1|CC2 LAB 13

Hormonal Assays Competitive Protein Binding (CPB)

Samples for Hormone Assay • Based on competition for protein binding

sites between tagged or labeled hormone
→ We may use different samples in hormone assay it as or and in competition with the unlabeled
depends on what specific type of hormone we would hormone in the patient’s sample
measure and of course depend on the type of method and o Ex: measurement of Total T4 →
instrument that we will be using for hormone assay Based on the measurement of
+Whole Blood – Ex: LH (luteinizing hormone), specific binding properties of TBG
testosterone (thyroxin binding globulin)

+Plasma → In plasma the anticoagulant that we’ll use Immunologic Assays→ hormone determination
depends on what type of hormone we are looking for → most widely used method for quantitating hormones in
• EDTA – ACTH, ADH, PTH (parathyroid which a labeled antibody would be used to determine
hormone) together with non-isotopic
• Heparin – Catecholamines, cortisol, ➢ depends on the specific type of immunologic
dopamine, FSH assay to be used
+Serum – aldosterone, androstenedione, DHEA
(dehydroepiandrosterone), estrogen, FSH, GH • RIA (radioimmunoassay) – a type of competitive
(growth hormone), HCG, progesterone protein binding technique which uses
radioactive isotopes as the label, it uses a radio
+Urine – estriol labelled hormone as the TAG hormone and an
→ In urine sample we may use different types of anti-sera which prepared against the specific
preservatives with varying volumes hormone which would act as a binding site
o CPB technique that utilizes radio-
• Boric Acid (1g/dL) – estriol and estrogen → labeled hormones as the tagged
used to preserves urine samples up to 7 hormone and anti-sera prepared
days against the specific hormone as a
• 10 mL of 6N HCl (hydrochloric acid) – 3-4 L binding site
container → for preserving urine samples for
catecholamines, VMA (vanillylmandelic acid), • IRMA (immunoradiometric assay) – which
5-HIAA uses Radiolabeled substance that is attached
• HCl (hydrochloric acid) → maintains pH of to the antibody instead of the hormone.
<3.0 → chemical testing (preferred o the means of detection is based on Ag-
preservative for chemical testing) Ab reaction. (Antigen-antibody reaction)

Different assay to use in hormone testing • ELISA (enzyme link immunosorbent

Classic Assays assay) – enzyme is attached to antibody; the
end product of the reaction can be measured
Bioassays spectrophotometrically
• Detection would be based on observations of
• EMIT (enzyme multiplied immunosorbent
the physiological responses specific for the
technique) – in which enzyme tags are
hormone being measured
used; and the enzyme is attached to the
• Involves injection of test materials into
prepared animals hormone or drug being tested

In this type of reaction:

→ MANINI METHOD- which is used for pregnancy
testing (we use toad or frog basically male toad are • Rate of NADH produced is directly
used for specific for determination of HCCR for proportional to the amount of drug tested
pregnancy testing) • This particular method, requires no
separation of bound and free antigen
2|CC2 LAB 13
Other method: Immunometric technique LABORATORY MEASUREMENT: HORMONES OF
THE THYROID GLAND
USES IN:
(MEASUREMENT) SERUM FREE
• TSH test TRIIODOTHYRONINE
• Sensitive test- example is
immunochemiluminescent assay • It measures the amount of free
triiodothyronine (FT3) present in the
Fluorescent Techniques blood sample
• FPIA (fluorescence polarization • could be measured using either radio
immunoassay) – in which it uses fluorescein- immunoassay or chemiluminometric assay
labeled drug, serum, and antibody are mixed • this type of method is used to evaluate and
and placed in the light path of a fluorometer manage disorders of the thyroid gland
which would serve as its analytic device or • related tests: TSH, FT4
instrument → Reference range which is use for different age group
• Antibody-bound conjugate (using as well as during the periof of pregnancy there would also
polarized fluorescence) - this particular be varying reference range according to what particular
conjugate is inversely proportional to serum trimester the mother is already in
drug concentration
→ FT3 is usually measured together with free FT4 and
HPLC (high performance liquid chromatography) total TSH
→ use for the separation of hormones in the other • Reference range:
components of the blood • Adults: 1.4-4.4 pg/mL (0.22-6.78
• It is based on the differential partitioning of pmol/L)
the compounds between the mobile phase • N>37 weeks (cord blood): 15-391
and the stationary phase pg/dL (0.2-6 pmol/L)
• Pregnancy, 1st: 211-383 pg/dL (3.2-
Colorimetry 5.9 pmol/L)
• Pregnancy, 2nd: 196-338 pg/dL (3-
→ chemical reaction producing color production is being
5.2 pmol/L)
used for the detection of hormones
• Pregnancy, 3rd: 196-338 pg/dL (3-
• Porter-Silber Method → used for the 5.2 pmol/L)
detection of 17-OHCS (hydrocortisone DIRECT EQUILIBRIUM DIALYSIS
steroid) → Porter Silber method phenyl
hydrazine would be mixed together with sulfuric → used as a direct or reference method for the
acid and alcohol to form a yellow end color determination of Free T4 or FT4
• Zimmerman Reaction → measures
→ this particular methodology, an undiluted serum would
steroids with 17-ketogenic steroids
be dialyzed for 16-18 hours at 37 °C and then, the product
structure
or dialysate would then be analyzed directly using RIA
→ specifically uses m-dinitrobenzene (meta
(radio immunoassay)
dinitrobenzene) to form a reddish-purple
complex • reference range: 2-128 ng/L (2.6 to 165
• Pisano Method → for quantitating pmol/L)
metanephrines and normetanephrines
ULTRACENTRIFUGATION
• Kober Method → for measurement of
estrogen using sulfuric acid and → This is the method on how ft3 and ft4 would be
hydroquinone that produces reddish brown separated from the sample
color reaction
o H2SO4 + hydroquinone = reddish- • Serum is adjusted to pH of 7.4
brown color • Incubated for 20 minutes at 37°C
• Afterwards it would be undergoing a spinning
using an ultracentrifuge for about 30 minutes
3|CC2 LAB 13
and must be maintained at 37°C and the ANTI-TSH RECEPTOR AUTOANTIBODY
ultracentrifuge must be set at 2000 rpm
• The ultrafiltrate would be collected and further
analyzed using immunoassay
• This particular method is PREFERRED since it is
less time consuming as compared with the
dialysis method

TRIIODOTHYRONINE MEASUREMENT
• Detection of autoantibody against the TSH
• Total triiodothyronine could also be measured. It receptor or the anti-TSH receptor
also known as the total T3 or the TT3 → Used for diagnosis of Grave’s disease
• This measures the level of total T3 in the blood
• Used for evaluating as well as management of • Autoantibodies will be detected based on
thyroid dysfunction, including hyperthyroidism the interference with the binding of the TSH-
• This one is typically measured together with the to-TSH receptor
free T3 and T4 as well as TSH and T3 uptake • detects autoantibodies that interfere with the
binding of TSH-to-TSH receptor
• Related tests: FT4, T3 uptake, ft3 and TSH • serum + TSH receptor + I125 labelled TSH
evaluation tracer
• Adults: 60-181 ng/dL (0.92-2.78 nmol/L)
→ The serum sample would be mixed with the reagent
• Pregnancy (last 5 mos): 116-247 ng/dL
containing the TSH receptor and an iodine labelled TSH
(1.79-3.8 nmol/L)
tracer
SERUM TOTAL T4 using COMPETITIVE
• MEASURED: amount of free tracer
IMMUNOASSAY
o expected is lower than 9 U/L tracer
• measures the total amount of to be considered normal
thyroxine/T4 → when we say total this means
THYROTROPIN RELEASING HORMONE (TRH)
both free and CHON bound (protein bound)
STIMULATION TEST
thyroxine present in blood
→ one of a many specific tests for determination of
→ compound which is being used for competitive thyroid disorder
immunoassay would be dependent on what it would
compete with • There would be an injection of TRH and
measurement of the output of TSH as a
• uses barbital buffers (vs TBPA) and 8-
reaction from the reaction made by the
anilino-1- naphthalene-sulfonic acid (vs TBG
injection of the TRH
or thyroxine binding globulin)
• specifically used in the diagnosis of combined
→ Barbital buffers would be used if the competitive pituitary thyroid disorders
immunoassay would be involving the used of TBPA • differentiates 2° secondary hypothyroidism to
(thyroxine binding prealbumin) while 8- anilino-1 3° tertiary hypothyroidism
napthalene-sulfonic acid would be used if the competitive TEST FOR THYROID FUNCTION
immunoassay would be involving TBG (thyroxine binding
globulin) TRH Stimulation Test

o Adults: 4.5-10.9 µg/dL (58-140 • The most specific and sensitive test for
nmol/L) the measurement / determination of the
relationship between TRH and TSH
• Measures relationship between TRH and
TSH
• Helpful in the detection of thyroid
hormone resistance syndromes.
• Dose needed: 500 ug or microgram of TRH
by administered by IV
4|CC2 LAB 13
Expected result: BMR: (basal metabolic rate) of the body

• Increased levels: an indicator of Primary • based on metabolic response measuring the

hypothyroidism O2 consumption in the resting fasting state
• Decreased levels: an indicator of
Hyperthyroidism RADIOACTIVE IODINE UPTAKE (RAIU)

OTHER LABORATORY TESTS: • Measure the ability of the thyroid gland to

trap iodine.
RAI uptake: (radioactive iodine) • Helpful in establishing the cause of
hyperthyroidism
• based on the ability of the thyroid to
• High uptake indicates metabolically active
concentrate, convert and release I2
gland
→ used to measure the ability of the thyroid gland to trap THYROXINE BINDING GLOBULIN (TBG)
iodine.
• Used to confirm results of FT3 and FT4
➢ based on the ability of the thyroid to and TT4 and THB (thyroid binding ratio)
concentrate, convert, and then release iodine • TBG excess expected to lead to increased
T3 and T4 but the unbound form will remain
• It is helpful in establishing the specific cause unchanged
of hyperthyroidism it is also useful in • Hormonal effect:
evaluating and monitoring the treatment o Estrogen increases TBG,
for thyroid cancer o androgens depress TBG
• In this method a specific percentage of iodine • Increased TBG:
dose would be taken up by the thyroid gland o Euthyroidism
which will be given orally, the high uptake such o Pregnancy
as the metabolic activity while low uptake would o estrogen surge
suggest metabolic inactivity • Decreased TBG:
o patients using anabolic steroids
→ since TSH stimulates iodine uptake the TSH levels
o patient who has nephrosis
must also be taken into account when performing
• 13-39 ug/ dL
radioactive iodine uptake
THYROID STIMULATING HORMONE
• Helpful in establishing hyperthyroidism IMMUNOASSAY TSH →significant test
• High uptake indicates metabolically active
gland • Thyroid stimulating hormone assay
• measures the amount of thyroid stimulating
TBI (traumatic brain injury:
hormone (TSH) in blood using
• based on the thyroid hormone transport chemiluminescence with low detection limit
system indirectly measuring the amount of (very sensitive assay) results are correlated
TBG with the results of T3 and T4 determination
• related tests: T3 and T4

reference range:

o Adults: 0.5-4.7 µunits/L

o Pregnancy (1st): 0.3-4.5 µunits/L
o Pregnancy (2nd): 0.5-4.6 µL
o Pregnancy (3rd): 0.8-5.2 µL
PBI: (protein bound iodine test)
TSH Test
• based on thyroid hormone concentration
representing the organic fraction of blood • The most important thyroid function test:
iodine that precipitates with serum proteins BEST SCREENING TEST → Which is done
together with the determination of t3 and t4
• Helps in the early detection of
hypothyroidism
5|CC2 LAB 13
• Used to differentiate primary hypothyroidism • Formed by removal of one iodine from the
from secondary hypothyroidism inner ring of (thyroxine) T4 and end product
• Used to adjust and monitor thyroid hormone of T4 metabolism
replacement therapy • Useful for identifying patient with
• TSH is expected to be increased with euthyroid sick syndrome in which the RT3
patient with primary hypothyroidism as would be expected to be elevated
well as to those who has HASHIMOTO • Assessing boarder line or conflicting
thyroiditis and thyrotoxicosis laboratory results involving T3, T4, FT3, FT4
• Also elevated in patients who has TSH
REFERENCE VALUES: 38- 44ng/dL
antibodies and those who has thyroid hormone
resistance FREE THYROXINE INDEX (FT4I)
→ Expected to be decreased or below the normal on • Indirectly assesses the level of free T4 in
patients with primary hyperthyroidism as well as those blood.
who has secondary and tertiary hypothyroidism • Based on the equilibrium relationship of
bound T4 and free T4
→ Also expected to be below normal or decreased in
• Expected to be → Elevated in patient with
patient who had treated GRAVE’s disease and euthyroid hyperthyroidism and decreased in
sick disease hypothyroidism
THYROGLOBULIN ASSAY → TG ASSAY FT4I = TT4 x100
T3U (%)
or TT4 x THBR (Thyroid hormone
• Also known as the TG assay binding ratio)
• Normally used as a postoperative marker of T3 UPTAKE
thyroid cancer.
• Used in monitoring course of metastatic or • Measures the number of available binding
recurrence of thyroid cancer. sites of the thyroxine binding proteins most
notably TBG.
→ It is expected that a well differentiated tumor will be o measurement of TBG, it doesn’t
displaying a tenfold increase in the thyroglobulin in measure the actual level of T3 but
response to a high TSH level rather it reflects the serum level of TBG
→ When this particular assay is used as a tumor marker, • Do not measure the level of thyroid hormone
in serum, reflects level of TBG.
it is also important to check / test for anti-thyroglobulin
• In inversely relationship with TBG
antibodies
• Decreased uptake of T3 results to an
→ This test could also use for differentiating subacute increase in TBG
thyroiditis in which there is an increased thyroglobulin as • Increased uptake of T3 results to decrease
compared with thyrotoxicosis in which there would be a in T3
decreased thyroglobulin
Increased levels: Hyperthyroidism
METHODS FOR TESTING: thyroglobulin uses
Decreased levels: Hypothyroidism
→ Double antibody RIA (radioimmunoassay), Groups of test and methods which used for the
ELISA (enzyme linked immunoassay, IRMA detection of tumors involving thyroid gland
(immunometric immunoassay), ICMA
(immunochemiluminescent assay) • Fine Needle Aspiration → often the first
step
REVERSE T3 → RT3 o Most accurate tool in the
• RT3 an end product of T4 metabolism evaluation of thyroid nodules,
allows prompt identification and
• Considered to be the third major
circulating thyroid hormone treatment of malignancies
involving the thyroid gland, it avoids
• Used to assessed borderline or conflicting
unnecessary surgeries in those who
laboratory results. Involving T3, T4, FT3 and
are having benign cases,
ft40020
6|CC2 LAB 13
→ A small gauge needle would be inserted • Increased- TSH
to a nodule and the cells will be further
Laboratory Measurement: Hormones Secreted by
aspirated for histopathologic examination
the Pituitary Gland
• Recombinant Human TSH – use to test
Tests for Growth Hormone
patients with thyroid cancers for the presence
of residual or recurrent disease. Patient Preparation
• Serum Calcitonin – Tumor marker for
detecting residual thyroid metastasis in • Complete rest for 30 minutes before blood
patients who have medullary thyroid collection
carcinoma, it is measured both before in and Specimen Requirement
six months after surgery
• Preferred specimen: at least 12hrs of
Pentagastrin Stimulation Test – for diagnosis of fasting for fasting serum sample
medullary thyroid carcinoma
Tests for Growth Hormone Deficiency
SUMMARY OF THYROID DISORDERS AND
LABORATORY TESTS Growth hormone deficiency- In childhood could be
defined if there is a failure of the serum growth hormone
to reach particularly defined level when at least two
pharmacological stimuli is used we have screening test
and confirmatory test for testing growth hormone
deficiency

Screening Test – Exercise test → also known as

Grave’s disease: Physical Activity Test → it would detect the growth
hormone activity after a physical activity
• Decrease- tsh
• Increase- t3, t4, ft4, rt3, tg • Expected result of the test → elevated
• Normal- tbg serum growth hormone
• If growth hormone fails to increase → time
Primary hypothyroidism:
when the test should proceed to confirmatory
• Decrease- t3, t4, ft4. Rt3, tg test
• Increase- tsh • If the growth hormone fails to increase,
• Normal- t3, tg, tbg you must proceed to confirmatory test

Hashimoto thyroiditis: Confirmatory Test

• Decrease= t3, t4, ft4, rt3, tg • Insulin Tolerance Test – gold standard for
• normal= may also be normal tg and tbg and testing the growth hormone deficiency
• increase- tsh • Arginine Stimulation Test - 2nd
Confirmatory Test
Nonthyroidal illness:
→ These two tests involve measurement or monitoring
• Decrease- t3, t4, growth hormone during nighttime or a 24 hour period
• Variation- TSH, ft4
• Increase- rt3 • Procedure: 24-hour or nighttime monitoring
of GH
• Normal- tg, tbg or normal t4, rt3
Interpretation:
Thyroid hormone resistance: all are expected to
increase except for tbg and tsh may be normal or • Confirmation of growth hormone
increase deficiency if → Failure of GH to rise above
Neonatal hypothyroidism: o > not beyond 5ng/mL (adults)
o > fails to increase by 10 mg/mL
• Decreased- t3, t4, ft4, rt3, tg may be (child) in all the tests is confirmed
decreased or normal GH deficiency
• Normal- tbg
7|CC2 LAB 13
Test for Acromegaly • Mab 1-hGH-Mab-HRP
• absorbance is measured after colorimetric
→ Acromegaly- is defined as a hormonal disorder which
reaction
could develop whenever the pituitary gland produces too
• Growth hormone concentration would vary
much growth hormone during adulthood depending on what time of the day the
Screening Test: Somatomedin C or insulin like immunoassay is being done because growth
growth factor 1 hormone is released at certain time of the
day
• IGF-1 is produced in the liver o Day: <0.2-10 uIU/mL
• Increased in patients with Acromegaly o Night: 30 uIU/mL
• Low in growth hormone deficiency (GHD)
Prolactin Immunoassay
Confirmatory Test: Glucose Suppression Test –
OGTT → which would be done just like the oral glucose → Principle: homologous competitive binding
tolerance test immunoassay/ sandwich technique

• Blood would collect every after 30 mins for 2 Sandwich technique → since it uses two or more
hours: fasting sample is required antibodies directed at different parts of the (PRL)
• This test method is considered to be the prolactin molecule
BEST METHOD in confirming acromegaly
• Affected by → hook effect- happens when
diagnosis and in people who does not have
there is an excess antigen during testing,
acromegaly it is expected that the glucose
dilution could be done to counteract this
strength drink should typically cause growth
phenomenon
hormone level to fall and vice versa to
o Adult male: 3-14.7 ng/mL or ug/L
patients who those has acromegaly
o Adult female: 3.8-23 ng/mL or ug/L
OTHER TEST FOR DETERMINING GROWTH o Pregnancy, 3rd tri: 95-473 ng/mL
HORMONE

Growth Hormone Immunoassay

→ Since this is an immunoassay, it would require the

use of an antibody, it uses specific GH (growth
hormone) antibody

• require multiple measurements

o drawn specimens every 20-30
minutes over a 12–24-hour period →
depending on what is being requested ACTH Immunoassay → Adrenocorticotropic
by the doctor immunoassay
• Insulin tolerance test: to produce
→ Useful for determination of hypercortisolism or
hypoglycemia and known and expected to
hypercortisolism
provoke GH release
o Basal: 2-5 ng/mL or ug/L Principle: chemiluminescence and ELISA or could
o If given insulin tolerance: >10 also be electrochemiluminescence immunoassay
ng/mL
o Arginine/L-dopa: >7.5 ng/mL • related test: cortisol
• Sample preferred: blood placed in EDTA
hGH-EASIA tube
• Patient preparation: must not take
→ Human growth hormone EAISA - a growth hormone
multivitamins or dietary supplements that
solid phase Enzyme Amplified Sensitivity
have vitamin B7 also known as biotin found
Immunoassay
in dietary supplements for hair, nail and skin.
→ It would use a monoclonal antibody the Mab 1 which is
→ The patient should not take any of this at least
coated in a micro titer well with a monoclonal antibody 2
12 hours prior to testing
(Mab) to which would be label with horse radish
peroxidase • reacts with intact ACTH and ACTH fragments
8|CC2 LAB 13
o Adults: 5-80 pg/mL (X 0.22= pmol/L) (TSH response to TRH is delayed: TSH
o Specimen: P, EDTA higher at 60’ than 20’)
o Hyperthyroidism: pituitary response
Dynamic Function Test to TRH is flat (TSH
→ Involves the use of an exogenous agent which would o Hypothyroidism: exaggerated
manipulate the body’s hormonal activity which would help response (>25 mU/L)
• TRH is given as an IV bolus
in diagnosis and characterization of an endocrine
disorder → Blood sampling done at 0 hours prior to giving the
dose IV bolus of TRH, 20 minutes after, and 60 minutes
→ Helpful in evaluation of endocrine conditions such as
after
problem with growth hormone, pubertal maturation, or
problems with sex development • Blood sampling done at 0, 20, 60 minutes.
• stimulating or suppressing a particular GnRH Test → Gonadotrophin releasing hormone
hormonal axis, and observing the appropriate
hormonal response • assesses hypogonadism
o If excess is suspected, conduct a • can be done together with anterior pituitary
suppression test function test (IST- inappropriate secretion of
o If deficiency is suspected, conduct thyrotropin, TRH – thyrotropin releasing
a stimulation test hormone, GnRH tests)
o Stimulus: exogenous analogue of a
Normally,
trophic hormone or a biochemical or
physiological stress like → • Adults: GnRH causes marked rise in LH
hypoglycemia or exercise (increments of >15 U/L) and smaller rise in
FSH (>2 U/L)
→ Examples of dynamic function test: stimulation for
• Children: GnRH causes marked rise in FSH
human gonotrophic stimulation test (hcg stimulation test,
and smaller rise in FSH
GNRH or gonadotrophin releasing hormone stimulation
test, agonist suppression test for GNRH)

Insulin Stress Test

• done when hypopituitarism is suspected

• also known as Insulin Tolerance Test
• insulin is administered to produce
hypoglycemic stress (<2.2 mmol/L)
• Tests the ability of Anterior Pituitary Gland to
produce ACTH and GH
o Normal GH increase more than >6
ug/L
o Normal Cortisol increase more than
> 500 nmol/L ACTH Stimulation Test
TRH Test → Thyrotropin releasing hormone test • Also known as cosyntropin test or
tetracosactide test
→ A stimulation test used in order to determine the effect • small amount of synthetic ACTH is injected to
of TRH or given TRH to the thyroid hormone patient, and amount of cortisol or aldosterone
→ Involved with an intake of intravenous dose of is measured
• distinguish whether the cause is adrenal
thyrotropin releasing hormone in order to assess its
disease whether (low cortisol and
specific effect on the thyroid hormone this could provide
aldosterone production) or if pituitary glands
useful information in distinguishing thyroid problem,
problem (low ACTH production)
• assesses the adequacy of Anterior Pituitary o Determine if the problem is adrenal
Reserve, or to evaluate hypothalamic or pituitary gland
diseases response whenever there are o cortisol should be increased by
changes in the concentration of the TRH twofold to threefold within 60
9|CC2 LAB 13
minutes after injection of synthetic o Absorbance proportional to LH
ACTH concentration → the more luteinizing
o preferred sample: patient hormone is being found on as a content
undergone fasting (8 hrs.) of the sample it is expected to have
higher absorbance reading
▪ Horseradish peroxidase – use
as a label if you will be using
enzyme immunoassay
▪ Iodine – as an alternative label
for immunoradiometric assay
- Mab1-LH-Mab2125I

Fertility test (male)

LH Ovulation Dip strip Urine Test
Primary: Semen analysis (semenalysis)
→ To determine levels of luteinizing hormone
qualitatively → also, determination of testosterone for fertility
- test approximately the same time each day • Testosterone 300-1000ng/dL
whatever time of the day the test would be • Prolactin 7-18ng/mL
done • Luteinizing Hormone (LH) 2-18Miu/mL
- reduce liquid intake two hours before testing • Follicle Stimulating Hormone (FSH):
2-18Miu/mL
→ used for determining ovulation cycle which could help
• Estradiol (Day 3): <50 pg/mL
in determining fertility of a human being
Fertility test (female)

Phase of Cycle

Hormone Follicular Day of LH surge

FSH < 10 mIU/mL > 15 mIU/mL
LH < 7 mIU/mL > 15 mIU/mL
PRL < 25 ng/mL
→ To determine what particular cycle
LH Immunoassay (EIA/IRMA)
• FSH: measures your ovarian reserve (to
→ quantitative assay detect ovarian function)
• low levels of FSH & LH: hypogonadotropic
→ PRINCIPLE: using enzyme immunoassay or immune hypogonadism
radio metric assay • high LH with a normal FSH level: PCOS or
PCOD (polycystic ovarian disease)
→ would be using monoclonal antibody for luteinizing
• high prolactin: hyperprolactinemia
hormone and another monoclonal antibody so its like the
sandwich method which would be labeled by horse radish HORMONE FOLLICULAR DAY OF LH
peroxidase SURGE
Estradiol < 50 pg/ml > 100 pg/ml
→ this would be measured using a colorimetric or Progesterone < 1.5 ng/ml >15 ng/ml
chromogenic reaction using spectrophotometer

→ The absorbance reading would be DIRECTLY • high estradiol (D3): poor ovarian reserve
PROPORTIONAL to LH concentration • estradiol rises as follicle matures; useful for
measuring follicular activity
→ more luteinizing hormone found = higher absorbance • mature follicles to release about > 200-300
reading pg/ml of estradiol
- Mab1-LH-Mab2HRP • more than P >15 ng/ml about 7 days after
o measured using chromogenic ovulation: corpus luteum is functioning
reaction normally → EXPECTED ACTIVITY / RELEASE
10 | C C 2 L A B 1 3
• low Day 21 Progesterone level → suggests o 270-280 mOsm/kg: <1.5 pg/mL
the cycles was anovulatory (no egg was (<1.4 pmol/L)
produced) o 280-285 mOsm/kg: <2.5 pg/mL
(<2.3 pmol/L)
Serum FSH Measurement (IRMA) o 285-290 mOsm/kg: 1-5 pg/mL (0.9-
4.6 pmol/L)
• uses immune radiometric assay
o 290-295 mOsm/kg: 2-7 pg/mL (1.9-
• sandwich method also using monoclonal
6.5 pmol/L)
antibody another monoclonal body and another
o 295-300 mOsm/kg: 4-12 pg/mL (3.7-
monoclonal body which would be labeled by
11.1 pmol/L)
radio metric iodine
• used to assess and manage disorders of the
endocrine gland involving suspected infertility
this is related to the previous tests discussed for
fertility testing (luteinizing hormone, prolactin,
estradiol, testosterone,

• measures the amount of follicle stimulating

hormone (FSH) in blood
• Mab1-serum-Mab2125I
• used to assess and manage disorders of the
endocrine glands, including suspected
infertility
• related tests: LH, PRL, testosterone,
estradiol

Normal Values for Serum FSH

• Female, menstruating:
o Follicular phase: 1.4-9.9 mIU/mL
(1.4-9.9 IU/L)
o Ovulatory phase: 0.2-17.2 mIU/mL
(0.2-17.2 IU/L)
o Luteal phase: 1.1-9.2 mIU/mL (1.1-
9.2 IU/L)
• Postmenopausal: 19.3-100.6 IU/L
• Male: 1-15.4 mIU/mL (1-15.4 IU/L)

ADH Measurement

• Also known as the method of the amount of

antidiuretic hormone or arginine vasopressin

➢ Done in order to detect, help in diagnosis and to

determine the prognosis of possible ADH
deficiency
➢ determine possible resistance of the kidney to
the effect of the anti-diuretic hormone
➢ Investigate why there is hyponatremia or low
sodium levels in an individual
➢ Significant in determining from the two types of
diabetes insipidus
• measures the amount of antidiuretic
hormone, or vasopressin, in blood

Related tests: sodium and osmolality