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IMMULITE Reproductive Hormone Assays: Multicenter Reference Range Data for Diagnostic Products Corporation Kits
Preface
This report is a compendium of reference range results and related information for several of the most important reproductive hormone assays available on DPC s automated, nonisotopic immunoassay platforms: the IMMULITE and IMMULITE 2000. The document represents one important aspect of DPC's commitment to the reproductive endocrinology community, not only to develop a comprehensive spectrum of clinically relevant assays that meet the demands of modern laboratory medicine for precision, accuracy and timeliness, but also to assist both laboratory and physician in making sense of patient results. This entails helping to disseminate in a clear, convenient format the best available information on the values to be expected for DPC assays in typical clinical settings. It also entails support for the kind of well-designed, carefully analyzed studies most likely to be of genuine benefit to the patient in the routine clinical application of these assays. We shall continue to gather relevant clinical data for these and other DPC reproductive hormone assays, and remain committed to updating this report as additional reference range results become available. Leo Vankrieken, Eur. Eng.
What s New
New to this edition are detailed results for testosterone, SHBG, and the free androgen index (FAI) in the menstrual cycle. Values for progesterone and prolactin applicable to nonpregnant women of reproductive age have been significantly updated. We have also added values for the estradiol/SHBG ratio (ESR) in the menstrual cycle, even though this index has no well-established application as yet. See page 18 for some recent publications bearing on this topic. Of greater practical interest, references to a number of DPC publications complementing this report on normal ranges have been added. These include poster presentations originally displayed at the AACC and now available as electronic documents on DPC s Web site showing individual subject trajectories throughout the menstrual cycle or pregnancy, against a backdrop of the reference range data displayed and summarized crosssectionally in this report.2,7,12,16 Another recent technical report summarizes a comparison of estradiol assays manufactured by DPC based on samples from the Multicenter Ovulatory Study (described in the next section) demonstrating, in the clinically most relevant way, that reference ranges established for the IMMULITE on daily samples collected throughout the menstrual cycle are applicable to the IMMULITE 2000 as well.13 (This was to be expected, after all, due to the close similarity, in both design and performance, of assays developed for these two systems.) Several tutorials are also cited in the list of references: one, for example, discusses SHBG and the FAI;4,11 another is on the use of FSH and estradiol assays during the luteal-follicular transition period.14 This report has been reorganized slightly. (See the adjoining table of contents.) Essential information on the IMMULITE and IMMULITE 2000 reproductive hormone assays including catalog numbers, calibration ranges, detection limits, conversion factors, and formulae for the FAI and ESR can now be found under Methods (page 4). As before, the report is being made available on DPC s Web site, www.dpcweb.com under Technical Documents, Technical Reports in Adobe Acrobat PDF format.
Table of Contents
Subjects...................................................................3 Methods...................................................................4 Data Analysis...........................................................5 Disclaimers ..............................................................5 References ............................................................17
Analytes
Follitropin (FSH).......................................................6 Lutropin (LH) ...........................................................7 LH/FSH Ratio...........................................................7 Estradiol (E2) ..........................................................8 Progesterone .....................................................9-10 Prolactin ...............................................................11 Total Testosterone ................................................12 Sex Hormone-Binding Globulin (SHBG) ................13 Free Androgen Index (FAI).....................................14 Estradiol/SHBG Ratio (ESR) ..................................15 Chorionic Gonadotropin (HCG) .............................16
Subjects
Although this report summarizes data from a variety of sources, three major studies deserve special mention. (Additional clinical studies will be summarized in future editions of this report.)
transferability to a representative IMMULITE 2000 assay of reference ranges established for the corresponding IMMULITE assay. Assays for estradiol were used in this study, which is summarized in another DPC technical report.13 The results provide experimental confirmation for expectations based on the close similarity of IMMULITE and IMMULITE 2000 assays in both design and performance.
Methods
This report summarizes reference range data generated with IMMULITE assays for eight analytes on serum samples. Based on the close similarity of their performance characteristics, the corresponding IMMULITE 2000 assays can be expected to have comparable reference intervals. Experimental verification of this claim for a representative analyte (estradiol) can be found in a related report.13 Listed below are the principal reproductive hormone assays available for the IMMULITE and IMMULITE 2000 platforms. (Other assays are under development.) For details, consult the package inserts. Also listed are conversion factors and the formulae adopted here for three derived parameters: the LH/FSH ratio, the free androgen index (FAI), and the estradiol/SHBG ratio (ESR).
1.4 g/dL 30 1,000 g/dL (0.038 mol/L) (0.81 27 mol/L) 10 pg/mL (37 pmol/L) 0.1 mIU/mL 0.4 mIU/mL 20 2,000 pg/mL (73 7,342 pmol/L) Up to 170 mIU/mL (2nd IRP 78/549) Up to 5,000 mIU/mL (3rd IS 75/537)
LH
L2KLH
Up to 200 mIU/mL 0.007 mIU/mL (1st IRP 68/40 & 2nd IRP 80/552) 0.2 ng/mL (0.6 nmol/L) 0.16 ng/mL (3.4 mIU/L) 0.02 nmol/L 10 ng/dL (0.3 nmol/L) 0.2 40 ng/mL (0.6 127 nmol/L) Up to 150 ng/mL (Up to 3,180 mIU/L, 3rd IS 84/500) Up to 180 nmol/L 20 1600 ng/dL (0.7 55 nmol/L)
Progesterone L2KPG
Prolactin SHBG
L2KPR L2KSH
2 g/dL 30 1,000 g/dL ( 0.054 mol/L) (0.81 27 mol/L) 12 pg/mL (44 pmol/L) 0.2 ng/mL (0.7 nmol/L) 0.1 mIU/mL 1.1 mIU/mL 20 2,000 pg/mL (73 7,342 pmol/L) 0.25 30 ng/mL (0.87 104 nmol/L) Up to 170 mIU/mL (2nd IRP 78/549) Up to 5,000 mIU/mL (3rd IS 75/537) Up to 200 mIU/mL (1st IRP 68/40 & 2nd IRP 80/552) 0.2 40 ng/mL (0.6 127 nmol/L)
Conversion Factors
Analyte DHEA-SO4 Estradiol Progesterone Prolactin Testosterone Conversion to Alternate Units g/dL 0.02714 mol/L pg/mL 3.671 pmol/L ng/mL 3.18 nmol/L ng/mL 21.2 mIU/L ng/dL 0.03467 nmol/L
LH
LKLH
Derived Parameters
LH/FSH Ratio: LH (in mIU/mL) / FSH (in mIU/mL) Free Androgen Index (FAI): 100 Total Testosterone (in nmol/L) / SHBG (in nmol/L) = 3.467 Total Testosterone (in ng/dL) / SHBG (in nmol/L) Estradiol/SHBG Ratio (ESR): Estradiol (in pmol/L) / SHBG (in nmol/L) = 3.671 Estradiol (in pg/mL) / SHBG (in nmol/L)
Progesterone LKPG
Prolactin SHBG
LKPR LKSH
Up to 150 ng/mL 0.5 ng/mL (Up to 3,180 mIU/L, (10.6 mIU/L) 3rd IS 84/500) 0.2 nmol/L 10 ng/dL (0.3 nmol/L) Up to 180 nmol/L 20 1600 ng/dL (0.7 55 nmol/L)
Data Analysis
S-PLUS 2000 (www.mathsoft.com) was used for most of the calculations, for data visualization, and for the graphs themselves.18
Centiles
The tables provide concentration estimates for relevant centiles, including the median (50th centile) and the central 95% range limits (2.5th and 97.5th centiles). Because distributions were often highly skewed rather than Gaussian or even symmetric, and in order to accommodate the presence of possible outliers, centiles were generally calculated using a robust nonparametric technique specifically, an S-PLUS implementation of the Harrell-Davis function, which is considered the nonparametric method of choice for univariate reference range analysis in clinical chemistry.3,19 In a few cases, parametric methods were used after a suitable transformation to improve symmetry. In the Multicenter Ovulatory Cycle Study, statistics were calculated for phases of the cycle, and sometimes for specific day ranges (relative to menstruation or the LH peak) which have often been singled out for special attention in the literature. In the Cross-Sectional Pediatric Fertility Study as well as in the Collaborative Study of HCG throughout Pregnancy, centiles were calculated after partitioning the data into age or gestational age brackets suggested by the data.
FSH (Follitropin)
Multicenter Ovulatory Cycle Study16
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. FSH, mIU/mL Ovulatory Cycles Follicular Phase Follicular Phase, Days 2 to 3 Midcycle Luteal Phase n* 54 (762) 54 (108) 54 (54) 54 (604) Median Central 95% 6.2 6.6 13.6 3.4 2.8 11.3 3.0 14.4 5.8 21 1.2 9.0
Additional Ranges
FSH, mIU/mL Group n 76 135 Median Central 95% 90.5 3.8 21.7 153 0.7 11.1 Postmenopausal* Males
*Preliminary
15
Follitropin
15
10
10
5 0
0 -24
-18
-12
-6
12
18
Follitropin
15
10
10
5 0
0 -8
-4
-1 1
LH (Lutropin)
Multicenter Ovulatory Cycle Study16
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. LH, mIU/mL Ovulatory Cycles Follicular Phase Midcycle Luteal Phase Perimenstrual, 9 days n* 54 (762) 54 (54) 54 (658) 54 (959) Median Central 95% 4.6 39 4.3 3.9 1.1 11.6 17 77 ND 14.7 ND 12.0
IMMULITE LH (LKLH)
mIU/mL
IU/L
Additional Ranges
60
60
LH, mIU/mL Group n 75 135 Median Central 95% 24.9 2.4 11.3 39.8 0.8 7.6 Postmenopausal* Males
*Preliminary
Lutropin
40
40
20
20
0 -24
-18
-12
-6
12
18
LH/FSH Ratio
LH / FSH Ratio Ovulatory Cycles Follicular Phase, Days 2 to 5 Follicular Phase, Days 2 to 9 Follicular Phase, Days 2 to 11 up to 5 days before LH Peak n* 54 (221) 54 (436) 54 (452) Median Central 95% 0.60 0.66 0.66 0.15 1.51 0.18 1.64 0.18 1.45
Estradiol
Multicenter Ovulatory Cycle Study16
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. Estradiol, pg/mL Ovulatory Cycles Follicular Phase Follicular Phase, Days 2 to 3 Periovulatory, 3 days Luteal Phase n* 54 (708) 54 (108) 54 (378) 54 (604) Median Central 95% 42 31 133 93 ND 160 ND 84
0 pg/mL
Estradiol
400
1000
200
500 0
-24
-18
-12
-6
12
18
34 400 27 246
Estradiol, pmol/L Ovulatory Cycles Follicular Phase Follicular Phase, Days 2 to 3 Periovulatory, 3 days Luteal Phase n* 54 (708) 54 (108) 54 (378) 54 (604) Median Central 95% 154 114 489 343 ND 587 ND 308 124 1468 101 905
pg/mL
Estradiol
400
1000
200
500 0
0 -8
-4
-1 1
Progesterone
14
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. Progesterone, ng/mL Ovulatory Cycles Follicular Phase Midfollicular Days 5 to 11 Midcycle Luteal Phase Midluteal, Days 7 to 8 of Luteal Phase n* 27 (382) 27 (186) 27 (27) 27 (323) 27 (54) Median Central 95% 0.47 0.43 1.06 8.9 13.1 ND 1.13 ND 0.98 0.48 1.72 0.95 21 6.0 24
21
ng/mL
Progesterone
20 0
0 -24
-18
-12
-6
12
18
Progesterone
20
ng/mL
60
Progesterone, nmol/L Ovulatory Cycles Follicular Phase Midfollicular Days 5 to 11 Midcycle Luteal Phase Midluteal, Days 7 to 8 of Luteal Phase n* 27 (382) 27 (186) 27 (27) 27 (323) 27 (54) Median Central 95% 1.5 1.4 3.4 28 42 ND 3.6 ND 3.1
10
30 0
0 -8
-4
-1 1
10
Nonpregnant Women
Analysis of the results for a total of 115 serum samples from nonpregnant women yielded a median of 9.4 ng/mL (199 mIU/L) and a central 95% range of 1.9 to 25 ng/mL (40 to 530 mIU/L). The data set used in this analysis included 62 results from two cross-sectional studies of nonpregnant adults, in addition to 53 results from the ovulatory cycle study summarized below specifically, the result obtained on the first sample collected from each subject.
Prolactin
ng/mL
Prolactin
40
400
20
200
10
100
2.5
5 -24
-18
-12
-6
12
18
16
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. Note: As explained on page 3, the increasingly stressful sample collection process entailed by the longitudinal design of this study can be expected to yield a higher frequency of prolactin elevations than a cross-sectional study like the one summarized immediately above. Prolactin, ng/mL Ovulatory Cycles Entire Cycle Follicular Phase Midcycle Luteal Phase n* 53 (803) 53 (53) 53 (699) Mean Central 95% 4.6 37 4.5 33 6.3 46 4.9 40 12.1 17 13.9 53 (1555) 13.0
Prolactin, mIU/L Ovulatory Cycles Entire Cycle Follicular Phase Midcycle Luteal Phase n* 53 (1555) 53 (803) 53 (53) 53 (699) Mean Central 95% 276 257 360 295 98 784 95 700 134 975 104 848 Group Females
Prolactin, mIU/L Age (yr) Cord 0.1 0.5 0.6 9 Males Cord 0.1 0.5 0.6 9 Combined Cord 0.1 0.5 0.6 9 n 28 28 55 27 36 55 55 64 110 Median 8,056 318 233 6,254 403 170 7,208 2,480 191 Central 95% 4,240 14,310 21 2,968 42 912 3,180 11,978 85 1,378 13 615 3,392 14,098 42 2,650 21 848
11
ng/dL
Total Testosterone
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. Testosterone, ng/dL Group Entire Cycle Follicular Phase Midcycle Luteal Phase n 26 (758) 26 (393) 26 (26) 26 (339) Median Central 95% 47 48 58 44 ND 118 ND 118 21 104 ND 119
Total Testosterone
40
1 0
0 -24
-18
-12
-6
12
18
Testosterone, nmol/L Group Entire Cycle Follicular Phase Midcycle Luteal Phase n 26 (758) 26 (393) 26 (26) 26 (339) Median Central 95% 1.6 1.7 2.0 1.5 ND 4.1 ND 4.1 0.7 3.6 ND 4.1
One of these sites also measured total testosterone levels in pregnant and postmenopausal women, with the following results. Testosterone, ng/dL Group Pregnant Females First Trimester Second Trimester Third Trimester Untreated 20 20 19 29 29 30 70 90 110 20 30 30 30 230 30 200 30 190 ND 100 ND 100 ND 60 n Median Abs Range
Postmenopausal Females
Additional Ranges
Testosterone, ng/dL Group Females Oral Contraceptives Postmenopausal Males 39 103 99 40 30 410 ND 110a ND 80L 200 810c n Median Central 95%
Treated Surgical
Testosterone, nmol/L Group Pregnant Females First Trimester Second Trimester Third Trimester Untreated Treated Surgical 20 20 19 29 29 30 2.43 3.12 3.81 0.693 1.04 1.04 1.04 7.97 1.04 6.93 1.04 6.59 ND 3.47 ND 3.47 ND 2.08 n Median Abs Range
Testosterone, nmol/L Group Females Oral Contraceptives Postmenopausal Males 39 103 99 1.39 1.04 14.2 ND 3.81a ND 2.77L 6.93 28.1c n Median Central 95%
Postmenopausal Females
12
SHBG
Multicenter Ovulatory Cycle Study16
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. SHBG, nmol/L Group Entire Cycle Follicular Phase Midcycle Luteal Phase n 26 (758) 26 (393) 26 (26) 26 (339) Median Central 95% 54 53 52 56 27 109 26 103 11 97 28 112
Additional Ranges
In an independent study, total testosterone, sex hormonebinding globulin (SHBG) and the free androgen index (FAI) were determined in normal cycling women, untreated postmenopausal women, women on oral contraceptives, women with mild to moderately severe hirsutism, and normal males. Note that the criteria adopted for selecting reference groups can have an effect on the resulting reference range limits. In this study, the following criteria were used to define "normal cycling" women: (1) a history of regular menstrual cycles of 25 to 34 days; (2) no obvious obesity, i.e. within about 20 percent of ideal body weight; and (3) no hyperandrogenic symptoms, such as acne, oily skin, or signs of hirsutism. SHBG, nmol/L Group
IMMULITE SHBG (LKSH)
n 18 29 24 50
nmol/L
90
SHBG
90
60
60
30
30
0 -24
-18
-12
-6
12
18
13
Additional Ranges
In an independent study, total testosterone, sex hormonebinding globulin (SHBG) and the free androgen index (FAI) were determined in normal cycling women, untreated postmenopausal women, women on oral contraceptives, women with mild to moderately severe hirsutism, and normal males. Note that the criteria adopted for selecting reference groups can have an effect on the resulting reference range limits. In this study, the following criteria were used to define "normal cycling" women: (1) a history of regular menstrual cycles of 25 to 34 days; (2) no obvious obesity, i.e. within about 20 percent of ideal body weight; and (3) no hyperandrogenic symptoms, such as acne, oily skin, or signs of hirsutism. Free Androgen Index (FAI) Group Females Oral Contraceptives Postmenopausal (untreated) Hirsute 18 29 24 1.2 1.5 5.6 ND 3.4a ND 6.6a 1.7 20.6a 14.8 94.8c n Median Range
15
15
10
10
0 -24
-18
-12
-6
12
18
14
20
10
10
0 -24
-18
-12
-6
12
18
15
10
100
1,000
10,000 100,000
HCG
16
32
Pregnant Females
Gestational Week
A total of 593 serum samples from apparently healthy pregnant women were processed by the IMMULITE HCG assay. The results are summarized below in mIU/mL [3rd IS 75/537] by gestational age (Gest.) equivalently, by weeks since last menstrual period (LMP). Weeks of Pregnancy Gest. 1.32 23 34 45 56 67 711 1116 1621 2139 LMP 3.34 45 56 67 78 89 913 1318 1823 2341 n 30 54 34 34 36 33 116 72 80 104 71 607 5,243 26,983 52,090 93,598 117,678 40,989 20,868 15,352 HCG, mIU/mL Median Central 95% 16 156 101 4,870 1,110 31,500 2,560 82,300 23,100 151,000 27,300 233,000 20,900 291,000 6,140 103,000 4,720 80,100 2,700 78,100
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References
1. Bodlaender P. IMMULITE progesterone: physiology and clinical utility. Los Angeles: Diagnostic Products Corporation, 1995. Technical report ZB134D. Castracane VD, Gimpel T, et al. Circulating human chorionic gonadotropin (hCG) throughout pregnancy as determined by the IMMULITE: comparison of combined cross-sectional statistics with individual trajectories [abstract 276]. Clin Chem 1999;45(S6):A81. Full presentation available at DPC's Web site, www.dpcweb.com, under Technical Documents, Scientific Posters. Harris EK, Boyd JC. Statistical bases of reference values in laboratory medicine. New York: Marcel Dekker, 1995. Kelly JA, Vankrieken L. Sex hormone binding globulin and the assessment of androgen status. Los Angeles: Diagnostic Products Corporation, 1997. Technical report ZB170B. National Committee for Clinical Laboratory Standards. How to define and determine reference intervals in the clinical laboratory; approved guideline. Wayne, PA: NCCLS, 1995. NCCLS Document C28-A. Reichstein E, Knapick M. Negligible HCG carryover on the IMMULITE and IMMULITE 2000. Los Angeles: Diagnostic Products Corporation, 1999. Technical report ZB191-A. Sibley PEC, Vankrieken L, et al. Impact of the menstrual cycle on BR-MA (CA15-3) and OM-MA (CA125) values, as determined by automated chemiluminescent assays on the IMMULITE Analyzer [abstract 385]. Clin Chem 1999;45(S6):A109. Full presentation available at DPC's Web site, www.dpcweb.com, under Technical Documents, Scientific Posters. Sibley PEC. IMMULITE tumor marker assays: multicenter reference range data. Los Angeles: Diagnostic Products Corporation, 1999. Technical report ZB148-D. Sibley PEC. OM-MA (CA125) and ovarian cancer. News & Views (DPC) 1999 Summer;13(3):12-4. Available at DPC's Web site, www.dpcweb.com, under News & Views, Summer 1999. Also available as DPC Technical report ZB195-A.
11. Vankrieken L. Testosterone and the free androgen index. Los Angeles: Diagnostic Products Corporation, 1997. Technical report ZB158A. 12. Vankrieken L, El Shami AS. IMMULITE FSH and Estradiol assays in the luteal-follicular transition period of normal ovulatory cycles [abstract 273]. Clin Chem 1999;45(S6):A80-1. Full presentation available at DPC's Web site, www.dpcweb.com, under Technical Documents, Scientific Posters. 13. Vankrieken L, Pregger, K. Comparison of estradiol results throughout the ovulatory cycle. Los Angeles: Diagnostic Products Corporation, 1999. Technical report ZB193-2 14. Vankrieken L, Reuben H. Hormonal levels during the early follicular phase of the menstrual cycle. Los Angeles: Diagnostic Products Corporation, 1999. Technical report ZB182-A. 15. Vankrieken L, Sibley PEC, Kelly JA. HCG and subunits: DPC assay specificities and clinical utility in obstetrical care and oncology. Los Angeles: Diagnostic Products Corporation, 1998. Technical report ZB174-A. 16. Vankrieken L, Van der Horst FAL, Castracane VD, et al. An international multicenter reference range study of normal menstrual cycles using IMMULITE reproductive hormone assays [abstract 322]. Clin Chem 1999;45(S6):A92. Full presentation available at DPC's Web site, www.dpcweb.com, under Technical Documents, Scientific Posters. 17. Vankrieken L. Choosing a window into the ovulatory cycle. News & Views (DPC) 1999 Winter;13(1):11. Available at DPC's Web site, www.dpcweb.com, under News & Views, Winter 1999, Fertility. 18. Venables WN, Ripley BD. Modern applied statistics with S-PLUS. 3rd ed. New York: Springer-Verlag, 1999. 19. Wilcox RR. Introduction to robust estimation and hypothesis testing. New York: Academic Press, 1997. 20. Wright EM, Royston P. Calculating reference intervals for laboratory measurements. Stat Methods Med Res 1999;8:93-112.
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10. Sibley PEC. Tumor marker assays; the significance of normal range studies. News & Views (DPC) 1999 Fall;13(4):6-8. Available at DPC's Web site, www.dpcweb.com, under News & Views, Fall 1999.
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