CC Hormone Assays

SERODIAGNOSTIC TECHNIQUES

Most of the hormone assay are ________

LH (luteinizing hormone) , testosterone

Samples for Hormone Assay:

Blood

➔ Preferred depending on the anticoagulant and hormone to be tested

➔ EDTA - ACTH, ADH, PTH

➔ Heparin - Catecholamines, cortisol, dopamine, FSH

Samples for Hormone Assay:

PLASMA

aldosterone, androstenedione, DHEA, estrogen, FSH, GH, HCG, progesterone

Samples for Hormone Assay:

SERUM

➔ 10 mL of 6N HCl - placed on 3-4 L container for preserving catecholamines, VMA, 5-HIAA

➔ HCl- maintains the urine pH of <3.0 for chemical testing

Samples for Hormone Assay:

URINE

Boric Acid (1g/dL) - estriol and estrogen for preserving urine sample for 7 days

We may use different types of preservatives for URINE specimen such as:

HCl

maintains the urine pH of <3.0 for chemical testing

Bioassays

Involves injection of test materials into prepared animals

physiological responses

BIOASSAYS

Based on observations of the __________ specific for the hormone being measured

Galli manini Method

example of BIOASSAY that is used for pregnancy testing

Male toad

BIOASSAY

is used for the determination of hCG or pregnancy testing

Competitive Protein Binding (CPB)

Based on competition for protein binding sites between tagged or labeled hormone and the unlabeled hormone in the patient's sample

IMMUNOLOGIC ASSAYS

Most WIDELY USED method for quantitating hormones in which a labeled antibody will be used to determine together with the non-isotopic labels

non-isotopic labels

IMMUNOLOGIC ASSAYS

Most WIDELY USED method for quantitating hormones in which a labeled antibody will be used to determine together with the __________

RIA - CPB

A Type of protein binding technique which uses a radioisotopes

RIA - CPB

technique that utilizes radio-labeled hormones as the tagged hormone and anti-sera

binding site

RIA (radioimmunoassay) is prepared against the specific hormone as a _________

IRMA (immunoradiometric assay)

Radiolabeled substance is attached to the antibody instead of the hormone

Ag-Ab reaction (means of determination)

reaction in IRMA (immunoradiometric assay)

EMIT (enzyme multiply immunosorbent technique

enzyme tags are used; enzyme is attached to the hormone or drug being tested

ELISA (enzyme linked immunosorbent assay

enzyme is attached to antibody; can be measured spectrophotometrically

DIRECTLY proportional

EMIT (enzyme multiply immunosorbent technique:

Rate of NADH produced is ______________ to the amount of drug tested

EMIT (enzyme multiply immunosorbent technique

Requires no separation of bound and free antigen

a. TSH test

b. Sensitive test (immuno chemiluminescence assay - very sensitive test)

test in immunometric

FPIA (fluorescence polarization immunoassay)

Antibody-bound conjugate (polarized fluorescence)

FLUORESCENT TECHNIQUES:

FPIA (fluorescence polarization immunoassay)

fluorescein-labeled drug, serum, and antibody are mixed and placed in the light path of a fluorometer

Fluorometer

serves as analytic device / analytic instrument

HPLC

Separation of hormones from other components of the blood

mobile and stationary phase

HPLC is based on the differential partitioning of the compounds between the _________ and ____________

1.) Porter-Silber Method

2.) Zimmerman Reaction

3.) Pisano Method

4.) Kober Method

4 COLORIMETRY METHODS

Porter-Silber Method → 17-OHCS

Phenyl hydroxyzine would be mixed with sulfuric acid and alcohol to form YELLOW END

Zimmerman Reaction

measures steroids with 17-keto structure

Zimmerman Reaction

Meta dinitrobenzene to form a REDDISH PURPLE COMPLEX

Pisano Method

For quantitating metanephrines and normetanephrines

Kober Method → for estrogen

H2SO4 + hydroquinone = reddish-brown color

for 30 minutes before blood collection

px must have complete rest for _________ blood collection

Fasting serum sample (preferably 12 hours of fasting)

Specimen requirement for GH

Screening test - exercise test → physical activity test

Confirmatory test

TEST FOR GROWTH HORMONE DEFICIENCY

failure of the serum

growth hormone deficiency in childhood could be defined if there is a ______________ growth hormone to reach particularly defined levels when at least 2 form ecological stimuli are used

o Insulin Tolerance Test

o Arginine Stimulation Test

CONFIRMATORY TESTS FOR GH

Insulin Tolerance Test

Gold standard confirmatory test for GH deficiency

Arginine Stimulation Test

2nd confirmatory test for GH deficiency

5ng/mL (adults)

>10 mg/mL

Failure of GH to rise > ____________ (adults) and _________ (child) in all the tests is confirmed GH deficiency

Somatomedin C or insulin like growth factor 1 (IGF-1)

SCREENING TEST FOR ACROMEGALY

Glucose suppression test - OGT

CONFIRMATORY TEST FOR ACROMEGALY

ACROGMEGALY

a hormonal disorder which could develop whenever the pituitary gland produces too much growth hormone during adulthood.

LIVER

IGF-1 is produced in the ________

INCREASED IGF-1

RESULTS IN IGF-1 TO PX WITH ACROMEGALY

LOW

LEVEL OF GHD IN IGF-1 TO PX WITH ACROMEGALY

30 mins for 2 hours

ACROMEGALY Confirmatory test: Glucose suppression test - OGT

Blood is collected every after __________ fasting sample is required

TRUE

FASTING SAMPLE IS REQUIRED IN ACROMEGALY CONFIRMATORY TEST GLUCOSE SUPPRESSION TEST

Glucose suppression test - OGT

is considered the best method for confirming acromegaly diagnosis and in people who does not have acromegaly

GROWTH HORMONE IMMUNOASSAY

uses specific GH antibody AND REQUIRE MULTIPLE MEASUREMENTS

20-30 minutes over a 12-24 hours period

GH IMMUNOASSAY must draw specimens every ________ minutes over a _______ hrs period

Insulin tolerance test

to produce hypoglycemia and provoke GH release

2-5 ng/mL or ug/L

GH IMMUNOASSAY: (ref value)

BASAL

>10 ng/mL

GH IMMUNOASSAY: (ref value)

INSULIN TOLERANCE TEST

>7.5 ng/mL

GH IMMUNOASSAY: (ref value)

ARGININE/L-DOPA

HGH-EASIA

solid phase Enzyme Amplified Sensitivity Immunoassay

HGH-EASIA

Mab 1-hGH-Mab-HRP

Mab 1-hGH-Mab-HRP

which is coated in micro titer well with a monoclonal anti body 2 which will be labeled horse radish peroxidase.

COLORIMETRIC REACTION

in HGH-EASIA absorbance is measured after the ______________

<0.2-10 uIU/mL

HGH-EASIA: REF VALUE

DAY

30 uIU/mL

HGH-EASIA: REF VALUE

NIGHT

PROLACTIN IMMUNOASSAY

Test for Pituitary hormone would be

homologous competitive binding immunoassay/sandwich technique

PRINCIPLE USED IN PROLACTIN IMMUNOASSAY

PROLACTIN IMMUNOASSAY

uses two or more antibodies directed at different parts of the PRL molecule. however, this type of test can be affected by hook effect

PROLACTIN IMMUNOASSAY

TEST THAT CAN BE AFFECTED BY HOOK EFFECT

HOOK EFFECT

commonly occurs when there is an excess antigen that is present during testing

3-14.7 ng/mL or ug/L

PRL IMMUNOASSAY: REF VALUE

ADULT MALE

3.8-23 ng/mL or ug/L

PRL IMMUNOASSAY: REF VALUE

ADULT FEMALE

95-473 ng/mL

PRL IMMUNOASSAY: REF VALUE

PREGNANCY; 3RD TRIMESTER

ACTH IMMUNOASSAY

this test is useful for determination of the cause of hypercortisolism or hypocortisolism.

chemiluminescence and ELISA

PRINCIPLE USED IN ACTH IMMUNOASSAY

CORTISOL

RELATED TEST FOR ACTH IMMUNOASSAY

ACTH IMMUNOASSAY

reacts with intact ACTH and ACTH fragments

5-80 pg/mL (X 0.22= pmol/L)

ACTH IMMUNOSSAY REF VALUE: ADULTS

EDTA

TUBE REQUIRED IN ACTH IMMUNOASSAY

DYNAMIC FUNCTION TEST

stimulating or suppressing a particular hormonal axis, and observing the appropriate hormonal response

SUPPRESSION TEST

test to conduct if excess is suspected in dynamic function test

STIMULATION TEST

TEST TO CONDUCT IF DEFICIENCY IS SUSPECTED IN DYNAMIC FUNCTION TEST

o Stimulation for Human Chorionic Gonadotropin/ HCG Stimulation Test

o GNRH/ Gonadotropin Releasing Hormone Stimulation Test

o Agonistic Suppression Test

Some Examples of Dynamic Function Test:

INSULIN STRESS TEST

done when hypopituitarism is suspected

hypoglycemic stress

insulin is administered to produce _________

INSULIN STRESS TEST

Tests the ability of Anterior Pituitary Gland to produce ACTH and GH

>6 ug/L

INSULIN STESS TEST: REF VALUE

GH

> 500 nmol/L

INSULIN STESS TEST: REF VALUE

CORTISOL

TRH TEST

assesses the adequacy of Anterior Pituitary Reserve or to evaluate hypothalamic disease

is delayed

TSH response to TRH IS ________

is flat (TSH<2mU/L)

trh test

Hyperthyroidism: pituitary response to TRH _______

exaggerated

trh test

Hypothyroidism: __________ response

IV bolus

TRH is given as an _______

0, 20,

TRH TEST

Blood sampling done at ________ and 60 minutes

GNRH TEST (GONADOTROPIN RELEASING HORMONE)

assesses hypogonadism

GNRH TEST (GONADOTROPIN RELEASING HORMONE)

can be done together with anterior pituitary function test (IST, TRH, GnRH tests)

ACTH SIMULATION TEST

cosyntropin test or tetracosactide test

SMALL AMOUNT

________ amount of synthetic ACTH is injected, and amount of cortisol or aldosterone is measured

ACTH SIMULATION TEST

distinguish whether the cause is adrenal or pituitary

2 HRS BEFORE TESTING

LH OVULATION DIPSTRIP URINE TEST

Patient are advised to reduced liquid intake _______ before testing

LH (LUTEINIZING HORMONE) OVULATION DIPSTRIP URINE TEST

mainly used for infertility test