Testosterone Analytical Problems Testosterone in Women

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Testosterone Analytical ProblemsTestosterone in
Women

• GP meeting CMH
• 9th July 2008

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Circulating Forms of Testosterone

• Total Serum Testosterone (nmol/l)
• Tightly Bound to Sex Hormone Binding Globulin
  (SHBG) (98) high affinity
• Loosely bound to Albumin
• Free Testosterone

Bioavailable Testosterone
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Free Testosterone

• Law of Mass Action
• Free T Protein ? PT

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Control of Testosterone/SHBG
Decreases in SHBG free testosterone
Increases in Free Testo SHBG Thyrotoxicosis
SHBG levels Free Testo Hypothyroidism
SHBG levels Free Testo Hyperprolactinaemia
SHBG levels Obesity SHBG levels
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Free Testosterone Estimations/Manipulations

• Direct or Indirect measurement very difficult
• FAI Free Androgen Index total testosterone
  x100
• SHBG
• Bioavailable testosterone
• Albumin Bound T Ka x (alb conc) x Free testo
• Total T FT x P1T x P2T P3T. . . . . 1PnT

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1970-80s
21 Century- automated
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Clin Chem 49.8 August 2003
Editorial Immunoassays for Testosterone in
Women Better than a Guess?
Article Testosterone Measured by 10 Immunoassays
and Isotope-Dilution GC-MS
J Clin Endocrinol Metab 89 February
2004 Editorial Serum Testosterone- Accuracy
Matters Article Measurement of Total Serum
Testosterone in Adult Men Comparison of Current
Laboratory Methods Versus Liquid
Chromatography-Tandem Mass Spectrometry
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Case StudyHancock et al Ann Clin Biochem
200845328-330

• Endocrine investigations
• serum cortisol 895 nmol/L ELEVATED
• Testosterone 18.8 nmol/L r r 03 nmol/L
• SHBG 10 nmol/L, r r 20120 nmol/L
• FTs, prolactin, E2, LH/FSH normal.
• O/N (1 mg) dexamethasone suppression test failed
  to suppress (0900 h cortisol 863 nmol/L).
• 2 day low dose dex test (0900 h cortisol 839
  nmol/L).

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Testostorone

• Measured on automated machine
• manufacturer A 18.8 nmol/l (rr 0 3.0)
• Manufacturer B 8.2 nmol/L (rr 0 3.0)
• (after extraction into organic solvent 7.4
  nmol/l)

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Is this interference problem known -YES
Other steroids implicated DHEAs Proven in
Quality Assessement samples
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THE ENDOCRINE SOCIETYPOSITION STATEMENT
Utility, Limitations, andPitfalls in Measuring
Testosterone An Endocrine SocietyPosition
StatementWilliam Rosner, Richard J. Auchus,
Ricardo Azziz, Patrick M. Sluss, and Hershel
RaffSUMMARY This review demonstrates that the
manner in which most assays for TT and FT are
currently performed is decidedly unsatisfactory.
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Know the type and quality of the assay that is
being used and the properly established and
validated reference intervals for that assay.
Reference intervals should be established by each
laboratory in collaboration with
endocrinologists, using well-defined and
characterized populations. In the absence of
other information, direct assays (those performed
on whole serum) perform poorly at low T
concentrations (i.e. in women, children, and
hypogonadal men) and should be avoided. Assays
after extraction and chromatography, followed by
either MS or immunoassay, are likely to furnish
more reliable results and are currently preferred.
Assays for T may behave differently in
controls and affected individuals, perhaps
reflecting differences in the endocrine milieu of
patients. Most assays will distinguish between
T concentrations in classic hypogonadism and
those in normal men. Serum TT, preferably
obtained on more than one morning sampling, is
the recommended screening test for hypogonadism.
Assuming a high-quality assay and well-defined
reference intervals, a serum TT, preferably drawn
during the early follicular phase of the
menstrual cycle, is recommended as the initial
test in seeking out androgen-producing tumors in
women. Calculated FT, using high-quality T and
SHBG assays with well-defined reference
intervals, is the most useful, clinically be
specific for the assay method, until a universal
standard is available. FT measurements in
children are of limited value. Evaluations of
androgen excess, virilization, intersex
disorders, or contrasexual maturation are the
only indications for T measurement in girls.
Several indications exist for T measurements in
boys, including assessment of gonadal failure,
disorders of sexual development or puberty,
and monitoring response to treatment. sensitive
marker of hyperandrogenemia in women and can be
used in concert with clinical end points in
the diagnosis and follow-up of such patients.
In the absence of pituitary insufficiency, the
use of T assays in the evaluation of sexual
dysfunction or fatigue in adult women is not
supported by published evidence and is strongly
discouraged. In children, reference intervals
must be adjusted for gender, age, and stage of
adolescent development and must

• Know the type and quality of the assay that is
  being used and the properly established and
  validated reference intervals for that assay.
• Direct assays (those performed on whole serum)
  perform poorly at low T concentrations (i.e. in
  women, children, and hypogonadal men) and should
  be avoided.
• Assays after extraction and chromatography,
  followed by either MS or immunoassay, are likely
  to furnish more reliable results and are
  currently preferred.

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• Calculated FT, using high-quality T and SHBG
  assays with well-defined reference intervals, is
  the most useful, clinically sensitive marker of
  hyper-androgenemia in women and can be used in
  concert with clinical end points in the diagnosis
  and follow-up of such patients.
• In the absence of pituitary insufficiency, the
  use of T assays in the evaluation of sexual
  dysfunction or fatigue in adult women is not
  supported by published evidence and is strongly
  discouraged.
• Most assays will distinguish between T concs in
  classic hypogonadism and those in normal men.
  Serum TT, preferably obtained on more than one
  morning sampling, is the recommended screening
  test for hypogonadism.

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NWLH Testosterone Assay
Chromatography Mass Spectrometry Meets the
criteria for a good assay one of only 4 in UK
(we receive quite a lot of samples from St
Elsewheres to correct their eroneous
results) Problems with establishing reference
ranges due to Ethics etc
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• Clinical Indications for Measuring Testosterone
  in Women
• Investigation of Infertility
• Investigation of virilisation
• ?investigation of differential diagnosis of
  syndromes presenting as PCOs
• ?? Monitoring of testosterone replacement in post
  menopausal women
• Other minor investigations which usually cause us
  problems
• Gender reassignment
• Androgen insensitivity syndrom

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Where does Testosterone come from in
Premenopausal Women?

• primarily Ovary
• Adrenals

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Ehrmann DA et al 2005 N Engl J Med 352 1223
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What tests should I request Testosterone LH/FSH P
rolactin SHBG Not any other androgens as a front
line screen
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2 patient clinical details Hirsuite
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Testosterone in Women We have a state of the art
assay for the measurement of testo in
women Difficult to set ref ranges for FAI If
testosterone is raised (above 5) we will phone
you,