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PREGNANCY TESTS

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

OVERVIEW 1. Normal pregnancy and levels of beta hcg 2. Pregnancy tests classification 3. beta hcg based tests a. Immunological assays - qualitative assays 1) immunoassays 2) agglutination inhibition assays - quantitative assays b. Biological assays 4. Other applications of beta hcg a. before prescribing certain medications b. part of quadruple/triple/double marker test c. follow up of germ cell tumors treatment d. early diagnosis of ectopic pregnancy e. threatned abortion f. diagnosis and management of gestational trophoblastic diseases

HCG (Human chorionic gonadotropin) HCG is synthesized first by syncitiotrophoblastic cells on outer surface of the conceptus and then by placenta. Main function of HCG is to maintain corpus leuteum during pregnancy so that progesterone is continuously produced by corpus leuteum. This progesterone prevents ovulation and maintains pregnancy. After 8-10 weeks of gestation, progesterone production from placenta starts. Hence HCG is no longer required and its levels start declining and the plateau.

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

* Normal pregnancy and levels of beta Hcg

(Day 14 after LMP)

The blastocyst implants at about 7 days after fertilization (21 days after last LMP)

The trophoblastic cells on the outer surface of the blastocyst penetrate the endometrium and develop into chorionic vili

Placental development occurs from these villi

Beta Hcg is synthesized by syncitiotrophoblasts of placenta That means beta hcg can be detectable in maternal blood 21 days after last LMP

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

In the first trimester, levels double each 2 days, with highest levels obtained at 8-10 weeks of about 1,00,000 mIU/ml

This is followed by a gradual fall

And after 15-16 weeks, a steady state of 10000-20000 mIU/ml is achieved

It becomes non detectable after about 2 weeks

HcG

1,00,000 90,000 80,000 70,000 60,000 50,000 40,000 30,000 20,000 10,000 0 2 12 Weeks 14 16 36 38

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

* Pregnancy tests classification

USG based Transvaginal 5 weeks after LMP Transabdominal 6 Weeks after LMP

Beta HCG based Serum HCG 3 weeks after LMP Urine HCG 5 weeks after LMP

* Tests for beta HCG - IMMUNOASSAYS - QUALITATIVE ASSAYS These tests are rapid and sensitive and can only tell whether beta hcg is present or absent. (i) sandwich ELISA

Principle: 1. Membranes are coated with mouse-anti beta hcg antibody in the test band region and with goat anti mouse antibody in the control region. 2. During testing, the urine specimen is allowed to react with the colored conjugate (mouse anti-hCG monoclonal antibody - colloidal gold conjugate) which was pre-dried on the test strip. 3. The mixture then moves upward on the membrane chromatographically by capillary action. 4. For a positive result, a pink-colored band with the specific antibody hCG- colored conjugate complex will form in the test band region of the membrane.

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

5. For a positive result, a pink-colored band with the specific antibodyhCG- colored conjugate complex will form in the test band region of the membrane. Absence of this pinkcolored band in the test band region suggests a negative result. 6. Regardless of the presence of hCG, as the mixture continues to move across the membrane to the immobilized goat anti-mouse, a pink-colored band at the control band region will always appear. 7. The presence of this pink-colored band serves as 1) verification that sufficient volume is added, 2) that proper flow is obtained, and 3) as a control for the reagents.

INTERPRETATION OF RESULTS: 1. Negative test results in patients suspected to be pregnant should be re-tested with a sample obtained 48 to 72 hours later, or by performing a quantitative assay. 2. When testing with a urine specimen, the first morning specimen would contain the highest concentration of hCG. 3. The shade of pink in the test band region (T) will vary depending on the concentration of hCG present. However, neither the quantitative value nor the rate of increase can be determined by a qualitative test. LIMITATIONs OF PROCEDURE: 1. A number of conditions other than pregnancy, including trophoblastic disease and certain nontrophoblastic neoplasms, cause elevated levels of hCG. These diagnoses should be considered if appropriate to the clinical evidence. 2. If a urine specimen is too dilute (drinking large amounts of water before testing i.e., low specific gravity) it may not contain representative levels of hCG. If pregnancy is still suspected, a first morning urine should be obtained from the patient 48-72 hours later and tested. 3. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the results of a single test, but should only be made by a physician after all clinical and laboratory findings have been evaluated. 4. Immunologically interfering substances such as those used in antibody therapy treatments may invalidate the test result. (ii) AGGLUTINATION INHIBITION ASSAYS (LATEX PARTICLE)

Scenario 1 (patient is pregnant) There is hcg in urine Urine sample + Anti hcg antibodies + hcg coated Latex particles

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

No clumping (Because hcg in urine binds to anti hcg and latex particles coated with hcg remain free)

Scenario 2 (patient is not pregnant) There is no hcg in urine Urine sample + Anti hcg antibodies + hcg coated Latex particles

clumping (Because ther is no hcg in urine, hence anti hcg binds to latex particles coated with hcg) Indirect Test: Suspected serum (containing antibodies) is incubated with donor sperms. Onwards the procedure is similar to direct test.

- QUANITITATIVE ASSAY These give an idea about level of beta hcg in serum/urine. Useful for 1. 2. 3. 4. estimation of gestational age diagnosis of ectopic pregnancy evaluation of threatned abortion management of Gestational trophoblastic diseases

- BIOLOGICAL ASSAYS Effect of hcg is tested on laboratory animals. These tests are outdated and not performed anymore. They are only mentioned here due to academic interests. 1. Ascheim Zondek test Urine from pregnant women injected into female mice. Formation of hemorrhagic corpus leutea in ovary after 4 weeks indicates positive result 2. Friedman test Similar except that female rabbit is used

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

3. Rapid rat test Look for hyperemia and hemorrhage in ovaries after injection of urine containing hcg.

* other applications of beta hcg 1. Exclusion of pregnancy before prescribing certain drugs to avoid teratogenic effects 2. part of triple test for downs syndrome and neural tube defects hcg, AFP and UE3 are measured at 14-16 weeks 3. follow up of ovarian/testicular germ cell tumors which produce beta hcg 4. early diagnosis of ectopic pregnancy Hcg Titre doubles every 2 days till 8-10 weeks to reach maximum of 1,00,000 mIU/ml @ 8-10 weeks This doubling of titre every two days is abnormally slow >6500 mIU/ml >6500 mIU/ml 1000-1500 mIU/ml 1000-1500 mIU/ml Transabdominal USG

Normal pregnancy

Ectopic gestation Normal pregnancy at 6 weeks Ectopic pregnancy at 6 weeks Normal pregnancy at 5 weeks Ectopic pregnancy at 5 weeks

Gestational sacs detected No gestational sac intrauterine Transvaginal USG can detect gestational sac Cant detect intrauterine gestational sac

5. detection of threatned abortion Lack of expected doubling detects threatned abortion 6. diagnosis and management of gestational trophoblastic diseases DIAGNOSIS: 1. GTDs produce more beta hcg as compared to mormal pregnancy at same gestational age 2. concentration of hcg parallels tumor load 3. hcg continues to rise beyond 10 weeks of gestation without plateauing.

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

MANAGEMENT: 1. After evacuation of v. mole, weekly estimations are indicated will three consecutive samples are negative for hcg. 2. Normally hcg becomes negative in three months after evacuation, if not, chemotherapy is indicated 3. once results are negative (by themselves or by chemo), regulary follow up every three months for 1-2 years.

Notes on pregnancy tests.. By Dr. Ashish Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes