Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin c...

four cases Sharma S, Pujani M, Pahuja S, Chandra J, Rath B, Labhchand - Indian J Pathol Microbiol

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CASE REPORT
Year : 2010 | Volume : 53 | Issue : 4 | Page : 820-823

Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin concentration in children: A series of four cases
Sunita Sharma1, Meenu Pujani1, Sangeeta Pahuja1, Jagdish Chandra2, B Rath2, Labhchand2
1 Department of Pathology, Lady Hardinge Medical College & Associated Hospitals, New Delhi - 110 001, India 2 Department of Pediatrics, Lady Hardinge Medical College & Associated Hospitals, New Delhi - 110 001, India

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Sharma S Pujani M Pahuja S Chandra J Rath B Labhchand

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27-Oct-2010

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Sharma S Pujani M Pahuja S Chandra J Rath B Labhchand

http://www.ijpmonline.org/article.asp?issn=0377-4929;year=2010;volume=53;issue=4;spage=820;epage=823;aulast=Sharma (1 of 6) [4/29/2012 9:17:49 PM]

Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin c... four cases Sharma S, Pujani M, Pahuja S, Chandra J, Rath B, Labhchand - Indian J Pathol Microbiol

Abstract
Mean corpuscular hemoglobin concentration (MCHC), a parameter that is reported as a part of a standard complete blood count by automated analyzer, is a measure of the concentration of hemoglobin in a given volume of packed red blood cell. Values of MCHC significantly above reference range are not physiologically possible due to limitations on solubility of hemoglobin. The high MCHC can give us a clue to certain type of hemolytic anemia and necessitate critical evaluation of peripheral smear to reach a definitive diagnosis. Here we are presenting a series of four cases with raised MCHC, emphasizing the importance of systematic and meticulous examination of the peripheral smear to render a definitive diagnosis.

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Keywords: Anemia, mean corpuscular hemoglobin concentration, peripheral smear

How to cite this article: Sharma S, Pujani M, Pahuja S, Chandra J, Rath B, Labhchand. Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin concentration in children: A series of four cases. Indian J Pathol Microbiol 2010;53:820-3 How to cite this URL: Sharma S, Pujani M, Pahuja S, Chandra J, Rath B, Labhchand. Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin concentration in children: A series of four cases. Indian J Pathol Microbiol [serial online] 2010 [cited 2012 Apr 29];53:820-3. Available from: http:// www.ijpmonline.org/text.asp?2010/53/4/820/72094

Abstract Introduction Case Reports Discussion References Article Figures Article Tables

Introduction
Mean corpuscular hemoglobin concentration (MCHC) is a measure of the concentration of hemoglobin in a given volume of packed red blood cells. It is reported as a part of a standard complete blood count by an automated analyzer. The normal reference range is 32-36 g/dl. Values of MCHC significantly above the reference range are not physiologically possible due to limitation on solubility of hemoglobin. Lipemia or other causes of turbidity in lysate can cause falsely high hemoglobin values which raise the apparent MCHC. Furthermore, hemolysis (in vitro or in vivo) causes lowering of hematocrit and increases the MCHC. Mild increase in MCHC can also result from cellular dehydration, like in spherocytosis and sickle cell anemia. The high MCHC can give us a clue to certain types of hemolytic anemia and necessitates the critical evaluation of peripheral smear to reach a definitive diagnosis. Here, we are presenting a series of four cases with raised MCHC, emphasizing the importance of systematic and meticulous examination of the peripheral smear to render a definitive diagnosis. All cases were analyzed on Sysmex KX-21 hematology analyzer (Transasia: Mumbai, India Diamed, Switzerland ) and immune-hematological tests were performed on gel cards (Diamed ID Microtyping System, India).

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Case Reports
Case 1 An 8-year-old male child presented in January 2008 with pallor, jaundice, passage of black colored urine and mild hepatomegaly. The clinical diagnosis of acute intravascular hemolysis was made. The hemogram findings [shown in [Table 1]] revealed unusually high MCHC and disproportionately low RBC count for the degree of anemia. This immediately alerted us to have a look at the sample. Gross examination of the sample revealed small clumps. The wet smear of the sample showed agglutinates of RBCs. On warming the sample at 37C, the MCHC got reduced from 41.4 to 37.4 g/dl. Careful examination of peripheral
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Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin c... four cases Sharma S, Pujani M, Pahuja S, Chandra J, Rath B, Labhchand - Indian J Pathol Microbiol

smear detected clumps of RBCs, spherocytes and a very occasional neutrophil and monocyte showing erythrophagocytosis [Figure 1]. Serum urea and creatinine were raised (101 and 1.6 mg/dl, respectively). Urine was cola colored and deposits showed granular pigmented casts. Thus, a diagnosis of intravascular hemolysis with acute renal failure probably due to paroxysmal cold hemoglobinuria (PCH) or cold agglutinin disease (CAD) was considered. Direct Coombs test (DCT) at 37C was negative for IgM, weakly positive for IgG and strongly positive for C3d (3+), while at 4C, only C3d was positive (3+). Test for Donath Landsteiner antibodies was positive with P+ve O group red cells. Venereal Disease Research Laboratory (VDRL) was negative. A final diagnosis of PCH was made. Patient was given blood transfusions, antibiotics and put on steroids (prednisolone 2 mg/kg for 2 weeks and then tapered gradually). He had improved and was discharged after 2 weeks.

Figure 1: Peripheral smear showing clumping of RBCs, microspherocytes and erythrophagocytosis (Wright's stain, 1000)
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Table 1: Hematological investigations of the four cases

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Case 2 A 9-year-old male child came in the month of December 2008 with a history of off-and-on fever and hyperpigmented lesions all over the body for 2 years. On examination, there was hepato-splenomegaly of 4 and 3 cm, respectively, below the costal margin. Hemogram findings [Table 1] showed pancytopenia with high MCHC of 42.6 g/dl, which corrected to 35.5 g/dl on warming the sample to 37C. Wet smear examination revealed clumps of RBCs in the smear. Peripheral smear examination showed clumps of RBCs with presence of normocytic, normochromic red cells and many microcytic, hypochromic cells. Thus, based on the above features, a possibility of CAD or cryoglobulinemia was considered. Further evaluation revealed antinuclear antibody (ANA), double stranded DNA (dsDNA) and antiphospholipid antibody (APLA) positivity. Test for CAD was positive with autoantibody specificity against I antigen. A final diagnosis of CAD secondary to Systemic Lupus Erythematosus (SLE) was made. Patient was treated with antibiotics, blood transfusions and methyl prednisolone for 3 days, but he did not respond and therefore was put on azathioprine for which he responded. Case 3 A 16-year-old female presented with pallor, jaundice and moderate splenomegaly. Hemogram findings [Table 1] revealed increased MCHC, red cell distribution width (RDW) and corrected reticulocyte count. Peripheral smear examination revealed many microspherocytes of uniform size admixed with normocytic, normochromic RBCs. Coombs test (direct and indirect both) was negative. Osmotic fragility showed shift to left with mean corpuscular fragility (MCF) of 4.8 g/l (control 4.0 g/l) and a tail of fragile cells. Her mother also revealed similar hemogram and smear findings, with MCF of 4.5 g/l (control 4.0 g/l). Based on the above findings, a diagnosis of hereditary spherocytosis (HS) was suggested. Case 4 A 2-year-old male child presented with pallor, icterus and mild splenomegaly. Hemogram [Table 1] showed increased MCHC, RDW and corrected reticulocyte count. Peripheral smear revealed the presence of numerous microspherocytes of variable size, admixed with normocytic normochromic RBCs. DCT was positive for IgG. A possibility of autoimmune hemolytic anemia was suggested. The patient was put on steroids and has been on regular follow up for the past 16 years.

Discussion
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Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin c... four cases Sharma S, Pujani M, Pahuja S, Chandra J, Rath B, Labhchand - Indian J Pathol Microbiol

MCHC estimates the average concentration of hemoglobin in average RBCs. There are a few known causes, as illustrated by Richard Ravel et al., can lead to increased MCHC in the results of the automated analyzer and alerts the hematologist to look for its specific cause by carefully and systematically analyzing the peripheral smear. These include:

[1]

which

Spherocytosis (hereditary/autoimmune), Free plasma hemoglobin (hemolysis), High titers of cold agglutinins, Severe plasma lipemia, Paraproteinemia/cryoglobulinemia and Very high WBC count.

An increased MCHC obtained by electronic cell counter is a characteristic finding of HS red cells. Presence of spherocytes is the hallmark of HS, and these are reliably present in 97% of patients with moderate/severe disease. However, 25-35% of mild HS patients lack the characteristic microspherocytes.
[2]

The average MCHC exceeds the upper limit normal, i.e. 36%, in approximately half of the HS patients due to red cell dehydration. The possible mechanisms of dehydrated HS red cells could be activation of K-Cl cotransport pathway or increased Na + /K + pump activity. The red cells of HS are smaller, lack the central pallor and have increased pigment density. Unlike autoimmune hemolytic anemia, HS spherocytes are fairly uniform in size and density. The combination of high MCHC, a wide RDW and a shift in distribution curves is often enough to suspect HS. Michaels et al.
[3]

conducted a study on 112 children with HS to evaluate the use of automated erythrocyte indices as a screening tool for HS. They found

that MCHC and RDW in HS group were significantly higher than in normal control subjects. Combining the two, the authors found the specificity to be 100%, the combination of two being an excellent predictor of HS. The two clinical syndromes manifested from cold autoimmune antibodies are CAD and paroxysmal cold hemoglobinuria. CAD typically occurs in adults and may be idiopathic/secondary to lymphoma, leukemia, infection with Mycoplasma pneumoniae, Epstein Barr Virus (EBV), Cytomegalovirus (CMV) and autoimmune diseases. Hemolytic anemia is found to occur in about 10% cases of SLE which is usually of warm antibody type, while CAD is very rare. RBC destruction is usually not severe in CAD. Case II also did not show any evidence of hemolysis. PCH is a rare variety of cold Autoimmune Hemolytic Anemia (AIHA) where IgG autohemolysin binds to RBCs at low temperatures and causes intravascular hemolysis on warming. one of the most common causes of acute hemolysis in children and usually occurs after an infectious illness. erroneously increased with falsely low RBC count. There are a number of practices right from collection of blood to analysis by an automated analyzer, which affect the accuracy of the final result. Successful venipuncture may be facilitated by keeping the subject's arm warm, applying tourniquet/sphygmomanometer cuff kept at approximately diastolic pressure and tapping the skin over the site of the vein a few times. If possible, compression of the vein should be completely avoided so as to prevent hemoconcentration. Hemolysis can be avoided/minimized by using a clean apparatus, withdrawing the blood slowly, not using too fine a needle, delivering the blood gently into the receiver and avoiding frothing during the withdrawal of the blood and subsequent mixing with anticoagulant.
[6] [5] [4]

Acute transient type of PCH is

In both these conditions, MCHC is

Recent use of evacuated tube systems ensures right amount of blood getting mixed with correct proportion of anticoagulant. The anticoagulant recommended by International Council for Standardization in hematology is dipotassium salt of ethylene diaminetetraacetic acid (EDTA) at a concentration of 1.50 0.25 mg/ml EDTA, in excess of 2 mg/ml, may result in a significant decrease in PCV, increase in MCHC, and red cell shrinkage and degenerative changes. The platelets are also affected; excess of EDTA causes them to swell and then disintegrate causing an artificially high platelet count.
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of blood.

[7]

[8]

Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin c... four cases Sharma S, Pujani M, Pahuja S, Chandra J, Rath B, Labhchand - Indian J Pathol Microbiol

There are significant effects of storage on blood cell morphology. By 3 hours, changes may be discernible and by 12-18 hours these become striking. Longer periods of standing lead to progressive crenation and sphering of RBCs. Wherever possible, the practice of making films of blood before adding anticoagulant is recommended, especially when screening for lead toxicity as the granules of punctuate basophilia may stain less obviously in anticoagulated blood.
[6]

Thus, to conclude, in a case of anemia with high MCHC, critical evaluation of peripheral smear should be done. The following flowchart suggesting a rational clinical laboratory approach for investigating a case of anemia with high MCHC may be followed [Figure 2].

Figure 2: Rational clinical laboratory approach for investigating anemia with high MCHC
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References
1.

Ravel R. Basic hematological tests and classification of anemia. In: Clinical Laboratory Medicine: Clinical application of Laboratory Data.6 th ed. Missouri, USA: Elsevier Health Sciences; 1995. p. 11-5. Brugnara C, Oski FA, Nathan DG. Diagnostic approach to anemia patient. In: Orkin SH, Nathan DG, Ginsburg D, Look AT, Fisher DE, Lux SE, editors. Nathan and Oski's hematology of infancy and childhood. 7th ed. Philadelphia: Elsevier Health Sciences; 2008. p. 456-63. Michaels LA, Cohen AR, Zhao H. Screening for Hereditary Spherocytosis by use of automated erythrocyte indices. J Pediatr 1997;130:957-60. Nair K, Pavithran K, Philip J, Thomas M, Geetha V. Cold haemagglutinin disease in Systemic Lupus Erythematosus. Yonsei Med J 1997;38:233-5. [PUBMED] [FULLTEXT] Greer JP, Foerrster J, Rodgers G. Hemolytic anemia. In: Greer JP, Foerster J, Rodgers GM, Paraskevas F, Glader B, editors. Wintrobe's Clinical hematology. Vol 1. 12th ed. London: Lippincott Williams and Wilkins; 2008. p. 915-20. Lewis SM, Bain J Barbara, Bates I. Dacie and Lewis Practical Hematology. In: Lewis SM, Bain BJ, Bates I, editors. 9th ed. London: Churchill Livingstone; 2001. p. 1-8. International Council for Standardisation in Hematology. Recommendations for EDTA anticoagulation of blood for hematology testing. Am J Clin Pathol 1993;100:371-2. Van Assendelft OW, Simmons A. Specimen collection, handling, storage and variability. In: Lewis SM, Koepke JA, editors. Hematology Laboratory management and practice. Oxford: Butterworth Heinemann; 1995. p. 109-27.

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Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin c... four cases Sharma S, Pujani M, Pahuja S, Chandra J, Rath B, Labhchand - Indian J Pathol Microbiol

Correspondence Address: Meenu Pujani Department of Pathology, Lady Hardinge Medical College & Associated Hospitals, New Delhi - 110 001 India

DOI: 10.4103/0377-4929.72094 PMID: 21045429

Figures
[Figure 1], [Figure 2]

Tables
[Table 1]

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2008 Indian Journal of Pathology and Microbiology | Published by Medknow Online since 5th April, 2008
ISSN: Print -0377-4929, Online - 0974-5130

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