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
What’s New
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.
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.
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.
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
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.
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.)
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
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
Methods...................................................................4 Data Analysis...........................................................5 Disclaimers ..............................................................5
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).
References ............................................................17
Analytes Follitropin (FSH).......................................................6 Lutropin (LH) ...........................................................7
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.
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
2
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.
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.
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.
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).
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.
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.)
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
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
IMMULITE® 2000 Reproductive Hormone Kits
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.
Kit
Catalog Number
Detection Limit
Calibration Range
DHEA-SO4
L2KDS
1.4 µg/dL 30 – 1,000 µg/dL (0.038 µmol/L) (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
Up to 200 mIU/mL 0.007 mIU/mL (1st IRP 68/40 & 2nd IRP 80/552)
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).
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)
IMMULITE® Reproductive Hormone Kits
SHBG
L2KSH
0.02 nmol/L
Up to 180 nmol/L
Total L2KTT Testosterone
10 ng/dL (0.3 nmol/L)
20 – 1600 ng/dL (0.7 – 55 nmol/L)
Kit
Catalog Detection Number Limit
DHEA-SO4
LKDS
2 µg/dL 30 – 1,000 µg/dL ( 0.054 µmol/L) (0.81 – 27 µmol/L)
LKE2
12 pg/mL (44 pmol/L)
20 – 2,000 pg/mL (73 – 7,342 pmol/L)
Unconjugated LKEF Estriol
0.2 ng/mL (0.7 nmol/L)
0.25 – 30 ng/mL (0.87 – 104 nmol/L)
Estradiol
Calibration Range
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
Up to 150 ng/mL 0.5 ng/mL (Up to 3,180 mIU/L, (10.6 mIU/L) 3rd IS 84/500)
SHBG
LKSH
0.2 nmol/L
Up to 180 nmol/L
10 ng/dL (0.3 nmol/L)
20 – 1600 ng/dL (0.7 – 55 nmol/L)
Total LKTT Testosterone
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) = 3.671 × Estradiol (in pg/mL) / SHBG (in nmol/L)
4
Notes and Disclaimers
Data Analysis
Some ranges should be considered preliminary; these are so flagged. Concentration levels below an assay's detection limit are tabulated as ND (not detectable).
S-PLUS 2000 (www.mathsoft.com) was used for most of the calculations, for data visualization, and for the graphs themselves.18
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.
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).
The tabulated centiles represent guidelines only. Each laboratory should establish or verify the appropriateness of adopting reference range limits suggested by this document.5
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
5
Cross-Sectional Pediatric Fertility Study
FSH (Follitropin)
Results for a cross-section of (not necessarily normal) infants and children at a pediatric hospital and wellness clinic in the southwestern US.
Multicenter Ovulatory Cycle Study16 Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.
FSH, mIU/mL
FSH, mIU/mL Ovulatory Cycles
n*
Group
Age (yr)
n
Females
Cord
30
ND
0.1 – 3
57
2.3
0.11 – 13
4–9
28
0.8
0.11 – 1.6
Cord
37
0.24
ND – 1.2
0.1 – 3
72
0.6
ND – 5.5
4–9
31
0.23
ND – 1.9
Cord
67
0.11
ND – 1.1
0.1 – 3
129
1.1
ND – 10
4–9
59
0.5
ND – 1.8
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
Males
Combined
*Number of subjects (total number of results)
Median Central 95%
Additional Ranges FSH, mIU/mL n
Postmenopausal*
76
90.5
21.7 – 153
Males
135
3.8
0.7 – 11.1
5
5
Group 10
10
15
15
Follitropin
IU/L
mIU/mL
IMMULITE FSH (LKFS)
*Preliminary
0
0
-24
-18
-12
-6
0
6
12
18
Cycle Day: Normalized to LH Peak
15
15 5
5
10
10 0
0
Follitropin
IU/L
mIU/mL
IMMULITE FSH (LKFS)
-8
-4
-1 1
4
8
Cycle Day: Luteal-Follicular Transition
6
Median Central 95%
Cross-Sectional Pediatric Fertility Study
LH (Lutropin)
Results for a cross-section of (not necessarily normal) infants and children at a pediatric hospital and wellness clinic in the southwestern US.
Multicenter Ovulatory Cycle Study16 Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.
LH, mIU/mL
LH, mIU/mL Ovulatory Cycles
n*
Group
Age (yr)
n
Females
Cord
31
ND
0.1 – 1.5
46
0.7
ND – 2.3
1.6 – 9
38
ND
ND – 1.3
Cord
36
ND
ND – 3.6
Median Central 95%
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
0.1 – 1.5
54
1.0
ND – 4.1
Perimenstrual, ± 9 days
54 (959)
3.9
ND – 12.0
1.6 – 9
46
ND
ND – 3.8
Cord
67
ND
ND – 3.5
0.1 – 1.5
100
0.7
ND – 3.7
1.6 – 9
84
ND
ND – 3.2
Males
Combined
*Number of subjects (total number of results)
mIU/mL
IU/L
60
60
40 20
Additional Ranges
40 20
-24
LH, mIU/mL
-18
-12
-6
0
6
12
18
Cycle Day: Normalized to LH Peak
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
Group
n
Postmenopausal*
75
24.9
11.3 – 39.8
Males
135
2.4
0.8 – 7.6
*Preliminary
0
0
Lutropin
IMMULITE LH (LKLH)
*Number of subjects (total number of results)
7
Median Central 95%
Estradiol
IMMULITE Estradiol (LKE2) pg/mL
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
0
42
-24
-12
-6
0
6
12
18
IMMULITE Estradiol (LKE2) pg/mL
pmol/L
Estradiol, pmol/L
54 (108)
114
ND – 308
Periovulatory, ± 3 days
54 (378)
489
124 – 1468
Luteal Phase
54 (604)
343
101 – 905
400
Follicular Phase, Days 2 to 3
200
ND – 587
500
154
1000
54 (708)
1500
Follicular Phase
Estradiol
Median Central 95%
0
0
n*
-18
Cycle Day: Normalized to LH Peak
*Number of subjects (total number of results)
Ovulatory Cycles
0
54 (708)
500
Follicular Phase
400
Estradiol
Median Central 95%
200
n*
1000
Estradiol, pg/mL
1500
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.
Ovulatory Cycles
pmol/L
Multicenter Ovulatory Cycle Study16
*Number of subjects (total number of results)
-8
-4
-1 1
4
Cycle Day: Luteal-Follicular Transition
Estradiol/SHBG Ratio (ESR) See pages 4 and 15.
8
8
Progesterone
IMMULITE Progesterone (LKPG) ng/mL
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
21 -24
-18
-12
-6
0
6
12
18
Cycle Day: Normalized to LH Peak
IMMULITE Progesterone (LKPG)
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
ng/mL 20
Progesterone
0
1.5
0
27 (382)
30
Follicular Phase
10
Median Central 95%
60
Progesterone, nmol/L n*
nmol/L
*Number of subjects (total number of results)
Ovulatory Cycles
14
Progesterone
0
27 (382)
0
Follicular Phase
7
Median Central 95%
20
n*
40
Progesterone, ng/mL
60
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.
Ovulatory Cycles
nmol/L
Multicenter Ovulatory Cycle Study16
-8
-4
-1 1
4
8
Cycle Day: Luteal-Follicular Transition
continued next page. . .
*Number of subjects (total number of results)
9
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
Age (yr)
n
Females
Cord
27
570
465 – 755
0.1 – 0.4
24
1.2
0.5 – 1
19
1.1 – 9 Males
Combined
Progesterone (nmol/L)
Median Central 95%
Group
Age (yr)
n
Females
Cord
27
1,813
1,479 – 2,401
0.25 – 17
0.1 – 0.4
24
3.8
0.8 – 54
0.8
0.2 – 1.6
0.5 – 1
19
2.5
0.6 – 5.1
38
0.4
ND – 1.4
1.1 – 9
38
1.3
ND – 4.5
Cord
27
520
345 – 650
Cord
27
1,654
1,097 – 2,067
0.1 – 0.4
33
1.5
0.3 – 14
0.1 – 0.4
33
4.8
1.0 – 45
0.5 – 1
14
0.8
ND – 2
0.5 – 1
14
2.5
ND – 6.4
1.1 – 9
42
0.4
ND – 1.3
1.1 – 9
42
1.3
ND – 4.1
Cord
54
550
350 – 750
Cord
54
1,749
1,113 – 2,385
0.1 – 0.4
57
1.5
0.25 – 17
0.1 – 0.4
57
4.8
0.8 – 54
0.5 – 1
33
0.8
ND – 2
0.5 – 1
33
2.5
ND – 6.4
1.1 – 9
80
0.4
ND – 1.3
1.1 – 9
80
1.3
ND – 4.1
Males
Combined
10
Median Central 95%
Prolactin
IMMULITE Prolactin (LKPR) ng/mL
mIU/L
Nonpregnant Women
20
400
10
200
Prolactin
40
800
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.
2.5
5
100
-24
-18
-12
-6
0
6
12
18
Cycle Day: Normalized to LH Peak
16
Multicenter Ovulatory Cycle Study
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.
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.
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
Prolactin, ng/mL Ovulatory Cycles
n*
Group
Age (yr)
n
Females
Cord
28
380
200 – 675
0.1 – 0.5
28
15
1 – 140
0.6 – 9
55
11
2 – 43
Cord
27
295
150 – 565
0.1 – 0.5
36
19
4 – 65
0.6 – 9
55
8
0.6 – 29
Cord
55
340
160 – 665
0.1 – 0.5
64
117
2 – 125
0.6 – 9
110
9
1 – 40
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
Males
Combined
*Number of subjects (total number of results)
Prolactin, mIU/L Ovulatory Cycles
n*
Median Central 95%
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
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
Cord
27
6,254
3,180 – 11,978
0.1 – 0.5
36
403
85 – 1,378
0.6 – 9
55
170
13 – 615
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
*Number of subjects (total number of results)
Males
Combined
11
Total Testosterone
IMMULITE Testosterone (LKTT) ng/dL
nmol/L
Group
n
Median Central 95%
80
2
Testosterone, ng/dL
40
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.
3
Total Testosterone
4
Multicenter Ovulatory Cycle Study16
1
47
ND – 118
Follicular Phase
26 (393)
48
ND – 118
Midcycle
26 (26)
58
21 – 104
Luteal Phase
26 (339)
44
ND – 119
0
26 (758)
-24
-12
-6
0
6
12
18
One of these sites also measured total testosterone levels in pregnant and postmenopausal women, with the following results.
Testosterone, nmol/L n
-18
Cycle Day: Normalized to LH Peak
*Number of subjects (total number of results)
Group
0
Entire Cycle
Testosterone, ng/dL
Median Central 95%
Entire Cycle
26 (758)
1.6
ND – 4.1
Group
Follicular Phase
26 (393)
1.7
ND – 4.1
Pregnant Females
Midcycle
26 (26)
2.0
0.7 – 3.6
Luteal Phase
26 (339)
1.5
ND – 4.1
*Number of subjects (total number of results)
n
Median
Abs Range
First Trimester
20
70
30 – 230
Second Trimester
20
90
30 – 200
Third Trimester
19
110
30 – 190
Postmenopausal Females
Additional Ranges Testosterone, ng/dL Group
n
Median Central 95%
Untreated
29
20
ND – 100
Treated
29
30
ND – 100
Surgical
30
30
ND – 60
Females Oral Contraceptives
39
40
ND – 110a
Postmenopausal
103
30
ND – 80L
99
410
200 – 810c
Males
Testosterone, nmol/L Group
Testosterone, nmol/L n
Median Central 95%
39
1.39
ND – 3.81a
Postmenopausal
103
1.04
ND – 2.77L
99
14.2
6.93 – 28.1c
Males
Abs Range
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
Females Oral Contraceptives
Median
Pregnant Females
a: absolute range, c: central 95% range, L: lower 95% range
Group
n
a: absolute range, c: central 95% range, L: lower 95% range
Free Androgen Index (FAI) See pages 4 and 14.
12
Untreated
29
0.693
ND – 3.47
Treated
29
1.04
ND – 3.47
Surgical
30
1.04
ND – 2.08
Additional Ranges
SHBG
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.
Multicenter Ovulatory Cycle Study16 Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.
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 Central 95%
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
SHBG, nmol/L
*Number of subjects (total number of results)
Group
nmol/L 90
90
60 30 0
0
-6
0
6
Oral Contraceptives
18
119
56.3 – 159a
Postmenopausal (untreated)
29
63.2
20.2 – 142a
Hirsute
24
40.6
19.9 – 84.8a
50
32.3
7.2 – 100c
a: absolute range, c: central 95% range.
30
-12
Range
Males
60
SHBG
nmol/L
-18
Median
Females
IMMULITE SHBG (LKSH)
-24
n
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.
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.
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 Study16
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.
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements. Free Androgen Index (FAI) Group
n
Median Central 95%
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
Free Androgen Index (FAI) Group
15
15
10
10
5
5 0
0
Free Androgen Index (FAI)
M/M
M/M
IMMULITE FAI (LKTT/LKSH)
-18
-12
-6
0
6
12
Median
Range
Oral Contraceptives
18
1.2
ND – 3.4a
Postmenopausal (untreated)
29
1.5
ND – 6.6a
Hirsute
24
5.6
1.7 – 20.6a
50 35.0 Males a: absolute range, c: central 95% range.
14.8 – 94.8c
Females
*Number of subjects (total number of results)
-24
n
18
Cycle Day: Normalized to LH Peak
14
Estradiol/SHBG Ratio (ESR) IMMULITE E2/SHBG Ratio (LKE2/LKSH) M/M
M/M
The Estradiol/SHBG Ratio is defined here as 1000 times the molar ratio of estradiol to SHBG.
Multicenter Ovulatory Cycle Study16
10 0
0
Results from an international study involving apparently normal, ovulating women who had daily reproductive hormone measurements.
20
= 3.671 × Estradiol (in pg/mL) / SHBG (in nmol/L)
10
Estradiol (in pmol/L) / SHBG (in nmol/L)
20
Estradiol / SHBG
Estradiol/SHBG Ratio (ESR):
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.
-24
n
Median Central 95%
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
-12
-6
0
6
Cycle Day: Normalized to LH Peak
Estradiol/SHBG Ratio (ESR) Group
-18
*Number of subjects (total number of results)
15
12
18
10,000 100,000 10
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.
1,000
Males and Nonpregnant Females
100
IMMULITE HCG, IU/L [3rd IS 75/537]
HCG
2
4
8
Gestational Week
2
Pregnant Females
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). HCG, mIU/mL
Weeks of Pregnancy 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
16
16
32
11. Vankrieken L. Testosterone and the free androgen index. Los Angeles: Diagnostic Products Corporation, 1997. Technical report ZB158–A.
References 1.
2.
Bodlaender P. IMMULITE® progesterone: physiology and clinical utility. Los Angeles: Diagnostic Products Corporation, 1995. Technical report ZB134–D.
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.
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.
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.
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.
17
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.
18
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