INTRODUCTION

Pregnancy test detects human chorionic gonadotropin (hCG), which is a hormone secreted by the viable placental tissue. The test may be employed for either qualitative or quantitative estimation of hCG. The term pregnancy test is a misnomer, as it has other diagnostic indications also. Indications for estimation of human chorionic gonadotropin are listed in Box54.1.

Sample: The fresh and first morning urine sample is preferred for testing. The urine should be clear and collected in a clean container (not to wash with soap). No preservatives should be added.

PREGNANCY TESTS

Pregnancy tests may be broadly classified as immunological or biological (outdated) tests.

Immunological Tests for Pregnancy

There are different immunoassays for detecting hCG. Compared to biological methods, they are more rapid, simple, accurate, economical and easily standardized. They are broadly divided into:

A. Immunoassays without radioisotopes

B. Immunoassays with radioisotopes


A. Immunoassays without Radioisotopes

Agglutination inhibition tests

Principle

It is based on latex particle agglutination inhibition. Latex particles coated with purified hCG serve as an antigen capable of reacting with an anti-hCG serum produced in rabbits (e.g. Gravindex test). It is a two stage procedure.

Procedure:

•• One drop of patient’s urine is mixed with one drop of a solution that contains hCG antibody(anti-hCG serum).

•• Then one drop of another solution that contains latex particles coated with hCG is added.

Interpretation:

•• Positive test: Test is considered as positive for hCG if there is no agglutination(homogeneous). If the patient is pregnant, hCG present in the urine will neutralize the antibodies in the antiserum, and added latex particles coated with hCG will not agglutinate.

•• Negative test: Test is interpreted as negative if there is agglutination. If the patient is not pregnant (no hCG in the urine), the antiserum will not be neutralized and will agglutinate the latex particles. The quantitative estimation can be done by serial dilutions of the patient’s urine (24-hour specimen).

Current pregnancy tests use immunoassays with monoclonal antibodies to measure human chorionic gonadotropin (hCG). Serum assays and sensitive urine assays can now detect pregnancy approximately 1 week after conception.

Direct agglutination test (hCG direct test)

Procedure:

Latex particles coated with anti-hCG monoclonal antibodies are mixed with the urine sample to be tested.

Interpretation:

Positive test: Agglutination develops if the urine sample contains hCG and is considered as a positive pregnancy test.

Negative test: Absence of agglutination (urine without hCG) indicates that the test is negative for pregnancy.

Enzyme-linked immunosorbent assay (ELISA)

Principle

This test is based on binding of one monoclonal antibody with hCG and a second antibody that is linked with an enzyme to ‘sandwich’ the hCG. Presence of hCG is detected by a color change. This is more sensitive and specific. Various kits in card forms (card tests) are available (Figs 54.1A and B).

Figs 54.1A and B: Card test showing: (A) Both control and test band (pregnancy test positive); (B) Only control band (pregnancy test negative)

Fluoroimmunoassay (FIA)

It is a highly precise sandwich assay. It uses a second antibody similar to ELISA, but is tagged with fluorescent dye. The fluorescence emitted is proportional to the amount of hCG. It is used not only to detect hCG, but also for follow up hCG concentrations.

B. Immunoassays with Radioisotopes

•• Radioimmunoassay (RIA):

This method employs radioactive isotopes (e.g. I125) to label either the antigen or antibody. It is more sensitive and can detect very small quantity of βsubunit of hCG (up to 0.002 IU/mL) in the serum.

•• Immunoradiometric assay (IRMA): 

This test uses sandwich principle to detect whole hCG molecule. IRMA uses I125 labeled hCG and is also very sensitive.

Biologic Pregnancy Tests

Biologic pregnancy tests employ animals. They are outdated and are of only historical interest.

Principle

Injection of hCG into certain animals produces characteristic anatomic changes either in ovary (production of corpora lutea, pronounced hyperemia) or secretion of sperm or ova in toads and frogs. The biologic effect of the hormone and the sensitivity of different animal species vary greatly. 

In addition, amphibians may also show seasonal variations in sensitivity. Various biological tests and their characteristics are presented in Table 54.1.


Causes of False Negative

•• If the urine sample is allowed to stand for prolonged period at room temperature (8 hours or more), it may result in loss of hCG activity.

•• Test done early in pregnancy may give false-negative result, but it can be prevented by using fresh/first voided morning sample of urine.

•• Ectopic pregnancy and threatened abortion are frequently associated with very low hCG levels.

Points to note:

•• Pregnancy test is employed for either qualitative or quantitative estimation of human chorionic gonadotropin hormone. Tests may be immunological or biological tests. There are several immunoassays which are rapid, simple, accurate and economical.