this is a series of notes on clinical pathology, useful for undergraduate and post graduate pathology students. Notes have been prepared from standard textbooks and are in a format easy to reproduce in exams.

1. 1
PREGNANCY TESTS
Notes on pregnancy tests.. By Dr. Ashish Jawarkar
Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

2. 2
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

3. 3
* 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

4. 4
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
14 16
36
Weeks
Notes on pregnancy tests.. By Dr. Ashish Jawarkar
Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
38

5. 5
* Pregnancy tests classification
USG based
Beta HCG based
Transvaginal
Transabdominal
Serum HCG
Urine HCG
5 weeks after
LMP
6 Weeks after
LMP
3 weeks after
LMP
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
T
C
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

6. 6
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
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7. 7
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

8. 8
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 down’s 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
Normal pregnancy at 6 weeks
Ectopic pregnancy at 6 weeks
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
Normal pregnancy at 5 weeks
1000-1500 mIU/ml
Ectopic pregnancy at 5 weeks
1000-1500 mIU/ml
Normal pregnancy
Ectopic gestation
Transabdominal USG
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

9. 9
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