D P C T e c h n i c a l R e p o r t

IMMULITE Reproductive Hormone Assays

Multicenter Reference Range Data for Diagnostic Products Corporation Kits Fourth Edition

Leo Vankrieken, Eur. Eng., International Marketing Manager, Reproductive Endocrinology

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.)

Multicenter Ovulatory Cycle Study16,12,7

Laboratories from five countries were involved in this international collaborative study: Belgium, Germany, The Netherlands, the United Kingdom, and the United States. Serum samples were collected from several volunteers at each center, every morning throughout one complete ovulatory cycle, beginning on the first day of menstruation and typically continuing one or even two days into the next cycle. A total of 60 women, all in apparent good health, were enrolled in the study. The results for six women were later eliminated from the analysis because they failed to exhibit basic characteristics of a normal ovulatory cycle: in one case, for example, there was no evidence of a midcycle rise in LH. The 54 women constituting the normal reference group had a median age of 31 years (range: 16 to 44 years), and a median cycle length of 29 days (range: 23 to 35 days). Results obtained by the IMMULITE FSH, LH, and Estradiol assays on the samples from these 54 subjects are summarized in this report. The prolactin results for 53 of the 54 subjects (there was one clear outlier) are also summarized here, even though it is arguable that having a blood sample drawn every day at the same time of day for an entire month makes the longitudinal study design less than ideal for this particular analyte, as a significantly higher frequency of transient and stress-induced prolactin elevations can be expected, compared to more routine clinical settings where there is both less stress and the possibility of repeat sampling should an elevated prolactin result be encountered. (Accordingly, we also report statistics for a cross-section of 115 nonpregnant women. The prolactin result obtained on the first sample collected from each of the subjects in the ovulatory study was included in the analysis. See page 11.) At a later date, the serum samples for half of the subjects (selected at random) were thawed and assayed by the IMMULITE Testosterone, SHBG and Progesterone assays. This made it possible to calculate two derived parameters: the free androgen index (FAI) and the estradiol/SHBG ratio (ESR). Results for these three analytes and the two indices are summarized in the current edition of this report. Nine of the subjects enrolled in the Multicenter Ovulatory Cycle Study were selected at random for a method comparison study. The goal was to assess the 3

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.

Cross-Sectional Pediatric Fertility Study

Serum samples from a pediatric hospital and "wellness" clinic in the southwestern US were processed by the IMMULITE FSH, LH, Progesterone and Prolactin assays. Assay results, along with information on age and sex, were assembled, reviewed and submitted for data analysis under the direction of Dr. William Byrd, University of Texas Southwestern Medical Center, Dallas. The study included 200 samples from children under 10 years of age, as well as 68 cord blood samples: over 80 percent had results by each of the four assays.

Collaborative Study of HCG in Pregnancy2

At one site in the southwestern United States, 145 serum samples were collected from normal singleton pregnancies, each from a different individual, spanning gestational ages from 4 weeks to term. Two additional sets of cross-sectional results obtained with the IMMULITE HCG assay were also used in the analysis: somewhat more recent data generated at a second location; and a data set compiled two or three years earlier from several different laboratories. Altogether, there was a total of 596 samples. An AACC poster presentation now available on DPC's Web site, www.dpcweb.com, under Technical Documents, Scientific Posters explores the consistency of the three data sets and also displays individual trajectories for women who were followed longitudinally throughout most of pregnancy at the first site, for a total of 18 to 23 results per subject.2

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).

IMMULITE 2000 Reproductive Hormone Kits

Kit DHEA-SO4 Estradiol FSH HCG Catalog Number L2KDS L2KE2 L2KFS L2KCG Detection Limit Calibration Range

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

IMMULITE Reproductive Hormone Kits

Kit DHEA-SO4 Estradiol Catalog Detection Number Limit LKDS LKE2 Calibration Range

Total L2KTT Testosterone

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

Unconjugated LKEF Estriol FSH HCG LKFS LKCG

LH

LKLH

0.7 mIU/mL 0.2 ng/mL (0.6 nmol/L)

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)

Total LKTT Testosterone

Data Analysis
S-PLUS 2000 (www.mathsoft.com) was used for most of the calculations, for data visualization, and for the graphs themselves.18

Notes and Disclaimers

Some ranges should be considered preliminary; these are so flagged. Concentration levels below an assay's detection limit are tabulated as ND (not detectable). The results summarized in this document are not all from the same centers or the same patient populations; hence care should be exercised when comparing results. The tabulated centiles represent guidelines only. Each laboratory should establish or verify the appropriateness of adopting reference range limits suggested by this document.5

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.

Menstrual Cycle Plots

The design of the Multicenter Ovulatory Cycle Study, which was based on collecting daily samples from a relatively large number of subjects, allows for displaying and analyzing the results in several complementary ways: that is, both cross-sectionally and longitudinally, and normalized to various points in the menstrual cycle, including midcycle (LH peak), beginning and/or end of cycle, or all three simultaneously. This report utilizes two methods for representing menstrual cycle data. There are conventional plots of concentration against cycle day, with day 0 representing the occasion of each woman's highest LH value. For three of the analytes FSH, estradiol and progesterone there are also plots focusing on the luteal-follicular transition period, with day 1 representing the first day of the new cycle.12,16,17 Examples of a third way of looking at menstrual cycle data (fully normalized Lucas plots), as well as plots of representative individual trajectories, can be found in AACC poster presentations, now available on DPC's Web site.16,7

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

Cross-Sectional Pediatric Fertility Study

Results for a cross-section of (not necessarily normal) infants and children at a pediatric hospital and wellness clinic in the southwestern US. FSH, mIU/mL Group Females Age (yr) Cord 0.1 3 49 Males Cord 0.1 3 49 Combined Cord 0.1 3 49 n 30 57 28 37 72 31 67 129 59 Median Central 95% ND 2.3 0.8 0.24 0.6 0.23 0.11 1.1 0.5 0.11 13 0.11 1.6 ND 1.2 ND 5.5 ND 1.9 ND 1.1 ND 10 ND 1.8

*Number of subjects (total number of results)

IMMULITE FSH (LKFS)

mIU/mL
IU/L

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

Cycle Day: Normalized to LH Peak

IMMULITE FSH (LKFS)

mIU/mL
IU/L 15

Follitropin

15

10

10

5 0

0 -8

-4

-1 1

Cycle Day: Luteal-Follicular Transition

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

Cross-Sectional Pediatric Fertility Study

Results for a cross-section of (not necessarily normal) infants and children at a pediatric hospital and wellness clinic in the southwestern US. LH, mIU/mL Group Females Age (yr) Cord 0.1 1.5 1.6 9 Males Cord 0.1 1.5 1.6 9 Combined Cord 0.1 1.5 1.6 9 n 31 46 38 36 54 46 67 100 84 Median Central 95% ND 0.7 ND ND 1.0 ND ND 0.7 ND ND 2.3 ND 1.3 ND 3.6 ND 4.1 ND 3.8 ND 3.5 ND 3.7 ND 3.2

*Number of subjects (total number of results)

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

Cycle Day: Normalized to LH Peak

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

*Number of subjects (total number of results)

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

IMMULITE Estradiol (LKE2)

pmol/L 1500

Estradiol

400

1000

200

500 0

-24

-18

-12

-6

12

18

34 400 27 246

Cycle Day: Normalized to LH Peak

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

*Number of subjects (total number of results)

IMMULITE Estradiol (LKE2)

pmol/L 1500

Estradiol

400

1000

200

500 0

*Number of subjects (total number of results)

0 -8

-4

-1 1

Cycle Day: Luteal-Follicular Transition

Estradiol/SHBG Ratio (ESR)

See pages 4 and 15.

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

Multicenter Ovulatory Cycle Study16

ng/mL

Progesterone

IMMULITE Progesterone (LKPG)

nmol/L 60 40

20 0

0 -24

-18

-12

-6

12

18

Cycle Day: Normalized to LH Peak

IMMULITE Progesterone (LKPG)

nmol/L

Progesterone

*Number of subjects (total number of results)

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

1.5 5.5 3.0 68 19 76

-4

-1 1

Cycle Day: Luteal-Follicular Transition

*Number of subjects (total number of results)

continued next page. . .

Cross-Sectional Pediatric Fertility Study

Results for a cross-section of (not necessarily normal) infants and children at a pediatric hospital and wellness clinic in the southwestern US. Progesterone, ng/mL Group Females Age (yr) Cord 0.1 0.4 0.5 1 1.1 9 Males Cord 0.1 0.4 0.5 1 1.1 9 Combined Cord 0.1 0.4 0.5 1 1.1 9 n 27 24 19 38 27 33 14 42 54 57 33 80 Median Central 95% 570 1.2 0.8 0.4 520 1.5 0.8 0.4 550 1.5 0.8 0.4 465 755 0.25 17 0.2 1.6 ND 1.4 345 650 0.3 14 ND 2 ND 1.3 350 750 0.25 17 ND 2 ND 1.3 Combined Males Group Females Age (yr) Cord 0.1 0.4 0.5 1 1.1 9 Cord 0.1 0.4 0.5 1 1.1 9 Cord 0.1 0.4 0.5 1 1.1 9 Progesterone (nmol/L) n 27 24 19 38 27 33 14 42 54 57 33 80 Median Central 95% 1,813 3.8 2.5 1.3 1,654 4.8 2.5 1.3 1,749 4.8 2.5 1.3 1,479 2,401 0.8 54 0.6 5.1 ND 4.5 1,097 2,067 1.0 45 ND 6.4 ND 4.1 1,113 2,385 0.8 54 ND 6.4 ND 4.1

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

IMMULITE Prolactin (LKPR)

mIU/L 800

40

400

20

200

10

100

2.5

5 -24

-18

-12

-6

12

18

Multicenter Ovulatory Cycle Study

16

Cycle Day: Normalized to LH Peak

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

Cross-Sectional Pediatric Fertility Study

Results for a cross-section of (not necessarily normal) infants and children at a pediatric hospital and wellness clinic in the southwestern US. Prolactin, ng/mL Group Females 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 Central 95% 380 15 11 295 19 8 340 117 9 200 675 1 140 2 43 150 565 4 65 0.6 29 160 665 2 125 1 40

*Number of subjects (total number of results)

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

*Number of subjects (total number of results)

11

Multicenter Ovulatory Cycle Study16

80

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

IMMULITE Testosterone (LKTT)

nmol/L 4 3 2

40

1 0

0 -24

-18

-12

-6

12

18

Cycle Day: Normalized to LH Peak

*Number of subjects (total number of results)

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

*Number of subjects (total number of results)

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

a: absolute range, c: central 95% range, L: lower 95% 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

a: absolute range, c: central 95% range, L: lower 95% range

Free Androgen Index (FAI)

See pages 4 and 14.

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)

*Number of subjects (total number of results)

n 18 29 24 50

Median 119 63.2 40.6 32.3

Range 56.3 159a 20.2 142a 19.9 84.8a 7.2 100c

Females Oral Contraceptives Postmenopausal (untreated) Hirsute Males

nmol/L

nmol/L

90

SHBG

90

60

60

a: absolute range, c: central 95% range.

30

30

0 -24

-18

-12

-6

12

18

Cycle Day: Normalized to LH Peak

FAI and ESR

For derived parameters the free androgen index and the estradiol/SHBG ratio see pages 4, 14 and 15.

13

Free Androgen Index (FAI)

The Free Androgen Index is defined here as 100 times the molar ratio of total testosterone to SHBG.
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)

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

Multicenter Ovulatory Cycle Study16

Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. Free Androgen Index (FAI) Group Entire Cycle Follicular Phase Midcycle Luteal Phase n 26 (758) 26 (393) 26 (26) 26 (339) Median Central 95% 2.9 3.1 3.6 2.8 0.8 10 0.8 9.3 1.3 17 0.8 11

*Number of subjects (total number of results)

IMMULITE FAI (LKTT/LKSH)

M/M
M/M

50 35.0 Males a: absolute range, c: central 95% range.

Free Androgen Index (FAI)

15

15

10

10

0 -24

-18

-12

-6

12

18

Cycle Day: Normalized to LH Peak

14

Estradiol/SHBG Ratio (ESR)

The Estradiol/SHBG Ratio is defined here as 1000 times the molar ratio of estradiol to SHBG.
Estradiol/SHBG Ratio (ESR): Estradiol (in pmol/L) / SHBG (in nmol/L) = 3.671 Estradiol (in pg/mL) / SHBG (in nmol/L)
Estradiol / SHBG

IMMULITE E2/SHBG Ratio (LKE2/LKSH)

M/M
M/M 20

Multicenter Ovulatory Cycle Study16

Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. Note: Because it was available as an obvious by-product of measuring estradiol and SHBG throughout the menstrual cycle, we have included results for a derived parameter analogous to the free androgen index (FAI), but based on the molar ratio of estradiol (rather than testosterone) to SHBG. The clinical usefulness of this parameter in any context remains to be established: it is clear from the literature that the estradiol/SHBG ratio (ESR) or free estrogen index has never achieved a central role in laboratory medicine comparable to that of the FAI, though it continues to figure in occasional studies of a diverse nature. A few representative articles are listed at the end of this document. DPC hopes that the ESR data summarized in this document may prove of some value in a research setting; and we welcome feedback on the potential use and limitations of this ratio. Estradiol/SHBG Ratio (ESR) Group Entire Cycle Follicular Phase Midcycle Luteal Phase n 27 (786) 27 (409) 27 (27) 27 (350) Median Central 95% 5.1 3.4 15.4 6.4 1.4 20 1.2 21 5.6 32 1.8 17

20

10

10

0 -24

-18

-12

-6

12

18

Cycle Day: Normalized to LH Peak

*Number of subjects (total number of results)

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IMMULITE HCG, IU/L [3rd IS 75/537]

Males and Nonpregnant Females

In a study performed with the IMMULITE HCG assay and involving serum samples from 797 adult volunteers, including 428 males and 369 nonpregnant females, 95% of the results for males were below the assay's detection limit (1.1 mIU/mL) and all were below 2.5 mIU/mL, while 83% of the results for nonpregnant females were below the detection limit, 95% were below 2.7 mIU/mL, and all were below 5.3 mIU/mL. The subjects were in apparent good health based on a questionnaire.

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.

2.

3.

4.

5.

6.

7.

8.

9.

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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Additional References: ESR

Listed below, in reverse chronological order by publication date, are few fairly recent articles which, in one way or another, looked at the estradiol/SHBG ratio (ESR) sometimes referred to as the free estrogen index. Gillberg P, Johansson AG, Ljunghall S. Decreased estradiol levels and free androgen index and elevated sex hormone-binding globulin levels in male idiopathic osteoporosis. Calcif Tissue Int 1999;64:209-13. Knochenhauer ES, Boots LR, Potter HD, Azziz R. Differential binding of estradiol and testosterone to SHBG. Relation to circulating estradiol levels. J Reprod Med 1998;43:665-70. Lyons A, Ng C, Fielding K, Hosking D, Selby C, Lawson N. Pituitary dysfunction in recently postmenopausal women. Nottingham EPIC Study Group. Clin Endocrinol (Oxf) 1997;47:431-8. Comment in: Clin Endocrinol (Oxf) 1998;48:124. Makarainen L, Ronnberg L, Kauppila A. Medroxyprogesterone acetate supplementation diminishes the hypoestrogenic side effects of gonadotropin-releasing hormone agonist without changing its efficacy in endometriosis. Fertil Steril 1996;65:29-34. Isojarvi JI, Laatikainen TJ, Pakarinen AJ, Juntunen KT, Myllyla VV. Menstrual disorders in women with epilepsy receiving carbamazepine. Epilepsia 1995;36:676-81. Morimoto I. [Gonadal dysfunctions in liver cirrhosis.] Nippon Rinsho 1994;52:132-7. [Japanese] Birkeland KI, Hanssen KF, Torjesen PA, Vaaler S. Level of sex hormone-binding globulin is positively correlated with insulin sensitivity in men with type 2 diabetes. J Clin Endocrinol Metab 1993;76:275-8. Comment in: J Clin Endocrinol Metab 1993;76:273-4. Chobanian NS, Kushlinskii NE, Bassalyk LS, Kuz'mina ZV, Savel'eva EV, Baryshnikov AIu, Kadagidze ZG. [Estradiol-17beta, sex steroid-binding globulin and the cellular and humoral immunity indices in young women with breast cancer.] Vopr Onkol 1992;38:439-46. [Russian] Daniel M, Martin AD, Drinkwater DT. Cigarette smoking, steroid hormones, and bone mineral density in young women. Calcif Tissue Int 1992;50:300-5. Sewdarsen M, Vythilingum S, Jialal I, Desai RK, Becker P. Abnormalities in sex hormones are a risk factor for premature manifestation of coronary artery disease in South African Indian men. Atherosclerosis 1990;83:111-7.

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