large doses of hCG are required, concentration by

alcohol precipitation or kaolin adsorption is necessary for

adequate sensitivity.

Rat Ovarian Hyperemia Test of Frank and Berman

Two immature female rats weighing 45 to 60 g each are

given two injections of urine/serum and sacrificed 24 hours

later using carbon monoxide. Positive result is indicated by

ovarian hyperemia due to capillary dilatation associated

with thecal cell hyperplasia.

Tests Using Male Toad/Male Frog

Galli-Mainini used male toad Bufo arenarum, while

Wiltberger and Miller used male frog Rana pipiens: 4 to

6 hours after injection with hCG, these animals release

sperm which can be detected microscopically. For

adequate sensitivity concentration is required.

Normal Values of hCG (Serum/Plasma)

hCG SI units

Males < 5.0 mlU/mL < 5.0 IU/L

Females

Nonpregnant < 5.0 mlU/mL < 5.0 IU/L

< 1 week gestation 5–50 mIU/mL 5–50 IU/L

2 weeks gestation 50–500 mlU/mL 50–500 IU/L

3 weeks gestation 100–10,000 mlU/mL 100–10,000 IU/L

4 weeks gestation 1000–30,000 mlU/mL 1000–30,000 IU/L

5 weeks gestation 3500–115,000

mlU/mL

3500–115,000 IU/L

6 to 8 weeks

gestation

12,000–27,000

mlU/mL

12,000–27,000

IU/L

12 weeks gestation 15,000–220,000

mlU/mL

15,000–220,000

IU/L

412 Concise Book of Medical Laboratory Technology: Methods and Interpretations

Comments

With all bioassays, drugs excreted in the urine may cause

decreased sensitivity and false negative tests or even

death of the animal. Quinidine, barbiturates, laxatives,

antihistamines, sulfonamides, salicylates, antibiotics,

ergot and morphine derivatives may cause interference.

So, the patient should discontinue all medication

3 to 4 days prior to the test. High concentrations of urinary

electrolytes (especially K+), bacteria and unspecified

endogenous substances, all of these may cause false

negative reactions.

Toxic substances may be removed partially or

completely from urine by: (i) dialysis in cellophane tubing

for 30 to 60 minutes under running water, (ii) acidification

with 0.1 N hydrochloric acid to a pH of about 6.0,

(iii) extraction with ether, or (iv) absorption with kaolin.

False positive reactions may be caused by high titers of LH

and/or FSH due to menopause or primary ovarian failure,

as well as number of drugs, especially phenothiazines,

prochlorperazine, and promazine.

Concentration of hCG is often expressed in animal units.

There are rat units, mouse units, male and female toads,

frog units, etc. These units are difficult to compare. More

accurate is the international unit (IU) of hCG, related to

specific gonadotropic activity of 0.1 mg of a dried standard

kept at the National Institute for Medical Research,

London. This is the amount of activity sufficient to cause

cornification of the vaginal epithelium of immature rats.

IMMUNOLOGIC METHODS

1. Hemagglutination inhibition test.

2. Latex particles agglutination inhibition test.

3. Radioimmunoassays for the β-subunit of hCG

(this is by far the most sensitive and specific method

available).

4. ELISA/Immunochromatography.

Most routine pregnancy tests have a sensitivity of

about 0.7–1.0 IU/mL. ELISA tests have sensitivity of up to

20 mIU/mL.

Immunologic Tests for Pregnancy

If purified hCG is injected into an animal, a specific hCGneutralizing antibody will be formed. Several tests for the

presence of hCG in urine depend on the specificity of an

antigen-antibody reaction.

Before proceeding with the test, perform the heat

coagulation test for proteinuria, if positive—a false positive

pregnancy test can be anticipated. The test is done in one

of the two main ways, which differ in the carrier used for

the external source of hCG. In one method, the patient’s

urine—1 drop—is mixed with 1 drop of an antiserum. The

mixture is then incapable of reacting with the hCG carried

on a suspension of latex particles which is added next and

no agglutination now occurs.

If the test urine does not contain hCG, the added

antiserum will not be neutralized, when the hCG-coated

latex principle suspension is added, the still active antiserum will react with the hCG on the particles causing

them to agglutinate. A positive result is thus shown by an

even suspension of the latex particles, a negative result, by

clumping. This, then, is a latex agglutination inhibition test

(False positive results may occur in proteinuria). In some

methods, the latex particles are coated with hCG antibody,

hence, the reversal of readings and interpretations should

be considered, i.e. agglutination implying a positive result.

In the other type of method, performed in a tube, the

external hCG, of the system is carried on red cells, which

are said to be sensitized to hCG. The test urine is mixed

with anti-hCG serum, and then with a suspension of

sensitized red cells. If the urine contains hCG, the antihCG serum will be neutralized and the sensitized red

cells will be unaffected, they fall to the bottom of the tube,