1. pregnancy tests

2. Serology of Human
Chorionic Gonadotrophin
(hCG) Hormone

3. From the earliest stage of development (9
days old),
the placenta produces hormones, either on
its own or in conjunction with the fetus.

4. The very young placental trophoblast
produces appreciable amounts of a
hormone(HCG) that is excreted in the
urine.

6. Human chorionic gonadotropin is
not found in the urine of normal,
young, nonpregnant woman.

7. hCG is composed of two sub units, alpha and beta. The
alpha sub unit is common to all glycoproteins and the beta
sub unit confers unique specificity to the hormone.

8. pregnancy tests are based on the detection of hCG
in serum or urine.
Its small size permits it to pass directly into the
urine from the circulation

10. Early in pregnancy, concentration of
hCG in maternal serum rises quickly,
with a doubling time of roughly two
days during the first few weeks as the
trphoblastic tissues increase in size.

11. Within 10 to 12 weeks, hCG values with
peak at 150,000 to 200,000 mIU/ml and
then gradually fall to normal plateau values
of 10,000 to 50,000 mIU /ml in the second
and 3rd trimesters

12. A later- term pregnancy in which there is a
sudden drop in hCG from the plateau may
indicate threatened abortion.
Ectopic pregnancies have much lower hCG
values .
Molar pregnancies and other trophoblastic
malignancies can have very high values of hCG,
considerably beyond those encountered in
normal pregnancy

14. Monoclonal based assay which uses
two different antibodies, one against the
α- subunit and one against the β sub unit
in a sandwich that capture the whole hCG
molecule on a solid phase is being
developed

15. Detection or quantitation of hCG is
then generally accomplished by a color
indicator reaction mediated by an
enzyme (e.g., alkaline phosphatase)
linked to the second antibody

17. Serology of HCG in Urine

18. HCG can be detected in the urine of pregnant
women 26 to 36 days after the first day of the last
menstrual period or 8 to 10 days after conception.
Pregnancy test should be negative 3 to 4 days
after delivery.

19. If the measured value in urine is negative,
but clinical examination indicates possible
pregnancy, the test should be repeated in
two days.
Urine pregnancy test may be negative even
though serum tested at the same time is
positive because the serum assay is more
sensitive.

21. is preferable because
early morning specimen
An
this is the most concentrated
If the specimen cannot be tested immediately, it
should be refrigerated at 4c, but for not longer
than 48 hours.

22. Urine pregnancy tests
Laboratory pregnancy tests are based on the
detection of rapidly rising levels of hCG in urine.
Immunologic pregnancy tests are done in one of
two ways.
Urine pregnancy testing kits can be divided in to

23. Rapid latex slide tests of the inhibition
indirect And direct type
Haemagglutinaton inhibition

24. latex slide test
indirect)
Inhibition (
in this type of test, two reagents are
supplied,
an antiserum containing anti hCG
antibody
a latex reagent consisting of polystyrene
particles sensitized (coated) with hCG,
positive and negative controls etc.

25. In the inhibition test, urine is first mixed with the antiserum,
and the latex reagent is added.
- If hCG is present in the urine, it will combine with the anti hCG antibody.
This will leave no antibody free to combine with the latex hCG and
therefore, there will be no agglutination of the latex particles.
- If there is no hCG in the urine, the antibody will be free to combine with
the latex hCG and cause agglutination of the latex particles. - In this test,
therefore, no agglutination indicates a positive test and agglutination
indicates negative test.

27. Direct latex slide test
Are more sensitive than inhibition tests.
In this test, the latex reagent consists of particles coated with
the anti- hCG antibody. This reagent is mixed directly with the
urine.
If hCG is present in the urine, it will combine with the
antibodies and cause agglutination of the latex
particles.
If no hCG is present in the urine, there will be no
agglutination of the latex particles. In this test,
therefore, agglutination of the particles indicates
positive test and no agglutination indicates a negative
test.

28. Inhibition tube haemagglutination
In this type of test, the principle is the same as in the latex slide
test except that the hCG is coated on red cells, not on polystyrene
particles.
The urine is reacted with anti hCG antiserum in the small tube
provided, and red cells coated with hCG are added. The contents of
the tube are mixed and then left at room temperature (20-28 OC)
for 1 ½ -2 hours to allow time for the red cells to settle.

29. If the urine contains hCG, it will combine with the antibody. This
will leave no antibody to react with the hCG on the red cells.
The non-agglutinated cells will settle and be seen as a red ring in
the bottom of the tube.
- If the urine contains no hCG, the anti hCG antibody will react
with the hCG on the red cells and cause their agglutination
(haemagglatination). The agglutinates will settle and be seen
covering evenly the bottom of the tube.
- In the inhibition (indirect) haemagglutinatan tube test,
therefore, a red ring of non-agglutinated cells in the bottom of
the tube indicates a positive test and a covering of agglutinated
cells indicates a negative test

31. Semiquantitative test
the amount of hCG in specimen can be measured
semiquantitatively by preparing serial dilutions of the specimen
in physiological saline and testing each dilution.
Most manufactures of slide and tube tests provide details of
how to perform a semiqntitative technique.
A more accurate result is obtained by using a tube technique.
Quantitative analysis of hCG aids in making a differential
diagnosis of a viable pregnancy versus a nonviable pregnancy,
twins or multiple gestations,

33. Factors that affect urine pregnancy test

34. The time in the pregnancy when the test is carried
out.
Interfering substance (drugs , red cell etc) & sensitivity
specificity of the assay.
Negative or inconclusive results may occur if the
concentration of HCG in the urine is below that which
the test is capable of detecting reliably.

35. The presence of excessive amounts of protein or
blood in the urine may cause false positive results
The presence of detergent can cause false positive
or false negative result.

36. Turbid specimens (due to amorphous debris or
epithelial cells) may give inconclusive results. Such
specimens should be filtered or centrifuged.
Bacterial contamination of the urine may cause unreliable
results. Heavily contaminated urine is unsuitable for testing.
Important- always read carefully the manufacturer's
information leaflet

37. Pregnancy test strip (RDT)

39. Reaction zone: contains soluble anti-hCG antibody-
Enzyme. These are mouse monoclonal antibodies linked
to an enzyme.
The test zone: contains immobilized polyclonal mixture
of hCG- antibody + dye substrate.
The control zone: the dye substrates + anti-(anti-HCG Ab-
Enzyme conjugates) Ab can recognize epitopes on the
mouse monoclonal

40. A few drops of urine is transferred to the specimen well
(loading well).
• Urine will flow by capillary action from loading well
towards zone R carrying along with it the HCG hormone.
• At R zone, the HCG hormone will react and bind with
the soluble anti-HCG Ab-enzyme conjugates forming a
complex of HCG hormone - HCG Ab - enzyme conjugate.
• This complex will migrate towards zone T.

41. • At zone T, this complex will react and bind with the
immobilized anti-HCG Ab, once it binds with the
immobilized Ab, this will activate the enzyme thus
allowing to act on the dye substrate and produce a
color that indicates a positive pregnancy result.
• The excess free HCG Ab - enzyme conjugates will
pass from zone T to zone C.