SHBG

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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

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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.

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

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 cross-sectionally 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.

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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 Study

16,12,7 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

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 Pregnancy

2 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

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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

®

Reproductive Hormone Kits

Kit Catalog Number Detection Limit Calibration Range DHEA-SO4 LKDS 2 µg/dL ( 0.054 µmol/L) 30 – 1,000 µg/dL (0.81 – 27 µmol/L) Estradiol LKE2 12 pg/mL (44 pmol/L) 20 – 2,000 pg/mL (73 – 7,342 pmol/L) Unconjugated Estriol LKEF 0.2 ng/mL (0.7 nmol/L) 0.25 – 30 ng/mL (0.87 – 104 nmol/L) FSH LKFS 0.1 mIU/mL Up to 170 mIU/mL (2nd IRP 78/549) HCG LKCG 1.1 mIU/mL Up to 5,000 mIU/mL (3rd IS 75/537) LH LKLH 0.7 mIU/mL Up to 200 mIU/mL (1st IRP 68/40 & 2nd IRP 80/552) Progesterone LKPG 0.2 ng/mL (0.6 nmol/L) 0.2 – 40 ng/mL (0.6 – 127 nmol/L) Prolactin LKPR 0.5 ng/mL (10.6 mIU/L) Up to 150 ng/mL (Up to 3,180 mIU/L, 3rd IS 84/500) SHBG LKSH 0.2 nmol/L Up to 180 nmol/L Total Testosterone LKTT 10 ng/dL (0.3 nmol/L) 20 – 1600 ng/dL (0.7 – 55 nmol/L)

IMMULITE

®

2000 Reproductive Hormone Kits

Kit Catalog Number Detection Limit Calibration Range DHEA-SO4 L2KDS 1.4 µg/dL (0.038 µmol/L) 30 – 1,000 µg/dL (0.81 – 27 µmol/L) Estradiol L2KE2 10 pg/mL (37 pmol/L) 20 – 2,000 pg/mL (73 – 7,342 pmol/L) FSH L2KFS 0.1 mIU/mL Up to 170 mIU/mL (2nd IRP 78/549) HCG L2KCG 0.4 mIU/mL Up to 5,000 mIU/mL (3rd IS 75/537) LH L2KLH 0.007 mIU/mL Up to 200 mIU/mL (1st IRP 68/40 & 2nd IRP 80/552)

Progesterone L2KPG 0.2 ng/mL_{(0.6 nmol/L)} 0.2 – 40 ng/mL_{(0.6 – 127 nmol/L)}

Prolactin L2KPR 0.16 ng/mL_{(3.4 mIU/L)} Up to 150 ng/mL (Up to 3,180 mIU/L, 3rd IS 84/500) SHBG L2KSH 0.02 nmol/L Up to 180 nmol/L Total Testosterone L2KTT 10 ng/dL (0.3 nmol/L) 20 – 1600 ng/dL (0.7 – 55 nmol/L)

Conversion Factors

Analyte Conversion to Alternate Units

DHEA-SO4 µg/dL × 0.02714 → µmol/L Estradiol pg/mL × 3.671 → pmol/L Progesterone ng/mL × 3.18 → nmol/L Prolactin ng/mL × 21.2 → mIU/L Testosterone ng/dL × 0.03467 → 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)

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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.

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

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

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FSH (Follitropin)

Multicenter Ovulatory Cycle Study

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Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.

FSH, mIU/mL Ovulatory Cycles n* Median Central 95% Follicular Phase 54 (762) 6.2 2.8 – 11.3 Follicular Phase, Days 2 to 3 54 (108) 6.6 3.0 – 14.4 Midcycle 54 (54) 13.6 5.8 – 21 Luteal Phase 54 (604) 3.4 1.2 – 9.0

*Number of subjects (total number of results)

IMMULITE FSH (LKFS)

Cycle Day: Normalized to LH Peak

-24 -18 -12 -6 0 6 12 18 mIU/mL Follitropin 0 5 10 15 IU/L 0 5 10 15 IMMULITE FSH (LKFS)

Cycle Day: Luteal-Follicular Transition

-8 -4 -1 1 4 8 mIU/mL Follitropin 0 5 10 15 IU/L 0 5 10 15

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 Age (yr) n Median Central 95%

Females Cord 30 ND 0.1 – 3 57 2.3 0.11 – 13 4 – 9 28 0.8 0.11 – 1.6 Males Cord 37 0.24 ND – 1.2 0.1 – 3 72 0.6 ND – 5.5 4 – 9 31 0.23 ND – 1.9 Combined Cord 67 0.11 ND – 1.1 0.1 – 3 129 1.1 ND – 10 4 – 9 59 0.5 ND – 1.8

Additional Ranges

FSH, mIU/mL

Group n Median Central 95%

Postmenopausal* 76 90.5 21.7 – 153

Males 135 3.8 0.7 – 11.1

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LH (Lutropin)

Multicenter Ovulatory Cycle Study

16

LH, mIU/mL Ovulatory Cycles n* Median Central 95% Follicular Phase 54 (762) 4.6 1.1 – 11.6 Midcycle 54 (54) 39 17 – 77 Luteal Phase 54 (658) 4.3 ND – 14.7 Perimenstrual, ± 9 days 54 (959) 3.9 ND – 12.0

IMMULITE LH (LKLH)

-24 -18 -12 -6 0 6 12 18 mIU/mL Lutropin 0 20 40 60 IU/L 0 20 40 60

LH/FSH Ratio

LH / FSH Ratio Ovulatory Cycles n* Median Central 95% Follicular Phase, Days 2 to 5 54 (221) 0.60 0.15 – 1.51 Follicular Phase, Days 2 to 9 54 (436) 0.66 0.18 – 1.64 Follicular Phase, Days 2 to 11 up to 5 days before LH Peak

54 (452) 0.66 0.18 – 1.45

Cross-Sectional Pediatric Fertility Study

LH, mIU/mL

Females Cord 31 ND 0.1 – 1.5 46 0.7 ND – 2.3 1.6 – 9 38 ND ND – 1.3 Males Cord 36 ND ND – 3.6 0.1 – 1.5 54 1.0 ND – 4.1 1.6 – 9 46 ND ND – 3.8 Combined Cord 67 ND ND – 3.5 0.1 – 1.5 100 0.7 ND – 3.7 1.6 – 9 84 ND ND – 3.2

Additional Ranges

LH, mIU/mL

Postmenopausal* 75 24.9 11.3 – 39.8

Males 135 2.4 0.8 – 7.6

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Estradiol

Multicenter Ovulatory Cycle Study

16

Estradiol, pg/mL Ovulatory Cycles n* Median Central 95% Follicular Phase 54 (708) 42 ND – 160 Follicular Phase, Days 2 to 3 54 (108) 31 ND – 84 Periovulatory, ± 3 days 54 (378) 133 34 – 400 Luteal Phase 54 (604) 93 27 – 246

Estradiol, pmol/L Ovulatory Cycles n* Median Central 95% Follicular Phase 54 (708) 154 ND – 587 Follicular Phase, Days 2 to 3 54 (108) 114 ND – 308 Periovulatory, ± 3 days 54 (378) 489 124 – 1468 Luteal Phase 54 (604) 343 101 – 905

Estradiol/SHBG Ratio (ESR)

See pages 4 and 15.

IMMULITE Estradiol (LKE2)

-24 -18 -12 -6 0 6 12 18 pg/mL Estradiol 0 200 400 pmol/L 0 500 1000 1500

IMMULITE Estradiol (LKE2)

-8 -4 -1 1 4 8 pg/mL Estradiol 0 200 400 pmol/L 0 500 1000 1500

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Progesterone

Multicenter Ovulatory Cycle Study

16

Progesterone, ng/mL Ovulatory Cycles n* Median Central 95% Follicular Phase 27 (382) 0.47 ND – 1.13 Midfollicular Days 5 to 11 27 (186) 0.43 ND – 0.98 Midcycle 27 (27) 1.06 0.48 – 1.72 Luteal Phase 27 (323) 8.9 0.95 – 21 Midluteal, Days 7 to 8 of Luteal Phase 27 (54) 13.1 6.0 – 24

Progesterone, nmol/L Ovulatory Cycles n* Median Central 95% Follicular Phase 27 (382) 1.5 ND – 3.6 Midfollicular Days 5 to 11 27 (186) 1.4 ND – 3.1 Midcycle 27 (27) 3.4 1.5 – 5.5 Luteal Phase 27 (323) 28 3.0 – 68 Midluteal, Days 7 to 8 of Luteal Phase 27 (54) 42 19 – 76

IMMULITE Progesterone (LKPG)

-24 -18 -12 -6 0 6 12 18 ng/mL Progesterone 0 7 14 21 nmol/L 0 20 40 60 IMMULITE Progesterone (LKPG)

-8 -4 -1 1 4 8 ng/mL Progesterone 0 10 20 nmol/L 0 30 60

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Cross-Sectional Pediatric Fertility Study

Progesterone, ng/mL

Females Cord 27 570 465 – 755 0.1 – 0.4 24 1.2 0.25 – 17 0.5 – 1 19 0.8 0.2 – 1.6 1.1 – 9 38 0.4 ND – 1.4 Males Cord 27 520 345 – 650 0.1 – 0.4 33 1.5 0.3 – 14 0.5 – 1 14 0.8 ND – 2 1.1 – 9 42 0.4 ND – 1.3 Combined Cord 54 550 350 – 750 0.1 – 0.4 57 1.5 0.25 – 17 0.5 – 1 33 0.8 ND – 2 1.1 – 9 80 0.4 ND – 1.3 Progesterone (nmol/L)

Females Cord 27 1,813 1,479 – 2,401 0.1 – 0.4 24 3.8 0.8 – 54 0.5 – 1 19 2.5 0.6 – 5.1 1.1 – 9 38 1.3 ND – 4.5 Males Cord 27 1,654 1,097 – 2,067 0.1 – 0.4 33 4.8 1.0 – 45 0.5 – 1 14 2.5 ND – 6.4 1.1 – 9 42 1.3 ND – 4.1 Combined Cord 54 1,749 1,113 – 2,385 0.1 – 0.4 57 4.8 0.8 – 54 0.5 – 1 33 2.5 ND – 6.4 1.1 – 9 80 1.3 ND – 4.1

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Prolactin

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.

Multicenter Ovulatory Cycle Study

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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 n* Mean Central 95%

Entire Cycle 53 (1555) 13.0 4.6 – 37 Follicular Phase 53 (803) 12.1 4.5 – 33

Midcycle 53 (53) 17 6.3 – 46

Luteal Phase 53 (699) 13.9 4.9 – 40

Prolactin, mIU/L

Ovulatory Cycles n* Mean Central 95%

Entire Cycle 53 (1555) 276 98 – 784

Follicular Phase 53 (803) 257 95 – 700

Midcycle 53 (53) 360 134 – 975

Luteal Phase 53 (699) 295 104 – 848

IMMULITE Prolactin (LKPR)

-24 -18 -12 -6 0 6 12 18 ng/mL Prolactin 2.5 5 10 20 40 mIU/L 100 200 400 800

Cross-Sectional Pediatric Fertility Study

Prolactin, ng/mL

Females Cord 28 380 200 – 675 0.1 – 0.5 28 15 1 – 140 0.6 – 9 55 11 2 – 43 Males Cord 27 295 150 – 565 0.1 – 0.5 36 19 4 – 65 0.6 – 9 55 8 0.6 – 29 Combined Cord 55 340 160 – 665 0.1 – 0.5 64 117 2 – 125 0.6 – 9 110 9 1 – 40 Prolactin, mIU/L

Group Age (yr) n Median Central 95%

Females Cord 28 8,056 4,240 – 14,310 0.1 – 0.5 28 318 21 – 2,968 0.6 – 9 55 233 42 – 912 Males Cord 27 6,254 3,180 – 11,978 0.1 – 0.5 36 403 85 – 1,378 0.6 – 9 55 170 13 – 615 Combined Cord 55 7,208 3,392 – 14,098 0.1 – 0.5 64 2,480 42 – 2,650 0.6 – 9 110 191 21 – 848

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Total Testosterone

Multicenter Ovulatory Cycle Study

16

Testosterone, ng/dL

Entire Cycle 26 (758) 47 ND – 118

Follicular Phase 26 (393) 48 ND – 118

Midcycle 26 (26) 58 21 – 104

Luteal Phase 26 (339) 44 ND – 119

Testosterone, nmol/L

Entire Cycle 26 (758) 1.6 ND – 4.1

Follicular Phase 26 (393) 1.7 ND – 4.1

Midcycle 26 (26) 2.0 0.7 – 3.6

Luteal Phase 26 (339) 1.5 ND – 4.1

Additional Ranges

Females

Oral Contraceptives 39 40 ND – 110a

Postmenopausal 103 30 ND – 80L

Males 99 410 200 – 810c

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

Testosterone, nmol/L

Females

Oral Contraceptives 39 1.39 ND – 3.81a

Postmenopausal 103 1.04 ND – 2.77L

Males 99 14.2 6.93 – 28.1c

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

IMMULITE Testosterone (LKTT)

-24 -18 -12 -6 0 6 12 18 ng/dL Total Testosterone 0 40 80 nmol/L 0 1 2 3 4

One of these sites also measured total testosterone levels in pregnant and postmenopausal women, with the following results.

Group n Median Abs Range

Pregnant Females First Trimester 20 70 30 – 230 Second Trimester 20 90 30 – 200 Third Trimester 19 110 30 – 190 Postmenopausal Females Untreated 29 20 ND – 100 Treated 29 30 ND – 100 Surgical 30 30 ND – 60 Testosterone, nmol/L

Group n Median Abs Range

Pregnant Females First Trimester 20 2.43 1.04 – 7.97 Second Trimester 20 3.12 1.04 – 6.93 Third Trimester 19 3.81 1.04 – 6.59 Postmenopausal Females Untreated 29 0.693 ND – 3.47 Treated 29 1.04 ND – 3.47 Surgical 30 1.04 ND – 2.08

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SHBG

Multicenter Ovulatory Cycle Study

16

SHBG, nmol/L

Entire Cycle 26 (758) 54 27 – 109

Follicular Phase 26 (393) 53 26 – 103

Midcycle 26 (26) 52 11 – 97

Luteal Phase 26 (339) 56 28 – 112

IMMULITE SHBG (LKSH)

-24 -18 -12 -6 0 6 12 18 nmol/L SHBG 0 30 60 90 nmol/L 0 30 60 90

FAI and ESR

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

Additional Ranges

In an independent study, total testosterone, sex hormone-binding 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 n Median Range

Females Oral Contraceptives 18 119 56.3 – 159a Postmenopausal (untreated) 29 63.2 20.2 – 142a Hirsute 24 40.6 19.9 – 84.8a Males 50 32.3 7.2 – 100c

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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)

Multicenter Ovulatory Cycle Study

16

Free Androgen Index (FAI)

Entire Cycle 26 (758) 2.9 0.8 – 10

Follicular Phase 26 (393) 3.1 0.8 – 9.3

Midcycle 26 (26) 3.6 1.3 – 17

Luteal Phase 26 (339) 2.8 0.8 – 11

IMMULITE FAI (LKTT/LKSH)

-24 -18 -12 -6 0 6 12 18

M/M

Free Androgen Index (FAI)

0 5 10 15 M/M 0 5 10 15

Additional Ranges

In an independent study, total testosterone, sex hormone-binding 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 n Median Range

Females Oral Contraceptives 18 1.2 ND – 3.4a Postmenopausal (untreated) 29 1.5 ND – 6.6a Hirsute 24 5.6 1.7 – 20.6a Males 50 35.0 14.8 – 94.8c

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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)

Multicenter Ovulatory Cycle Study

16

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)

Entire Cycle 27 (786) 5.1 1.4 – 20

Follicular Phase 27 (409) 3.4 1.2 – 21

Midcycle 27 (27) 15.4 5.6 – 32

Luteal Phase 27 (350) 6.4 1.8 – 17

IMMULITE E2/SHBG Ratio (LKE2/LKSH)

-24 -18 -12 -6 0 6 12 18 M/M Estradiol / SHBG 0 10 20 M/M 0 10 20

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HCG

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.

Pregnant Females

2

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 HCG, mIU/mL

Gest. LMP n Median Central 95%

1.3–2 3.3–4 30 71 16 – 156 2–3 4–5 54 607 101 – 4,870 3–4 5–6 34 5,243 1,110 – 31,500 4–5 6–7 34 26,983 2,560 – 82,300 5–6 7–8 36 52,090 23,100 – 151,000 6–7 8–9 33 93,598 27,300 – 233,000 7–11 9–13 116 117,678 20,900 – 291,000 11–16 13–18 72 40,989 6,140 – 103,000 16–21 18–23 80 20,868 4,720 – 80,100 21–39 23–41 104 15,352 2,700 – 78,100 Gestational Week 2 4 8 16 32 IMMULITE HCG, IU/L [3rd IS 75/537] 10 100 1,000 10,000 100,000

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References

1. Bodlaender P. IMMULITE® progesterone: physiology and clinical utility. Los Angeles: Diagnostic Products Corporation, 1995. Technical report ZB134–D.

2. 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.

3. Harris EK, Boyd JC. Statistical bases of reference values in laboratory medicine. New York: Marcel Dekker, 1995.

4. Kelly JA, Vankrieken L. Sex hormone binding globulin and the assessment of androgen status. Los Angeles: Diagnostic Products Corporation, 1997. Technical report ZB170–B.

5. 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.

6. Reichstein E, Knapick M. Negligible HCG carryover on the IMMULITE® and IMMULITE® 2000. Los Angeles: Diagnostic Products Corporation, 1999. Technical report ZB191-A.

7. 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.

8. Sibley PEC. IMMULITE® tumor marker assays: multicenter reference range data. Los Angeles: Diagnostic Products Corporation, 1999. Technical report ZB148-D.

9. 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.

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.

11. Vankrieken L. Testosterone and the free androgen index. Los Angeles: Diagnostic Products Corporation, 1997. Technical report ZB158–A.

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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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 post-menopausal 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,

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Diagnostic Products Corporation 5700 West 96th Street Los Angeles, CA 90045-5597 Tel: 800.372.1782 Tel: 310.645.8200 Fax: 310.645.9999

SHBG

IMMULITE

Reproductive Hormone Assays

Multicenter Reference Range Data

for Diagnostic Products Corporation Kits

Fourth Edition

IMMULITE

Reproductive Hormone Assays: Multicenter Reference Range Data

for Diagnostic Products Corporation Kits

Preface

Table of Contents

Analytes

What’s New

Subjects

Multicenter Ovulatory Cycle Study

Cross-Sectional Pediatric Fertility Study

Collaborative Study of HCG in Pregnancy

Methods

IMMULITE

Reproductive Hormone Kits

IMMULITE

2000 Reproductive Hormone Kits

Conversion Factors

Derived Parameters

Data Analysis

Centiles

Menstrual Cycle Plots

Notes and Disclaimers

FSH (Follitropin)

Multicenter Ovulatory Cycle Study

Cross-Sectional Pediatric Fertility Study

Additional Ranges

LH (Lutropin)

Multicenter Ovulatory Cycle Study

LH/FSH Ratio

Cross-Sectional Pediatric Fertility Study

Additional Ranges

Estradiol

Multicenter Ovulatory Cycle Study

Estradiol/SHBG Ratio (ESR)

Progesterone

Multicenter Ovulatory Cycle Study

Cross-Sectional Pediatric Fertility Study

Prolactin

Nonpregnant Women

Multicenter Ovulatory Cycle Study

Cross-Sectional Pediatric Fertility Study

Total Testosterone

Multicenter Ovulatory Cycle Study

Additional Ranges

SHBG

Multicenter Ovulatory Cycle Study

FAI and ESR

Additional Ranges

Free Androgen Index (FAI)

Multicenter Ovulatory Cycle Study

Additional Ranges

Estradiol/SHBG Ratio (ESR)

Multicenter Ovulatory Cycle Study

HCG

Males and Nonpregnant Females

Pregnant Females

References

Additional References: ESR

_{Reproductive Hormone Assays: Multicenter Reference Range Data}