react with GH is very high. Similarly, LH cross-reacts to a very high degree with hCG because of similar alpha chains. Hence, LH estimates are invalid in pregnant women or persons with hCG-secreting tumors. Immunoassays should

 

Hormonal Tests for Diagnosing the Cause of Anovulation

Prolactin Pituitary Hormone

Excessive prolactin can suppress pituitary output (LH

and FSH) and can act directly on the ovary to suppress

follicular growth.

Thyroid Hormone

Hyper and hypothyroidism can interfere with hormonal

metabolism (the rate at which hormones are used up

by the body) and with the delicate hormonal balance

between the pituitary and ovary. In addition, through an

intriguing mechanism, hypothyroidism may contribute to

excess prolactin production.

FSH and LH Pituitary Hormones

Elevated FSH almost always indicates ovarian failure. If

FSH and LH are depressed, suspect one of three things: that

a faulty hormonal feedback mechanism is inappropriately

telling the pituitary to cut back production; that the

hypothalamus is not “beating the drum” to stimulate

the pituitary to function; or that a pituitary inadequacy

prevents the gland from functioning normally.

Adrenal Androgens (DHEAS and Testosterone)

In the presence of excessive hair (hirsutism) or male

secondary sex characteristics (enlarged clitoris

or ambiguous genitalia), elevated male hormone

(testosterone), elevated DHEAS, or elevated adrenal

androgens may indicate a congenital enzymatic defect,

polycystic ovaries, or a tumor in the pituitary gland,

adrenal gland, or ovary. Testosterone or adrenal androgens

can suppress ovulation as well as cause a number of other

problems discussed later.

Conditions that can Interfere with

Ovulation and Menstruation

¾ Pregnancy

¾ Hypothalamic malfunction:

• Emotional stress (endorphins?)

• Amenorrhea

• Athletics (extreme exercise)

• Dieting, poor nutrition, weight loss, low body fat

• Anorexia

• Idiopathic (drugs, toxins, medications?).

¾ Pituitary gland malfunction:

• Hyperprolactinemia

• Tumor

• Surgery

• Trauma

• Empty sella syndrome

• Sheehan’s syndrome

• Cushing’s disease.

¾ Hormonal feedback problems affecting pituitary gland:

• Hepatorenal disease

• Adrenal disease

• Cushing’s syndrome

768 Concise Book of Medical Laboratory Technology: Methods and Interpretations • Congenital adrenal hyperplasia

• Polycystic ovary

• Hypo/hyperthyroidism

• Obesity (excess estrogen).

¾ Ovarian abnormalities:

• Ovarian cysts

• Endometriosis

• Infection.

¾ Premature ovarian failure

¾ Incidental fertility findings:

• Asherman’s syndrome (adhesions in the uterus)

• Cervical stenosis (cervix closed from surgery).

¾ Idiopathic (no identifiable cause).

Practical Evaluation of Hormonal Status

¾ The patients with a disorder of reproductive function

serve as their own bioassay

¾ The history and physical examination are most

important in evaluating any patient with reproductive

dysfunction

¾ Evaluating the female patient with normal pubertal

development as a reference is often useful in

determining the cause of the reproductive dysfunction

¾ Laboratory tests are used to confirm what is suspected

on the basis of the initial evaluation

¾ Immunoassays of WHO recommended standards are

recommended for proper clinical correlation

¾ Measurements of basal FSH, LH, PRL and TSH are

warranted in all amenorrheic patients once pregnancy

has been excluded

¾ Radiographic studies of the sella turcica are indicated

in amenorrheic women with low levels of circulating

LH and FSH, whether prolactin is elevated or not. High

sensitive immunoassays are required to ascertain the

lower end values

¾ Individuals with hypothalamic or pituitary tumors

and those with presumptive hypopituitarism should

undergo dynamic testing of pituitary function

¾ Individuals with hirsutism should have serious etiologic

factor eliminated for appropriate laboratory testing.

ALGORITHM FOR EVALUATING

AMENORRHEA (FIGS 24.12 to 24.18),

IMMUNOASSAYS FOR LH, FSH AND PRL

Sensitivity

The LH and FSH levels can drop to very low concentrations

as low as 1 mIU/mL. Hence, assays have to be with very

high sensitivity.

Sensitivity is of diagnostic importance in hypogonadism particularly when it is very essential to differentiate between the low and normal values of LH and FSH.

Detection Limits of Various Immunoassays

¾ 2nd Gen ELISA 2.0 mIU/mL

¾ RIA 0.2 mIU/mL

¾ Chemiluminisence 0.2 mIU/mL

¾ 3rd Gen RIAC ELISA 0.02 mIU/mL

Conditions in Which LH/FSH Levels can go

Below 2.0 mIU/ mL

¾ Hypopituitarism

LH < 1.5 mIU/mL

FSH < 1.0 mIU/mL.

¾ Pituitary tumor + Hypopituitarism

• LH < 2.0 mIU/mL

• FSH < 1.5 mIU/mL.

¾ Non-functional pituitary tumor

• LH < 1.2 mIU/mL

• FSH < 1.0 mIU/mL.

¾ Hypogonadism

• LH < 1.5 mIU/mL

• FSH < 1.5 mIU/mL.

Streptavidin-Biotin Assay System

The strength and speed of the binding between avidin and

biotin is used to provide amplification of signal and as the

basis of generic signal generation reagents.

As compared to low sensitivity assays, streptavidinbiotin based assay systems offer an increase in signal ratio

as well as improvement in rate of change of the measured

signal, which in turn offers the immunoassay users greater

accuracy from the test system in question (Fig. 24.19).

Calibrator Matrix

Calibrators should ideally be prepared by using a base

material identical to that in the test samples. For clinical

FIG. 24.12: Antibody binding sites and streptavidin

The Endocrine System 769

FIG. 24.13:

applications, human serum is the preferred base matrix

(Fig. 24.20).

WHO Std. Reference

The World Health Organization’s International Laboratory

for Biological Standards is now providing qualified investigators with an International Reference Preparation

of Human Pituitary Gonadotropins (LH and FSH) for

Immunoassay (coded WHO 1st IRP 68/40 and WHO

2nd IRP 78/549). Data compared with these standard

preparations can be reported in terms of International

Units. These units differ from those obtained with use of

the 2nd IRP-hMG).

Thus, the importance of knowing the “standard

preparation” that is used and the “normal range” for

any given laboratory is obvious. Also important is that

commercially available assays may use different standards,

and some of the kits do not even state what reference

preparation is provided.

770 Concise Book of Medical Laboratory Technology: Methods and Interpretations FIG. 24.14:

FIG. 24.15:

The Endocrine System 771

FIG. 24.16:

FIG. 24.17:

772 Concise Book of Medical Laboratory Technology: Methods and Interpretations Cross Reaction

LH, FSH and PRL are pituitary hormones and are structurally

similar to other pituitary hormones. Therefore, the tendency

to cross react is very high when it comes to the estimation

of these assays. For example, the tendency of PRL to cross

react with GH is very high. Similarly, LH cross-reacts to a

very high degree with hCG because of similar alpha chains.

Hence, LH estimates are invalid in pregnant women or

persons with hCG-secreting tumors. Immunoassays should

be highly specific with minimal cross reaction.

FIG. 24.19: Streptavidin-biotin assay system

FIG. 24.20: Difference between ELISA generations

FIG. 24.18:

Monoclonal Capture

Monoclonal captures are better compared to traditional

polyclonal capture as it is more specific.

FIGS 24.13 TO 24.18: Algorithm for evaluating amenorrhea

The Endocrine System 773

Assays having polyclonal capture antibodies give

rise to different results. Monoclonal antibodies, which

react against one highly specific antigenic determinant

of a given hormone (epitype characterization), can help

alleviate interlaboratory differences in results (Fig. 24.21).