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

2

Preface

This report is a compendium of reference range results andrelated 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, convenientformat—the best available information on the values to beexpected 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.

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

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 nowavailable 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 themenstrual 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;

4,11another

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 PDFformat.

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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.)

Mul ticenter 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 thenext 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, andEstradiol 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 dayat 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 threeanalytes 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 methodcomparison study. The goal was to assess the

transferability to a representative IMMULITE 2000 assay

of reference ranges established for the correspondingIMMULITE 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 Pediatr ic Ferti li ty Study Serum samples from a pediatric hospital and "wellness"

clinic in the southwestern US were processed by theIMMULITE 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 setcompiled 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 threedata 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® 2000assays 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/FSHratio, the free androgen index (FAI), and the

estradiol/SHBG ratio (ESR).

IMMULITE ®

Reproductive Hormone Ki ts

KitCatalogNumber

DetectionLimit

CalibrationRange

DHEA-SO4 LKDS2 µg/dL

( 0.054µmol/L)

30 – 1,000 µg/dL

(0.81 – 27 µmol/L)

Estradiol LKE212 pg/mL(44 pmol/L)

20 – 2,000 pg/mL(73 – 7,342 pmol/L)

UnconjugatedEstriol

LKEF0.2 ng/mL(0.7 nmol/L)

0.25 – 30 ng/mL(0.87 – 104 nmol/L)

FSH LKFS 0.1 mIU/mLUp to 170 mIU/mL(2nd IRP 78/549)

HCG LKCG 1.1 mIU/mLUp to 5,000 mIU/mL(3rd IS 75/537)

LH LKLH 0.7 mIU/mLUp to 200 mIU/mL(1st IRP 68/40 &2nd IRP 80/552)

Progesterone LKPG0.2 ng/mL(0.6 nmol/L)

0.2 – 40 ng/mL(0.6 – 127 nmol/L)

Prolactin LKPR0.5 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

TotalTestosterone

LKTT10 ng/dL(0.3 nmol/L)

20 – 1600 ng/dL(0.7 – 55 nmol/L)

IMMULITE ®

2000 Reproductive Hormone Ki ts

KitCatalogNumber

DetectionLimit

CalibrationRange

DHEA-SO4 L2KDS1.4 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/mLUp to 170 mIU/mL(2nd IRP 78/549)

HCG L2KCG 0.4 mIU/mLUp to 5,000 mIU/mL(3rd IS 75/537)

LH L2KLH 0.007 mIU/mLUp to 200 mIU/mL(1st IRP 68/40 &2nd IRP 80/552)

Progesterone L2KPG0.2 ng/mL(0.6 nmol/L)

0.2 – 40 ng/mL(0.6 – 127 nmol/L)

Prolactin L2KPR0.16 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

TotalTestosterone

L2KTT10 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

Deri ved 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)

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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 suitabletransformation to improve symmetry.

In the Multicenter Ovulatory Cycle Study, statistics werecalculated 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 displayingand 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—thereare 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; hencecare should be exercised when comparing results.

The tabulated centiles represent guidelines only. Eachlaboratory should establish or verify the appropriateness of

adopting reference range limits suggested by this

document.5

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FSH (Follitropin)

Mul ticenter Ovulatory Cycle Study 16

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

m I U / m L

F o

l l i t r o p

i n

0

5

1 0

1 5

I U / L

0

5

1 0

1 5

IMMULITE FSH (LKFS)

Cycle Day: Luteal-Follicular Transition-8 -4 -1 1 4 8

m I U / m L

F o

l l i t r o p

i n

0

5

1 0

1 5

I U / L

0

5

1 0

1 5

Cross-Sectional Pediatr ic Ferti li ty 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

Additi onal Ranges

FSH, mIU/mL

Group n Median Central 95%

Postmenopausal* 76 90.5 21.7 – 153

Males 135 3.8 0.7 – 11.1

*Preliminary

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LH (Lutropin)





LH, mIU/mL

Ovulatory Cycles n* Median Central 95%


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)


m I U / m L

L u

t r o p

i n

0

2 0

4 0

6 0

I U / L

0

2 0

4 0

6 0

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 Pediatr ic Ferti li ty Study Results for a cross-section of (not necessarily normal)



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

Additi onal Ranges

LH, mIU/mL


Postmenopausal* 75 24.9 11.3 – 39.8

Males 135 2.4 0.8 – 7.6

*Preliminary

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Estradiol





Estradiol, pg/mL


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



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)


p g / m L

E s

t r a d i o l

0

2 0 0

4 0 0

p m

o l / L

0

5 0 0

1 0 0 0

1 5 0 0

IMMULITE Estradiol (LKE2)


p g / m

L

E s

t r a d i o l

0

2 0 0

4 0 0

p m

o l / L

0

5 0 0

1 0 0 0

1 5 0 0

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Progesterone





Progesterone, ng/mL


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



Midfollicular

Days 5 to 1127 (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)


n g / m L

P r o g e s

t e r

o n e

0

7

1 4

2 1

n m

o l / L

0

2 0

4 0

6 0

IMMULITE Progesterone (LKPG)


n g / m L

P r o g e s

t e r o n e

0

1 0

2 0

n m

o l / L

0

3 0

6 0

continued next page. . .

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10

Cross-Sectional Pediatr ic Fertil ity Study Results for a cross-section of (not necessarily normal)



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.





Note: As explained on page 3, the increasingly stressfulsample collection process entailed by the longitudinal

design of this study can be expected to yield a higher frequency of prolactin elevations than a cross-sectionalstudy 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*Number of subjects (total number of results)

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)


n g / m L

P r o

l a c t

i n

2 . 5

5

1 0

2 0

4 0

m I U

/ L

1 0 0

2 0 0

4 0 0

8 0 0

Cross-Sectional Pediatr ic Ferti li ty Study

Results for a cross-section of (not necessarily normal)

infants and children at a pediatric hospital and wellnessclinic in the southwestern US.

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


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





Testosterone, ng/dL


Entire Cycle 26 (758) 47 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


Midcycle 26 (26) 2.0 0.7 – 3.6

Luteal Phase 26 (339) 1.5 ND – 4.1


Addit ional Ranges

Testosterone, ng/dL


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



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

Free Androgen I ndex (FAI ) See pages 4 and 14.

IMMULITE Testosterone (LKTT)


n g / d L

T o

t a l T e s

t o s

t e r o n e

0

4 0

8 0

n m

o l / L

0

1

2

3

4

One of these sites also measured total testosterone levels

in pregnant and postmenopausal women, with the

following results.

Testosterone, ng/dL

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


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





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)


n m o l / L

S H B G

0

3 0

6 0

9 0

n m

o l / L

0

3 0

6 0

9 0

FAI and ESR For derived parameters—the free androgen index and the

estradiol/SHBG ratio—see pages 4, 14 and 15.

Additi onal 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

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

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14

Free Androgen I ndex (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)


Results from an international study involving apparentlynormal, ovulating women who had daily reproductive


Free Androgen Index (FAI)


Entire Cycle 26 (758) 2.9 0.8 – 10


Midcycle 26 (26) 3.6 1.3 – 17

Luteal Phase 26 (339) 2.8 0.8 – 11


IMMULITE FAI (LKTT/LKSH)


M / M

F r e e

A n

d r o g e n

I n d e

x ( F A I )

0

5

1 0

1 5

M / M

0

5

1 0

1 5

Additi onal 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 oralcontraceptives, 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 rangelimits. 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

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

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15

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)


Results from an international study involving apparentlynormal, ovulating women who had daily reproductive


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 isclear 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)


M / M

E s

t r a d i o l / S H B G

0

1 0

2 0

M / M

0

1 0

2 0

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16

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, byweeks 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 Week2 4 8 16 32

I M M U L I T E H C G

, I U / L [ 3 r d

I S 7 5 / 5 3 7 ]

1 0

1 0 0

1 , 0

0 0

1 0 , 0

0 0

1 0 0 , 0

0 0

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17

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 humanchorionic 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 TechnicalDocuments, Scientific Posters.

3. Harris EK, Boyd JC. Statistical bases of referencevalues in laboratory medicine. New York: Marcel

Dekker, 1995.

4. Kelly JA, Vankrieken L. Sex hormone binding

globulin and the assessment of androgen status. LosAngeles: 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 TechnicalDocuments, Scientific Posters.

8. Sibley PEC. IMMULITE®

tumor marker assays:multicenter reference range data. Los Angeles:

Diagnostic Products Corporation, 1999. Technicalreport 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 availableat DPC's Web site, www.dpcweb.com , under Technical Documents, Scientific Posters.

13. Vankrieken L, Pregger, K. Comparison of estradiolresults throughout the ovulatory cycle. Los Angeles:


report ZB193-2

14. Vankrieken L, Reuben H. Hormonal levels during theearly 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:


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.

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cycle. News & Views (DPC) 1999 Winter;13(1):11.Available at DPC's Web site, www.dpcweb.com,under News & Views, Winter 1999, Fertility.

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20. Wright EM, Royston P. Calculating reference

intervals for laboratory measurements. Stat MethodsMed Res 1999;8:93-112.

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18

Addit ional 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. Decreasedestradiol levels and free androgen index and elevated

sex hormone-binding globulin levels in male

idiopathic osteoporosis. Calcif Tissue Int1999;64:209-13.

• Knochenhauer ES, Boots LR, Potter HD, Azziz R.

Differential binding of estradiol and testosterone to

SHBG. Relation to circulating estradiol levels. JReprod 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 Steril1996;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 2diabetes. 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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