Urine Pregnancy Test (UPT): Principle, Procedure, Interpretation and Limitations

There are two types of tests for pregnancy testing: one uses blood sample and other uses urine. Both tests detect the presence of beta subunit of a hormone called human chorionic gonadotropin (hCG). The Urine pregnancy test (UPT) is an easy and rapid chromatographic immunoassay for the qualitative detection of human chorionic gonadotropin (hCG) in urine for the early detection of pregnancy.

Human Chorionic Gonadotropin (hCG)

Human chorionic gonadotropin (hCG) is a hormone produced by the developing placenta shortly after fertilisation. In normal pregnancy, hCG can be detected in urine as early as 7 to 10 days after conception. Levels of hCG rise rapidly, frequently exceeding 100mIU/mL by the first missed menstrual period, and peaking at 100,000-200,000 mIU/mL about 10-12 weeks into pregnancy. The appearance of hcG soon after conception and it’s subsequent rise in concentration during early gestational growth make it excellent marker for the early detection of pregnancy.

Principle of Urine Pregnancy Test

Most of the urine pregnancy test kits are based on lateral-flow technology. Most of them qualitatively detect the presence of hCG in urine specimen at the sensitivity of 25 mIU/mL. The test uses two lines to indicate results. The test line utilizes a combination of antibodies including a monoclonal hCG antibody to selectively detect elevated levels of hCG. The control line is composed of goat polyclonal antibodies and colloidal gold particles.

The assay is conducted by adding a urine sample to the sample well of the test device and observing the formation of colored lines. The sample migrates via capillary action along the membrane to react with the colored conjugate.

Positive samples react with the specific antibody-hCG-colored conjugate to form a colored line at the test line region of the membrane. Absence of this colored line suggests a negative result. A colored line will always appear in the control line region if the tests has been performed properly.

Specimen Collection

Collect urine into a clean, dry container. The first morning urine specimen is preferred since it generally contains the highest concentration of HCG; however, urine specimens collected at any time of the day may be used. Refrigerate specimens at 2° to 8°C (36° to 46°F) for up to 72 hours, if the testing is not performed immediately. If samples are refrigerated, bring them to room temperature before testing.


  1. Allow the Pregnancy Test Strip and urine sample to reach room temperature (15-30°C) before opening the foil pouch.
  2. Remove the Pregnancy Test Strip from the pouch and use it as soon as possible.
  3. Place the test device on clean and level surface. Hold the dropper vertically and transfer 3 full drops to the specimen well and start the timer. Avoid air bubble formation.
  4. Wait for the colored line(s) to appear. Read the result after 5 minutes. Do not read the result after 15 minutes.

Results and Interpretation

POSITIVE: Two coloured lines appear. One line should be in the Control region (C) and another line should be in the Test region (T). This means there is a strong possibility that patient is pregnant.

NEGATIVE: One coloured line in the Control region (C). No apparent colored line appears in the Test region (T). This means patient is either not pregnant or has tested too early.

INVALID: The result is invalid if the Control Line (C) fails to appear. Insufficient volume of urine or incorrect procedure are the most likely reasons for an invalid result.


  1. In cases where very high levels of HCG are present (>500,000 mlU/ml) a false negative result can occur due to a “Prozone” effect. If pregnancy is still suspected, simply dilute specimen 1:1 with deionized water and retest.
  2. If a urine sample is too dilute (ie: low specific gravity) it may not contain a representative level of HCG. If pregnancy is still suspected, a first morning urine sample should be obtained and retested 48 hours later.
  3. A number of conditions other than pregnancy, including trophoblastic disease and certain non-trpohoblastic neoplasms including testicular tumors, prostate cancer, breast cancer and lung cancer cause elevated levels of hCG. Therefore, the presence of hCG in urine should not be used to diagnose pregnancy unless these conditions have been ruled out.
  4. Drugs containing hCG may interfere with the test, and produce misleading results.
About Dhurba Giri 33 Articles
Dhurba Giri is the founder and content creator of LaboratoryTests.org. He is a Medical Laboratory Technologist and Scientific Blogger from Pokhara, Nepal. Connect with him:


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