Test Methodology 121 Qualitative Human Chorionic Gonadotropin By Immunoassay

Qualitative tests provide results that are either positive or negative for the substance to be tested, while quantitative tests provide numerical values as results. In the case of qualitative hCG, a positive test result means the patient is most likely pregnant and a negative result indicates the patient is most likely not pregnant. This test, performed using a urine specimen, is usually less sensitive than a quantitative test but also is a simpler to perform and less expensive. This type of test, used in satellite laboratories or physician office laboratories, is categorized as waived testing under federal regulations and is available for patients as a home test kit. The test requires only a few steps and the results are read within a few minutes by color change reaction. The result will be positive when the hCG concentration is at least 30 IU/L, which is often only a day or two after the first missed menstrual period. In terms of gestational age, this is around day 30 of the last menstrual period.

The Reaction Principle

Antibody specific to whole molecule hCG or the beta polypeptide chain is bound to a nitrocellulose filter paper and reacts with patient hCG from the urine sample. Unbound materials are washed away with the flow of urine sample over the filter paper while peroxidase-labeled antibody that is specific to the hCG-anti-hCG complex flows into the area. In this step, the patient hCG becomes sandwiched between the two antibodies. A benzedrine substrate and buffers are then added, and an insoluble colored product forms on the filter paper to indicate the presence of patient hCG.

Interferences

False-positive results occur because of interference from some proteins, drugs, bacteria, or erythrocytes or leukocytes in the urine. Using centrifuged urine can eliminate interference from particles. Positive hCG results should correlate with the patient's history and physical examination. Negative results may be due to a very dilute urine or urine that has been exposed to heat. Generally a negative result indicates a need to retest the patient urine in a few days if menses do not begin.

The Specimen

The first morning urine, centrifuged if necessary to remove cells and particulates, is used. The specimen should be stored at refrigeration temperature if not tested within a few hours or frozen if not tested within 24 hours.

Reference Range

Negative for the nonpregnant woman.

ovarian ligaments. This condition appears to be on the increase but is most likely reported more frequently due to improved diagnostic tools. Ectopic pregnancy remains one of the most common causes of maternal death during the first trimester, typically characterized by severe pain and hemorrhage as with clotting disorders, a more severe complication that may lead to death. The cause of ectopic pregnancy is most likely scarred or obstructed fallopian tubes secondary to sexually transmitted disease or repeated pelvic inflammatory disease.6

The ectopic pregnancy can be visualized with ultrasound by about 6 weeks after the LMP when hCG levels are 1500 IU/L or higher.7 Ultrasound imaging is generally used to verify the presence of ectopic pregnancy, and surgical intervention is usually required to stop the bleeding and repair the site.7

TEST METHODOLOGY 12-2. QUANTITATIVE hCG BY ENZYME IMMUNOMETRIC ASSAY

Quantitative hCG tests are more sensitive than qualitative tests, detecting hCG levels at 5 to 10 IU/L, and are necessary when ruling out pregnancy before the first menstrual period. This information regarding very early pregnancy is helpful prior to surgery or other potentially harmful medical interventions. Quantitative hCG tests require an automated instrument to read results, often by some labeled immunoassay method, and standard solutions of hCG for instrument calibration and calculation of results. Serial hCG levels are helpful to rule out abnormalities in the pregnancy; however, for accurate interpretation of test results, such testing is best performed by the same laboratory using the same methodology.5 In a healthy pregnancy, hCG will double about every 2 days during weeks 2 to 5, while in abnormal pregnancies such as ectopic pregnancies or in impending miscarriage, the hCG levels do not double as quickly or may even decline. Measurement of hCG at 16 to 20 weeks' gestation is used in combination with other tests (e.g., chromosomal tests) for prediction of Down syndrome, in which the hCG is generally higher than the reference range.

The Reaction Principle

This is a double-antibody technique in which first antibody to hCG is bound to the whole molecule, followed by an enzyme-labeled antibody specific to the beta polypeptide chain (anti-hCG:anti-hCG beta sandwich). Following addition of substrate and buffers, the colored product forms in proportion to the patient's hCG level and is measured photometrically.

Interferences

Hemolysis, lipemia, or turbidity may account for interfering absorbances. The Specimen

Fresh serum free from hemolysis, lipemia, or turbidity should be tested within a few hours or stored at -20°C until testing can occur.

Reference Ranges

Not Pregnant <5 IU/L

Spontaneous abortion, more commonly referred to as miscarriage, is failure to maintain the pregnancy to full term or until a viable baby can be delivered. This sudden, unplanned disruption in the pregnancy is associated with vaginal bleeding, cramping pain, a previous positive hCG test, and decreasing hCG levels over a 48hour period. History, physical examination, and possibly imaging techniques are often necessary to rule out ectopic pregnancy.8 A normally progressing pregnancy will result in a doubling of hCG in 2 to 3 days. Likewise, an intrauterine pregnancy can usually be visualized accurately with ultrasound by about 5 to 6 weeks after the LMP, when hCG levels are 6500 IU/L or higher.1 Newer ultrasound techniques may detect pregnancies at earlier stages,7 but there are higher false-positive and false-negative rates.1 There are many causes of miscarriage, but the most common is an abnormally developing blastocyst or embryo. Over half of the spontaneous abortions that occur in the first trimester result from an abnormality in the

4 Weeks' gestation after LMP

5 Weeks' gestation after LMP

6 Weeks' gestation after LMP 15-26 Weeks after LMP

5-100 IU/L 200-3000IU/L 10,000-80,000 IU/L 5000-80,000 IU/L

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