neat sample, generate the first result after 14 minutes and include dedicated software for data management. Onboard and calibration stabilities were tested by storing two lots of reagent uncapped on the RX Series analysers for a period of 28 days. Stressing studies were also carried out for the reagents, calibrators and controls. All were stored at 37oC for 3 weeks and then performance was compared to fresh material. Within-run and total precision were assessed by testing serum samples at defined medical decision levels, 2 replicates twice a day for 11 days. A correlation was conducted with 40 serum samples using commercially available cystatin C assay. Results: The liquid assay reagent presents an on-board stability of 28 days at approximately 80C and a calibration frequency of every 7 days. The liquid calibrators and controls are stable to expiry at 2 - 80C.Evaluation of the performance parameters shows an assay sensitivity of 0.40mg/L and linearity up to 10mg/L. The within-run precision and total precision for three different concentration levels (n=44) and expressed as %C.V. were typically<6.5. Correlation with other commercially available assay generated the following linear regression equation: Y= 0.90x + 0.07; r =1.0. Conclusion: Data shows optimal analytical performance of the assay for the measurement of cystatin C on the fully automated RX series analysers with the added advantage of the stability of the liquid reagent, calibrators and controls. This is of value in the accurate determination of this analyte in human serum/plasma for clinical and therapeutic applications.
D-165 Multiplexing combined with immuno-mass spectrometry: An approach to simultaneous quantification of multiple analytes in biological fluids V. Kulasingam1, I. Batruch2, C. R. Smith2, T. Martz3, D. A. Jeffery3, A. Buckler3, T. Rezai4, M. F. Lopez4, E. P. Diamandis5. 1University of Toronto, Toronto, ON, Canada, 2Mount Sinai Hospital, Toronto, ON, Canada, 3Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, 4Thermo Fisher Scientific, Cambridge, MA, 5University of Toronto; Mount Sinai Hospital, Toronto, ON, Canada
Currently, a major bottleneck in the verification phase of putative biomarkers is the lack of methods/reagents to quantify low levels of analytes in biological fluids. The aim of this study was to establish a high-throughput multiple reaction monitoring (MRM) assay capable of quantifying 4 analytes simultaneously. HER-2/neu, IGFBP2, VEGF and IL-8 were selected to demonstrate the feasibility of this approach since these molecules are present in low pg/mL to ng/mL range in serum. Digested human recombinant proteins for the 4 analytes were used to select proteotypic peptides as well as MRM transitions on the TSQ Quantum Ultra mass spectrometer (triple quadrupole, Thermo). Peptides ASPLTIEGR (m/z 472), LIQGAPTIR (m/z 484), FMDVYQR (m/z 479) and ENWVQR (m/z 416) corresponding to HER-2/neu, IGFBP2, VEGF and IL-8, respectively, were used for quantification. An additional 1-2 peptides per protein were used for verifying the identity of the protein. The experimental procedure consisted of an immunoextraction step on a 96-well microtiter plate of the 4 analytes from a complex mixture. Specifically, each well on the plate was coated with 100 ng/well of each of four monoclonal antibodies (400ng/well in total), followed by immunocapture of the analytes in the sample, trypsin digestion in-well, with subsequent MRM on the triple quadrupole mass spectrometer. In-house ELISA assays were developed for all 4 analytes using commercially available reagents and used to validate the findings of the immuno-MS approach. The amount of spiked "heavy" peptide for each analyte was kept constant across the samples so that quantification was performed by generating a calibration curve using a ratio of the light:heavy peptide. This method demonstrated a limit of detection of 0.1 - 1 ng/mL with coefficient of variation < 20%. We conclude that
D-167 Effectiveness of a predictive algorithm combining total Tau, p-Tau181, A42 in CSF/plasma to discrimate early Alzheimer Disease from ageassociated MCI and secondary dementias A. SCOGNAMIGLIO1, M. Tondelli2, P. Nichelli3, T. Trenti1. 1Clinical Pathology Department - Nuovo Ospedale S.Agostino Estense, Baggiovara,Modena, Italy, 2Neuroscience Department- Nuovo Ospedale S.Agostino Estense, Baggiovara,Modena, Italy, 3Neuroscience Department - Nuovo Ospedale S.Agostino Estense, Baggiovara,Modena, Italy
Diagnostic biomarkers for Alzheimer Disease (AD) would be especially valuable as aids in the early diagnosis in the disease course, when correct diagnosis is difficult and when therapeutic drugs have the greatest effective potential. It is well known that total Tau protein, p-Tau protein (p-Tau181) and -amyloid peptides (A40 and A42) in CSF/plasma are laboratory tests of moderate sensitivities and specificities when
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Proteins/Enzymes
performed alone to diagnose AD. The strategy of combining the biomarkers for AD diagnosis is appealing as the combination might result in increased sensitivity and specificity. These markers may be useful to discriminate early or incipient AD from age-associated memory impairment, depression, and some secondary dementias. The aim of the present study was to evaluate the role of total Tau, p-Tau (p-Tau181) amyloid peptides (A40 and A42) in CSF and -amyloid peptides (A40 and A42) in plasma as combined biomarkers able to distinguish AD in patients when the clinical presentation required to differentiate AD from some problematic differential diagnoses, especially age-associated memory impairment, depressive pseudodementia, Parkinsons disease, alcoholic dementia, etc. Analysis of CSF t-tau, p-tau and CSF/plasma b-amyloid (A1-42) was performed using quantitative enzyme-linked-immunoassay (ELISA) (Innotest Innogenetics,Ghent Belgium) applied on Biomeck 3000 (Beckman Coulter USA). Microsoft Excel Macro was set up to visualize the results obtained with the INNOTEST -amyloid (1-42), INNOTEST hTau Ag, and INNOTEST PHOSPHOTau in cerebrospinal fluid. These Macro program, based on up to date literature, combine biomarker results to increase sensitivity and specificity for discriminating AD from healthy controls and other neurologic disorders. The study protocol will follow The STARD Iniziative-Towards Complete and Accurate Reporting of Studies on Diagnostic Accuracy Checklist. Results: CSF t-tau, p-tau and CSF/plasma b-amyloid (A1-42) were determined on all consecutive patients admitted to our neurological department for investigation of cognitive disturbances when it was not possible a definitive diagnosis nevertheless the use of clinical information and brain-imaging techniques i.e. SPECT and MRI. The studied patients were reviewed after approximately 1 year period and the reviewed diagnosis was the diagnostic golden standard used in this research, also in this case, when necessary were used brain-imaging techniques together clinical information to a definitive diagnosis. 33 patients were evaluated, 15 female end 18 male aged 43- 84. 8 out of 33 patients showed a full concordance between the laboratory biomarker index and the AD ultimate diagnosis, 22 out of 33 patients showed a full agreement in the exclusion of AD between laboratory data and clinical final evaluation (the most common diagnosis performed were: Parkinsons atypical disease, depression, frontotemporal dementia, normal pressure hydrocephalus). 3 out of 33 patients showed laboratory data not in agreement with the clinical finding, however up to now it was not possible a definitive diagnosis. In conclusion, in this study, nevertheless the limited but selected patients, the biomarkers for Alzheimer Disease diagnostic use, when combined by the means of a predictive algorithm, exhibit effectiveness in the diagnosis of early AD and they have the potential to help to differentiate AD from some problematic differential diagnoses.

D-169 Urinary KIM-1: a Novel Sensitive Marker of Renal Tubular Damage for Confidence in Safety A. McGuinty, P. Cleall, R. Allavena, E. Horsley, D. Brees, A. Rossi, R. Walley, S. Sultana. Pfizer, Sandwich, United Kingdom
INTRODUCTION: There is a need for early and more sensitive markers of kidney damage. Traditional markers, serum urea and creatinine lack sensitivity to mild kidney injury leading to a delay in diagnosis and treatment. Kidney Injury Molecule-1 (KIM-1) is elevated in urine in response to proximal tubular damage due to nephrotoxicity and ischaemia. In order to assess the utility of Kim-1 as a translatable biomarker of kidney injury we validated a pre-clinical assay using a rat model of nephrotoxicity and tested a novel assay for human KIM-1 in healthy male volunteers. METHODS: Pre-clinical study -A novel therapeutic known to cause tubular degeneration and necrosis in rats was administered at doses of 0, 50 and 250 mg/kg/ day for 2 weeks (n=10/group) followed by a 2 week and 4 week (n=5/group) recovery. Urine samples from various time points were analyzed for Kim-1 levels with a commercially available ELISA assay using Mesoscale Discovery (MSD) technology. Clinical stud y- 28 healthy males [21-54 yrs] volunteered in a non-drug study each providing 4 urine samples over 4 days. Urinary KIM-1 levels were measured with a prototype ELISA assay using MSD technology, and urinary creatinine measured on the Advia 1650 analyzer. RESULTS: Pre-clinical study - Prior to study start the rat Kim-1 assay was validated with results as follows: Linear Range 0.006 - 2.59 ng/ml, Precision (Intra = 5.0%, Inter = 9.5%), Recovery = 88.5 - 94.2%. A dose related increased incidence of renal tubular degeneration/necrosis was observed after 14 days of dosing. The microscopic findings were associated with statistically significant (p<0.05) dose related increases in urinary Kim-1 of up to 4.5 fold (day 4) in 250 mg/kg group, and to 2.5 fold (day 1) in 50 mg/kg group. In contrast, no changes were noted in the traditional biomarkers, serum creatinine and urea over the same time course. During the recovery phase, no microscopic findings were observed at 2 or 4 weeks; indicating recovery. Recovery could also be monitored with Kim-1, as levels returned to baseline shortly after cessation of treatment (day 18). Clinical study - Prior to study start the human KIM-1 assay was validated with results as follows: Linear Range 0.0365 - 10.0 ng/ml, Precision (Intra = 5.4%, Inter = 15.2%), Recovery = 104.3 - 121.8%. In the healthy male volunteer study, the geometric mean creatinine-corrected urinary KIM-1 was 0.426 ng/mg, a range of 0.113 ng/mg -1.182 ng/mg. Based on published data, a cutoff of 1.6 ng/mg was established, giving 100% specificity for these data. CONCLUSION: In the rat study Kim-1 correlated well with histopathology, proving a predictive and sensitive marker of renal proximal tubular damage and recovery. The novel assay for human KIM-1 performed well in healthy volunteers. Further work is needed to establish assay performance in patients with acute renal injury and chronic kidney disease but these data along with published literature suggest utility in clinical monitoring for acute proximal tubular damage. The findings give greater confidence in renal safety monitoring, allowing the clinical assessment of compounds known to cause preclinical nephrotoxicity.
D-168 Evaluation of the VITROS 5600 Integrated System for IgA, IgG and IgM determination in serum J. Teixeira, A. Denooz, J. P. Chapelle. University of Lige, Lige, Belgium
Background : The VITROS 5600 Integrated System (Ortho Clinical Diagnostics) merges five technologies (MicroSlide, MicroTip, MicroWell, MicroSensorTM and Intellicheck) into a single system, integrating a menu of chemistry, immunodiagnostic and infectious disease assays for both routine and STAT testing. Objective : We evaluated the performance of the system for measuring immunoglobulin (Ig) concentrations in serum by immunoturbidimetry. Methods : IgA, IgG and IgM concentrations were determined according to the assay protocols on the VITROS 5600 System. The comparison method was immunonephelometry on the BN II analyser (Siemens Diagnostics). The evaluation was performed using CLSI protocols. Results : The new immunoglobulin assays showed excellent precision at all concentration levels on serum pools and OCD controls. Intra-assay CVs ranged from 1.30 to 2.13 %. Inter-assay CVs were 1.67 - 3.55 % for IgA at concentrations from 97 to 687 mg/dL, 1.80 - 3.83 % for IgG (384 - 1850 mg/dL) and 1.79 - 3.92 % for IgM (40 - 227 mg/dL). Determination of successive dilutions of high concentration samples (n = 10) in kit buffer showed excellent agreement between expected and measured values (r > 0.99 for the three Ig). The correlation with the comparison technique (r = 0.99 for IgA, 0.98 for IgG, 0.99 for IgM) was assessed from data obtained on 90 patient samples covering the whole concentration range (up to 948 mg/ dL for IgA, 2210 mg/dL for IgG, 292 mg/L for IgM). Serum concentrations > 800 mg/ dL (IgA), 2700 mg/dL (IgG), 400 mg/dL (IgM) are automatically diluted by the VITROS 5600. We verified that values exceeding these limits (up to 1050 mg/dL for IgA, up to 3200 mg/dL for IgG and up to 575 mg/dL for IgM) were correctly processed by the system. Conclusions : These results indicated that the VITROS 5600 has excellent analytical performance for immunoglobulin determinations in serum.
D-170 Quantification of C-reactive protein in human serum with affinity purification and isotope-dilution tandem mass spectrometry E. L. Kilpatrick1, W. Liao2, I. V. Turko2, J. E. Camara2, N. G. Dodder2, D. M. Bunk2. 1National Institute of Standards and Technology, Charleston, SC, 2National Institute of Standards and Technology, Gaithersburg, MD
Objective: Characterize the performance of affinity purification coupled with isotopedilution tandem mass spectrometry in the quantification of low abundance proteins in serum. Relevance: Certified reference materials are presently not available for C-reactive protein (CRP) at normal and moderately elevated levels primarily due to the lack of definitive methods. This study advances the development of a higher order method necessary to generate future reference materials with levels better matched to clinical diagnostic cut-off points, thereby reducing issues related to commutability and increasing interlaboratory harmonization. Methodology: The coding sequence for mature recombinant C-reactive protein (rCRP) was inserted into a vector with a hexahistidine leader sequence and expressed in ArcticExpress (DE3) strain of E. coli with media containing 15N-ammonium chloride as the sole nitrogen source. 15N-labeled rCRP (15N-rCRP) was purified sequentially by immobilized metal affinity and phosphatidylcholine binding with a yield of
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approximately 60 micrograms. Tosyl-activated superparamagnetic polystyrene beads were conjugated with monoclonal antibody against CRP and incubated with human serum spiked with either 15N-rCRP alone or with 15N-rCRP and a 5 fold spike of purified human CRP. Following elution, the CRP was trypsin-digested and the protein quantified by analyzing five tryptic peptides by isotope-dilution tandem mass spectrometry with both external and standard addition calibration . The serum sample analyzed was a commercially available stock commonly used for control purposes with ~1.6 mg/L concentration ( n = 3) as determined by the manufacturer by immunoturbidimetric assay. Validation: Protein purity of 15N-rCRP was greater than 99% as assessed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Analysis by matrix-assisted laser desorption ionization mass spectrometry indicates that the principle product has a mass consistent with CRP with 15N incorporation along with the hexahistidine leader sequence. Incorporation as a percent of total nitrogen was estimated to be greater than 99% as determined by isotopic profile analysis. CRP binding was linear with r2 regression coefficient > 0.99. Mixtures of CRP and 15N-rCRP in specific ratios subjected to affinity purification, digestion and LC-MS/MS analysis were linear (r2 > 0.99) demonstrating similar binding and digestion characteristics. External calibration curves for quantification of CRP in serum were linear (r2 > 0.99) and a produced value of 1.3 mg/L (%CV = 19%). Standard addition calibration produced a similar value (1.3 mg/L (%CV = 18%)). Conclusions: The incorporation of isotope-dilution mass spectrometric quantification into the affinity purification of CRP from human serum requires the generation of a stable-isotope labeled internal standard. The present study demonstrated the generation of 15N-rCRP intact protein of sufficient purity, label incorporation and quantity to perform small scale analysis of CRP in human serum utilizing affinity purification in combination with isotope-dilution tandem mass spectrometry. This technique will play a significant role in the future development of higher order methods for the measurement of CRP and other low abundance proteins in serum. Present work is focused on the development of a yeast protein expression system in order to increase yields and generate recombinant protein structurally identical to native forms.
Proteins/Enzymes
D-172 Tissue-based Quality Control Materials for Tests of the Intestinal Disaccharidases J. Lu1, D. G. Grenache2. 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 2Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT
Background: Carbohydrate malabsorption is due to a deficiency of one or more intestinal disaccharidases. Disaccharidase activity is determined by measuring the glucose liberated from a disaccharide substrate after incubation of the substrate with a homogenate made from a biopsy of duodenal mucosa. The lack of tissue-based quality control material is a significant limitation for clinical laboratories that perform intestinal disaccharidase tests. Objective: To develop tissue-based quality control materials for intestinal disaccharidase tests. Methods: Duodenal mucosa from freshly slaughtered adult pigs was utilized. Samples (~5 mg) were homogenized in 500 L of saline for 2 minutes using a Bullet BlenderTM (Next Advance, Inc., Averill Park, NY). Individual substrate solutions (56 mM lactose, maltose, palatinose, and sucrose) were prepared in 0.1 M sodium maleate buffer, pH 6.0. Equal volumes (30 L) of homogenate and each substrate were combined and incubated at 37C for 60 minutes, with the exception of maltose, which was incubated for 30 minutes. Following incubation, the enzyme reactions were stopped by heating at 100C for 5 minutes. The Roche Cobas c501 analyzer (Roche Diagnostics, Indianapolis, IN) was used to measure both glucose (hexokinase) and total protein (benzethonium chloride). Enzyme activities were expressed in units of mol/min/g protein. Unheated tissue samples (N=15) were used as baseline controls. Test samples were incubated at variable temperatures for 5-10 minutes prior to homogenization. The percent of remaining activity in the test samples was calculated relative to that of baseline controls. Results: Shown in the table below. Percent of enzyme activity after incubation at various temperatures Mean Activity of Unheated Tissue (mol/min/g protein) Lactase 47.2 Maltase 432.8 Palatinase 14.1 Sucrase 36.7 50C 65C 70C 80C 10 min 10 min 10 min 10 min 146% 143% 151% 141% 108% 63% 96% 20% 31% 72% 105% 17% 0% 20% 17% 0% 100C 5 min 0% 0% 0% 0%
D-171 Evaluation of a New Olympus CRP Assay on the Olympus AU400TM, AU640 TM, AU2700 TM and AU680TM Immunochemistry Analyzers. M. M. D. L. P. S. T. Bertsch3, C. Aschenneller3. 1Olympus Life Science Europa GmbH (Irish Branch), O'Callaghan's Mills, Ireland, 2York Hospital, York, United Kingdom, 3Klinikum Nurnberg, Nurnberg, Germany
CRP is one of the most sensitive acute-phase reactants providing information about acute and chronic inflammatory processes. CRP levels in serum can be elevated to orders of magnitude above the normal level (<5mg/L) following infection, myocardial infarction, trauma, inflammation or surgery. The new CRP Latex assay, OSR61157, provides a dynamic assay range from 4480mg/L. This assay principle utilizes sample mixed with R1 buffer and R2 reagent containing latex material coated with anti-human CRP antiserum. Turbidimetric absorbance [600(660)nm] of complexes formed during the antigen-antibody reaction is measured at 37oC for approximately 5 minutes. The reaction is a calibrated spline method, utilizing 6 liquid stable calibrators (ODC00032) traceable to ERM-DA470. Prozone hook effect does not occur until >750mg/L. Imprecision CV values are <5% (4-10 mg//L) and <3% (10-480 mg/L) for within run and <5% total CV for all analysers tested using NCCLS EP5-T2. Interferences measured at app10 mg/L are as follows: lipemic and haemolytic <5% (1000mg/L Intralipid, 5g/L hemoglobin), icteric <10% (684 mol/L bilirubin) (NCCLS EP7-P). Reagent displays 90 day on board stability with open claim recalibration. Two comparisons with current Olympus CRP reagents were performed: OSR6147 in York District Hospital, UK and OSR6199 in Klinikum Nrnberg, Germany. The following table shows the results of the comparison of the assay on AU2700. Method Comparison Test method Range (mg/L) Slope Intercept Correlation coefficient Specimen type No. of samples OSR6147 OSR61157 (new assay) 5-300 0.990 2.1 mg/L 0.996 Serum 959 OSR6199 NA OSR61157 (new assay) 4 - 480 1.014 6.4 mg/L 0.998 LiHe plasma 997
Burkhard1,
McCusker1,
O'Gorman1,
Turley2,
Mellen2,
Conclusions: Sucrase is the most heat-labile and palatinase is the most heat-stable of the 4 enzymes. Porcine mucosal samples can be heated prior to homogenization to create a low-activity quality control material while unheated tissue can serve as a normal activity control.
D-173 Validation of the SEBIA Mini-Capillary System (MINICAP) for Serum Protein Electrophoresis and Hemoglobinopathy Analysis R. L. Fitzgerald1, C. B. Alday2, E. A. Coker2, D. A. Herold1. 1VAMC/ UCSD, San Diego, CA, 2VAMC, San Diego, CA
Objective: SEBIA has recently developed a mini-capillary system that uses two capillary electrophoresis (CE) columns which is designed for the analysis of serum proteins and hemoglobin variants in mid sized laboratories. The objective of this study was to compare the CE system for the analysis of serum proteins and hemoglobin variants with gel electrophoresis and high performance liquid chromatography, respectively. Relevance: Analysis of serum proteins is performed to confirm a variety of disease states but is primarily used as a screen for monoclonal populations of immunoglobulins. Hemoglobinopathy analysis is performed to identify thalassemias as well as structural variants. Methodology: For CE protein analysis, serum was loaded onto the SEBIA Minicap, diluted with buffer, and injected into the capillaries. Migration occurs for 4 minutes. Proteins are detected at 200 nm. The data system calculates each fraction; albumin, alpha 1, alpha 2, beta 1, beta 2 and gamma. Beta 1 and beta 2 were combined as one fraction using a manual overlay with the reference pattern. For proteins, the comparison method was Hydrasys gel electrophoresis. For CE hemoglobinopathy analysis, red blood cells from EDTA anticoagulated blood are loaded into the mini-capillary system, hemolyzed, and injected into the capillaries. Migration takes place for 8 minutes and hemoglobin is detected at 415 nm. Hemoglobin variants are tentatively identified based on component migration patterns
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relative to reference windows. The comparative method for hemoglobinopathy analysis was the Bio-Rad Variant. Validation: For proteins, the CE method was linear for the 5 fractions (albumin, alpha 1, alpha 2, beta, and gamma) at concentrations typically encountered in a clinical laboratory. For a patient with a monoclonal gammopathy, within and between run coefficient of variations of all fractions were less than 5.5%. A paired t-test demonstrated that the CE and gel electrophoresis system were not significantly different for quantifying alpha 2, beta or gamma regions, while albumin and alpha 1 were significantly different for 55 patient samples. On the CE system, albumin was lower and alpha 1 was higher as compared with gel electrophoresis. Most importantly, all monoclonal bands detected by CE were confirmed by gel electrophoresis. For CE hemoglobinopathy analysis, the method was linear for A2, S and C for concentrations typically encountered in a clinical laboratory. Within run coefficient of variations of Hb A, F, S and C was < 4 % for CE. The run-to-run precision of Hb A2 was 2.9 %. A paired t-test on 10 AS trait specimens demonstrated that CE reported significantly higher values for hemoglobin S. One hemoglobinopathy case had elevated hemoglobin F (6.9%) by HPLC and no detectable hemoglobin F by CE. This specimen appeared to be a rare hemoglobin variant, otherwise specimens from 42 patients run on the CE hemoglobinopathy program generally agreed well with HPLC. Conclusions: This is the first clinical evaluation of the Sebia mini-capillary system for proteins and hemoglobin variant analysis. Our results demonstrated comparable performance of CE with gel electrophoresis and HPLC. For hemoglobinopathy analysis, the Sebia mini-capillary system provided complementary information for rare hemoglobin variants.

conclusion, the FAICEA for detection of TRACP 5b demonstrates good analytical performance and less influences of TRACP 5a and inactive TRACP 5b fragments compare to the other immunoassay kit.
D-175 Phenotyping and Quantitation of -1-Antitrypsin by LC-MS/MS: A Novel Methodology for the Laboratory Evaluation of -1-Antitrypsin Deficiency M. Snyder, Y. Chen, L. M. Benson, J. A. Katzmann, H. R. Bergen. Mayo Clinic, Rochester, MN
Background: -1-antitrypsin (A1AT) is the primary inhibitor of neutrophil elastase in the lungs, functioning to prevent tissue damage that would result from uncontrolled protease activity. Mutations in the A1AT gene lead to decreased concentrations of the protease inhibitor, resulting in A1AT deficiency disease. The two most common alleles associated with A1AT deficiency are the S and Z alleles. Diagnosis of this disorder requires quantitation of A1AT in conjunction with identification of the specific deficiency alleles. The current algorithm of laboratory testing for A1AT deficiency uses a combination of quantitation, genotyping, and/or phenotyping. Here, we describe a novel multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of A1AT and identification of the specific deficiency alleles. Method: Peptides containing the common S and Z deficiency alleles, as well as an invariant peptide, were identified by LC-MS/MS of a purified A1AT tryptic digest. Each of the identified peptides was synthesized in unlabeled and 13C/15N-labeled forms. Serum samples (n=18) were denatured and digested with trypsin. After addition of labeled internal standard peptides, the digested plasma samples were analyzed by LC-MS/MS on a Finnigan TSQ Quantum Ultra mass spectrometer. The S, Z, and wild-type alleles were identified based on detection of the specific peptides and comparison to the internal standard. The results were compared to the current phenotyping assay performed by isoelectric focusing (IEF) electrophoresis using the Hydragel 18 A1AT Isofocusing system (Sebia) according to the manufacturers instructions. For quantitation, varying concentrations of unlabeled invariant peptide were used to construct a standard curve. For each sample, the ratio of the invariant peptide peak area to the internal standard peak area was calculated, which was then compared to the standard curve. The quantitation results obtained by LC-MS/MS were compared to those determined by immunonephelometry as performed on a Dade Behring II nephelometer according to manufacturers instructions. Results: For the A1AT allele identification, in 17 out of the 18 samples, the LC-MS/ MS results were identical to those obtained by IEF gel electrophoresis, resulting in a concordance of 94%. The single discrepant result was due to administration of replacement therapy to a patient with known A1AT deficiency. A1AT quantitation was assessed for precision and accuracy. The inter- and intra-assay CVs were <5% for 2 peptide standards and <10% and <5%, respectively, for 3 serum samples. To assess accuracy, quantitation of the serum samples by LC-MS/MS was compared to the current nephelometric assay. This comparison demonstrated a linear relationship, with a correlation coefficient of 0.93 and a slope near unity. Conclusion: We have developed a novel multiplex LC-MS/MS method for A1AT quantitation and allele identification. This method demonstrates excellent correlation with current phenotyping and nephelometric assays and is analytically robust. It is a promising method that offers numerous improvements over the current testing methodologies used for the diagnosis of genetic A1AT deficiency.
D-174 Evaluation of analytical performance of a novel immunoassay system for determination of serum tartrate-resistant acid phosphatase type 5b T. Miura1, Y. Igarashi2, Y. Mochizuki3, W. Kikuchi1, K. Ito1, K. Sasagawa1, R. Kojima1, K. Katayama1, F. Nomura4. 1Biochemical Laboratory, Nitto Boseki Co., Ltd., Fukushima, Japan, 2Tsuchiya Children's Hospital, Saitama, Japan, 3Department of Obstetrics and Gynecology, Dokkyo Medical University, Tochigi, Japan, 4Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University., Chiba, Japan
Tartrate-resistant acid phosphatase type 5b (TRACP 5b) is a reliable bone resorption marker derived specifically from osteoclasts and its serum levels are not affected by feeding and renal dysfunction. The development of TRACP 5b specific assays has been complicated by the presence of the highly similar isoform TRACP 5a which is derived from macrophages and an abundant inactive TRACP 5b fragments in serum. Recently we have developed a new ELISA which detects the enzyme activity of TRACP 5b specifically based on a novel assay system called fragments absorbed immunocapture enzymatic assay (FAICEA, OsteolinksTRAP-5b: Nitto Boseki Co., Ltd, Fukushima, Japan). The assay uses two different monoclonal antibodies, Trk49 and Trk62, generated with immunization of TRACP 5b from human bone. The antibody Trk49 is highly specific to inactive TRACP 5b fragments. In contrast the Trk62 is highly specific to intact active TRACP 5b. The microwell plate coated with both antibodies is used for the assay. At the immunoreaction, the Trk49 binds inactive TRACP 5b fragments firstly, thereby making Trk62 more available to bind active TRACP 5b in reaction mixture. After the immunoreaction, binding TRACP 5b activity is measured by 2-chloro-4-nitrophenyl phosphate (CNPP) as a substrate. The CNPP is more specific substrate for TRACP 5b than traditional substrate, 4nitrophenyl phosphate (PNPP). In this study, we examined analytical performance of the FAICEA system. Then the influences of TRACP 5a and inactive TRACP 5b fragments for FAICEA were compared with that for another commercially available immunoassay kit for TRACP 5b (BoneTrap: IDS, Boldon, UK). Sensitivity: The minimum detection limit of the FAICEA is 0.1 U/L, determined by the upper 3 SD limit in a zero standard precision study. Recovery: Spike recovery was determined by adding a known quantity of purified TRACP 5b to serum samples with different levels of endogenous TRACP 5b. The recovery was 92-103%. Precision: Intra-assay was determined for 8 replicate of 2 sera. Inter-assay was determined for 8 replicate for 7days for 2 sera. The CVs of Intra-assay were 2.0% and 2.2%. The CVs of inter-assay were 1.6% and 1.9%. Assay Range: The assay range of FAICEA is 0.1-15 U/L. In the spike test of TRACP 5a into serum, increasing amounts of purified TRACP 5a was added to normal human serum. The FAICEA measurements were not affected by increasing TRACP 5a levels. In contrast, TRACP 5b levels measured by BoneTrap as increased by 67%. To investigate possible inactive fragment interference, serum samples (three TRACP levels) were diluted serially with saline. The FAICEA demonstrated excellent linearity (93-102% recovery for highest dilutions), whereas BoneTrap measured highly increased values after dilution (127-164% recovery for highest dilutions), suggesting fragment interference in the undiluted samples. In
D-176 Changes in serum free light chain values do not precede changes in Mspike in a series of patients with relapsed/refractory intact immunoglobulin multiple myeloma S. N. Uljon1, P. G. Richardson2, C. Grudzien1, P. H. Schur1, M. J. Tanasijevic1, N. I. Lindeman1. 1Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 2Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
BACKGROUND: Serum free light chains (SFLC) are used to diagnose and follow multiple myeloma, particularly for patients with light chain or hyposecretory disease. SFLC may also, however, be useful in monitoring patients with intact immunoglobulin multiple myeloma (IMM), where the short circulating half-life of free light chains relative to intact immunoglobulin may make the SFLC test valuable as an earlier indicator of treatment response than quantification of M-spikes from
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scanned serum protein electrophoresis (SPEP) gels or heavy chain nephelometry measurements. To test the hypothesis that the SFLC test detects changes in IMM disease status before M-spike, we reviewed SFLC, M-spike, and heavy chain results obtained from individual patients at multiple time points during the course of their disease. Further, we tested the specificity of the SFLC assay for free light chains in the presence of abundant intact monoclonal immunoglobulin, as is seen in patients with IMM. METHODS: 630 samples from 17 patients with relapsed/refractory myeloma participating in a phase I clinical trial were reviewed; mean follow-up was 24 mos (range, 8-36 mos). Each patients SFLC, M-spike, and heavy chain results were reviewed along with bone marrow biopsy, medication records, and clinical notes. SFLC assay specificity was assessed by direct measurement of intact IgG-kappa immunoglobulin and by spike-and-recovery studies with normal serum; intact IgGkappa was purified from pooled sera from 12 patients with IMM. SFLC and heavy chains were measured on a Siemens BN2 analyzer; Helena SPIFE 3000 was used for SPEP. RESULTS: 15/17 (88%) cases showed M-spike, total heavy chain, and SFLC measurements that rose and fell in near-synchrony throughout the patients course. In one patient, changes in serum free light chain preceded changes in M-spike and heavy chain by an average of 3 mos, while in another patient, SFLC results lagged behind the M-spike and heavy chain measurements by 1 mo. In eight patients, the SFLC concentrations showed short-term fluctuations in the absence of corresponding changes in M-spike, heavy chain, or clinical treatment or status. Specificity studies demonstrated minimal (<0.5%) cross-reactivity of the free light chain assay for the intact IgG-kappa immunoglobulin. DISCUSSION: In 16 of 17 refractory myeloma patients tested over ~2 yrs, SFLC measurements did not show changes earlier than M-spike measurements other than short-term fluctuations that reversed on subsequent measurement and that did not correlate with other laboratory values or changes in treatment or clinical condition. The synchrony between the SFLC and M-spike results was not attributable to crossreactivity in the SFLC assay, which showed >99% specificity for free light chains in the presence of abundant intact immunoglobulin. For practical clinical management in this population of patients with refractory/relapsed IMM, SFLC measurements did not add value as an adjunct to conventional SPEP with M-spike quantification.
Proteins/Enzymes
D-178 SIGNIFICANT DIFFERENCES WHEN USING CREATININE, MDRD OR CYSTATIN C FOR ESTIMATING GLOMERULAR FILTRATION RATE IN ICU PATIENTS M. Lipcsey1, M. Furebring2, L. A. Hansson3, S. Rubertsson4, A. Larsson5. 1Section of Anaesthesiology & Critical Care, Department of Surgical Sciences Uppsala University Hospital, Uppsala, Sweden, 2Sections of Infectious Diseases, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden, 3Laboratory Medicine, Karolinska Institute, Stockholm, Sweden, 4Section of Anaesthesiology & Critical Care, Department of Surgical Sciences, Uppsala University Hospital, Uppsala, Sweden, 5Clinical Chemistry, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden
Background. Renal dysfunction is associated with increased morbidity and mortality in intensive care patients. In most cases the glomerular filtration rate (GFR) is estimated based on serum creatinine and the Modification of Diet in Renal Disease (MDRD) formula but cystatin C estimated GFR is being used increasingly. The aim of this study was to compare creatinine and MDRD and cystatin C estimated GFR in intensive care patients (ICU). Methods. Retrospective observational study was performed, on patients treated within the general ICU during 2004-2006, in a Swedish university hospital. Results. GFR markers are frequently ordered in the ICU. Ninety-two percent of the patient test results had Cystatin C estimated GFR (eGFRCystatin C) 80 mL/min/1.73 m2, 75 % had eGFR 50 mL/min/1.73 m2 and 30 % had eGFR 20 mL/min/1.73 m2. In contrast, only 46% of the patients had reduced renal function assessed by plasma creatinine alone and only 47% had eGFRMDRD 80mL/min/1.73 m2. The mean difference between eGFRMDRD and eGFRCystatin C was 39 mL mL/min/1.73 m2 for eGFRCystatin C values 60 mL/min/1.73 m2. Conclusions. GFR is commonly assessed in the ICU. Cystatin C estimated GFR yields markedly lower GFR results than plasma creatinine and eGFRMDRD. Many drugs are eliminated by the kidneys and their dosage is adjusted for kidney function. Thus, the differences in GFR estimates by the methods used indicate that the GFR method used in the intensive care unit may influence the treatment.
D-177 CYSTATIN C AND MDRD ESTIMATED GLOMERULAR FILTRATION RATE DIFFER DURING NORMAL PREGNANCY A. Larsson1, L. Hansson2, M. Palm3, O. Axelsson3. 1Department of Medical Sciences, Clinical Chemistry, Uppsala, Sweden, 2Laboratory Medicine, Karolinska Institute, Sweden, 3Department of Womens and Childrens Health, Obstetrics and Gynecology, Uppsala, Sweden
Background. Estimation of the glomerular filtration rate (eGFR) is used to monitor patients with suspected kidney disease and to optimize the dosage of drugs that are eliminated by the kidneys. Plasma creatinine and cystatin C are the two most widely used GFR markers, Both markers are recommended to be reported as estimated GFR but there is a lack of reference intervals for pregnant females. Methods. We have studied creatinine (eGFRMDRD) and cystatin C (eGFRcystc) estimated GFR during 52 normal pregnancies from pregnancy week ten to delivery and postpartum. Each woman was sampled repeatedly and the samples were grouped according to gestational age into the following periods: week 7 - 16; week 18 - 24; week 24 - 28; week 28 - 31; week 31 - 34; week 34 - 38; -2 - 0 weeks prior to delivery and postpartum (>6 weeks after delivery). The 2.5 and 97.5 percentiles for these markers were calculated according to the recommendations of the International Federation of Clinical Chemistry on the statistical treatment of reference values. Results. In healthy pregnant females eGFRcystc was higher in the first two trimesters and lower prior to delivery in comparison with eGFRMDRD. eGFRcystc and eGFRMDRD give different results. No significant correlations between the two estimates were found in any of the time groups. Conclusions. It is important to distinguish between the two GFR estimates and use separate gestational specific reference intervals for pregnant females.
D-179 The effect of the acute phase response on ceruloplasmin concentrations P. J. Twomey, C. A. Hockings. The Ipswich Hospital, Ipswich, United Kingdom
Introduction: Wilsons Disease (WD), also known as hepatolenticular degeneration, is an inborn error of copper metabolism affecting approximately 1 in 30,000 live births. A significant reduction in the ATP7B P-type ATPase activity results in decreased excretion of copper into bile by hepatocytes leading to hepatic injury and copper deposition in the brain, cornea and kidneys. An additional consequence is the failure to incorporate copper into ceruloplasmin, reducing its half-life in plasma. Accordingly, serum ceruloplasmin concentrations are often employed to screen for WD. However, ceruloplasmin is also an acute phase response protein. The objective of this study was to use routine clinical data to determine the relationship between ceruloplasmin and C-reactive protein (CRP). Methods: Using the laboratory information system, all serum requests that had both ceruloplasmin and CRP analysed were retrospectively obtained since this laboratory introduced in-house ceruloplasmin. Requests for patients <18 years of age were excluded as were subsequent requests for the same individual patients. Both ceruloplasmin and CRP were analysed on an AU2700 (Olympus Diagnostica, County Clare, Ireland) using Olympus reagents in line with the manufacturers instructions. Least squares linear regression analysis was carried out using MS Excel to determine the exact relationship (slope, intercept and correlation co-efficient) between ceruloplasmin and CRP. The t-distribution was employed to calculate the p-value for the correlation co-efficient. Results: There were 312 result sets that met the criteria. The linear regression equation was ceruloplasmin = 0.4706 x CRP + 260.56 with r2 = 0.0743. The p-value for r was <<0.001. Discussion: The main desirable attribute of a screening test is a high clinical sensitivity, that is, not to miss a diseased patient. An acute phase response is seen much more frequently in routine clinical practice than a WD case. Patients with abnormal liver function tests are often tested for ceruloplasmin to exclude WD, many of whom will have an inflammatory response. The relevant clinical question is - what
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is the chance that an acute phase response will result in a patients ceruloplasmin going from below the lower reference interval to above it? A CRP of 22 mg/L would be associated on average with a ceruloplasmin about 10 mg/L higher than if the CRP was 1 mg/L. Therefore, many patients with concurrent viral infections or minor injury will have their serum ceruloplasmin concentrations increased by approximately 10 20 mg/L. A CRP of 150 mg/L would be associated on average with a ceruloplasmin about 70 mg/L higher. While patients with such CRP values are not common, they are more common the patients with WD. Thus, the acute phase response may result in some WD patients whose healthy level is just below the lower reference limit having a normal serum ceruloplasmin concentration due to an acute phase response. Conclusion: Laboratories should consider measuring CRP with serum ceruloplasmin requests to increase the clinical sensitivity of ceruloplasmin for the detection of WD.
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Ferritin As An Indices of Body Iron Stores and Acute Myocardeal Infarction in Diabetics

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-0.50 0.017 22 -0.63 0.001 23 -0.40** 0.018 34