pregnancy test

1. shaibana
PREGNANCY TESTS

2. • Pregnancy in simple terms is the period from
conception to birth. This period is usually 40 weeks
after the Last Menstrual Period (LMP)
• Pregnancy Test (PT) attempts to determine whether
a woman is pregnant.
INTRODUCTION

3. • Pregnancy test is a misnomer as most of the
methods employed measure human chorionic
gonadotropin(hCG) and not the presence of fetus
• Different methods had been designed since the
early times for the detection of pregnancy

4. Indications
Diagnosis and confirmation of early normal uterine
pregnancy.
Evaluation of ectopic pregnancy.
To monitor pregnancy following in vitro fertilization and
embryo transfer.
o Diagnosis and follow up of
Gestational trophoblastic disease:
• Hydatidiform(vesicular) mole
• Invasive mole
• Choriocarcinoma(gestational or nongestational)

5. • Records of attempts at pregnancy testing have been
found as far back as the ancient Greek and ancient
Egyptian cultures.
• The ancient Egyptians watered bags
of wheat and barley with the urine of a possibly
pregnant woman. Germination indicated pregnancy.
HISTORY

6. • Hippocrates suggested that a woman who had
missed her period should drink a solution of honey in
water at bedtime: resulting abdominal distention
and cramps would indicate the presence of a
pregnancy.
• Selmar Aschheim and Bernhard Zondek introduced
testing based on the presence of human chorionic
gonadotropin (hCG) in 1928.

7. • In the Aschheim and Zondek test, an infantile
female mouse was injected s/c with urine to be tested,
and the mouse later was killed and dissected. Presence
of ovulation indicated that the urine contained hCG
and meant that the person was pregnant.
• A similar test was developed using immature rabbits.
Here, too, killing the animal to check her ovaries was
necessary.

8. • By the arrival of frog test, introduced by Lancelot
Hogben, used still 1950s and allowed the frog to
remain alive and be used repeatedly: a female
frog was injected with serum or urine of the patient;
if the frog produced eggs within the next 24 hours,
the test was positive.

9. • Hormonal pregnancy tests such as Primodos
and Duogynon were used in the 1960s and. These
tests involved taking a dosed amount of hormones,
and observing the response a few days later.
• A pregnant woman does not react, as she is
producing the hormones in pregnancy; a woman not
pregnant responds to the absence of the hormone
by beginning a new menstrual cycle.

10. • Immunologic pregnancy tests were introduced in
1960 when Wide and Gemzell presented a test
based on in-vitro hemagglutination inhibition.
• Direct measurement of antigens, such as hCG, was
made possible after the invention of the
radioimmunoassay in 1959. Radioimmunoassays
require sophisticated apparatus and special
radiation precautions and are expensive.

11. • Hormone secreted by the chorionic cells after
fertilization of ovum.
• Primary function is to support the corpus luteum until
the placenta produces amounts of progesterone
sufficient to support the pregnancy.
• The hormones appears in urine and serum after 9 days
of conception, reaches a peak between 7th and 12th
week of gestation, after which it decreases
HUMAN CHORIONIC GONADOTROPIN

12. • It has 2 subunits:-
– α – LH, FSH and TSH
– β- specific and used for diagnosis.
• Other positive conditions
– Chorio carcinoma
– Renal cell carcinoma
– Hydatidiform mole

13. • Urine first morning sample should be tested.
• If stored in refrigerator, test within 72 hours.
• For longer storage sample should be frozen at -20°C.
• hCG concentration is expressed as mIU/ml.
SPECIMEN

14. 1. Bio-assays
2. Immuno-assays
3. Radio immuno-assays
4. Radio receptor assays
5. Immuno metric assays
METHODS FOR MEASURING β-Hcg LEVEL

15. • In bio assays the test are performed by using
experimental animals.
• The biological test for pregnancy can be performed
only after 2-3 weeks of conception, so that the
concentration of hCG in urine is sufficient to show
results.
Bio assays

16.  Aschheim- Zondak test.
 It depends upon the ovarian changes in immature mice
caused by hCG.
 2 ml of urine from the women suspected to be pregnant is
injected daily for 2 days into immature mice.
 5 days after injection of urine the mice are killed.

17. • The ovaries are examined
for the presence of
corpus leuteum and
haemorrhage which
indicates ovulation.

18.  Kupperman test
• This test is the modification of aschheim-zondak
test in order to save the animals.
• In this an immature rat is used.
• About 2 ml of urine is injected sub cutaneously into
immature rat and ovarian changes observed after 6
hours.
• If urine is injected intraperitoneally the ovarian
changes can be observed within 2 hours.

19.  Freidman test
In this tests, 10-15ml
of urine is injected intra
venously into rabbit and
ovulation is observed by
examining the ovaries
after 48 hours

20.  Hogben test.
In this test, about
20-30 ml of urine is
concentrated and
injected into the dorsal
lymph sac of south
african toad, xenopus
levis. If hCG is present in
urine it causes ovulation
after 12 hours.

21.  Galli-mainini test
In this test, 2 ml of urine is injected into the male
amphibian (toad or frog). hCG in urine causes
expulsion of spermatozoa with in 2 hours.

23. 1. The biological test require animals
2. Tests can be performed only after 2 to 3 weeks of
pregnancy so that sufficient quantity of hCG is
excreted in urine
3. Results are not obtained quickly; one has to wait for 2
to 48 hours
4. Tests involve tedious procedures such as sacrificing
the animals
Disadvantages of Biological Tests

24. • Simple, expensive and rapid test
• Not very sensitive, therefore more often used for
qualitative or semi quantitative detection of hCG
Immuno assay

25.  Latex particle agglutination inhibition assay
• In his assay, latex particles coated with hCG serum
and patients urine.
• In pregnancy, the hCG in urine will neutralize with
hCG, so there will be no agglutination with hCG
coated latex particle.
• Thus no agglutination indicates a positive test (ie.,
presence of hCG in patients urine) and agglutination
indicates a negative test.

26. • The test performed on a slide and requires only a few
minutes.
• Sensitivity :- 500-4000mIU/ml

27.  Haem agglutination inhibition assay(HAI)
Similar to LAI, except that erythrocytes are
coated with hCG instead of latex particles.
More sensitive then LAI test and can detect 150-
4000 mIU/ml.

28.  Direct agglutination test
In this test Ab to hCG is coated either on latex
particles or on erythrocytes. There for agglutination
occurs in the presence of hCG in the patients urine.
Agglutination indicates a positive test.

29. • The RIA’s for hCG detection are based on the
principle of competitive binding.
• hCG in test serum and a radio labeled hCG compete
for binding sites on anti hCG Ab.
• The bound radio labeled hCG is related to the
concentration of hCG in the specimen.
• Sensitivity : very sensitive quantitative test and can
detect hCG less than 5mIU/ml
Radio immuno assay

31. • In this assay, a radio labeled target tissue such as
testis or ovary of rats, pigs, or cows is used for
binding with hCG from the specimen.
• The resultant radioactivity is proportional to amount
of hCG in the specimen.
• Sensitivity : 20-50mIU/ml
Radio receptor assay

33. • This type of assay uses hCG Ab labeled with enzyme/
radioisotope for the detection of hCG in the
specimen.
• The principle is similar to the sandwich technique for
detection of Ag by ELISA.
• Sensitivity : 25-50mIU/ml.
Immunometric assay

34. 1. Immunological tests are more accurate
2. Result is obtained quickly within few minutes
3. These tests can be carried out very easily. The
procedure is not cumbersome, as in the case of
biological tests
4. Immunological tests can be performed on 5th day of
conception.
5. More sensitive and involve single step method
ADVANTAGES OF IMMUNOLOGICAL TESTS
FOR PREGNANCY

35. • In a normal pregnancy, trophoblast secrete a
detectable amount of hCG almost immediately on
implantation.
• The level of hCG rises rapidly until reaching a peek of
appx. 1 lakh mIU/ml of serum after 60-80 days.
• It will decline from peek to plato ie., 10000-
20000mIU/ml at 15-16 week and maintain it for
remaining period of pregnancy.
DIAGNOSIS OF NORMAL PREGNANCY

36. • Results depends on the sensitivity of test used.
• Serum hCG values ranges from 100-100000mIU/ml.

37. • Serum hCG values are significantly lower than normal
uterine pregnancy.
• Serum hCG values range from 20-20000 mIU/ml after
4-8 weeks of ectopic gestation.
• In serum specimens collected at intervals over 48
hours period normal pregnancy shows 60% rise in
hCG where as in ectopic pregnancy, the rise wil be <
30%.
DIAGNOSIS OF ECTOPIC PREGNANCY

38. • In trophobastic disease very high values of hCG is
seen( 3-100 times higher than normal pregnancy.

39. Principle
• Inhibition of agglutination:-
The urine is 1st treated with anti hCG reagent and
then reacted with the latex particles coated with hCG.
If the urine contain hCG the anti hCG will be
neutralised. When latex coated hCG is added
afterwards, agglutination reaction will not take place
since anti hCG is already neutralized. Hence the
reaction is indicated by lack of agglutination.
Gravindex method

40. Specimen
 First morning sample/ random urine sample is
collected in a clean dry container. It must be
collected at least 12 days after the first missed
menstrual cycle.
 In the case of negative result repeat the test after
1 week.

41. Requirements
 Granvindex test kit contains
anti hCG serum
hCG latex Ag
test slide
dispossible applicator stick

42. Procedure
1. Bring all the reagents to room temperature.
2. Place 1 drop of urine on the ring of the test slide
3. Add 1 drop of anti hCG reagent. Mix well by using
applicator stick.
4. After about 30 seconds, add 1 drop of hCG latex Ag.
5. Mix again with the applicator stick and spread out the
pool of liquid uniformly with in the entire area of the
ring on the testslide. Observe for 2 min.
6. Simultaneously perform the test on negative control.

43.  Observation
Patient urine Latex particle
agglutination with in 2
min
Negative
Patient urine Homogenous suspension,
no agglutination.
Positive
Negative
control urine
Latex particle
agglutination within 2 min
negative

44.  Direct test
Urine drop Latex reagent 1 drop
which contains hCG Ab
coated particles.
agglutination
Presence of
hCG Ag
hCG Ag cambines with Ab positive
Urine drop Latex reagent 1 drop
which contains hCG Ab
coated particles.
No
agglutination
hCG Ag
absent
No hCG Ag to combine
with Ab
negative

45.  Indirect test
Urine drop Antiserum 1
drop which
contain hCG Ab
Latex reagent 2
drops which contain
hCG Ag coated
particle
No
agglutination
Presence of
hCG Ag
hCG Ag
combines with
hCG Ab
No hCG Ab to
combine with latex
particles
positive
Urine drop Antiserum 1
drop which
contain hCG Ab
Latex reagent 2
drops which contain
hCG Ag coated
particle
agglutination
Absence of
hCG Ag
hCG Ab remains
free
hCG Ab combines
with latex particles
negative

46. Principle
 This is 2 side sandwich assay in which anti serum
hCG is used both in conjugate form as well as in
native form.
 It is a quantitative test for the determination of
hCG in urine. The test is based on the ELISA
method.
Visipreg strip and card method

47.  Procedure
Strip containing dry conjugate of anti-hCG is drop in
the urine upto the indicator level.
If hCG is present, it will react with ant hCG conjugate
to form complex of anti-hCG conjugate hCG.
This complex migrates forward on the membrane
predisposed with anti serumanti-hCG in native form

48. Immobilized anti hCG is able to capture the hCG
molecule of migration causing agglutination of anti
serum
A variable purple band is formed along the exact
location of immobilized anti-hCG antiserumis the
positive result
No colored band indicates negative result

50. • The test for pregnancy which can give the quickest
result after fertilisation is a rosette inhibition
assay for early pregnancy factor (EPF).
• EPF can be detected in blood within 48 hours
of fertilization.
• Testing for EPF is expensive and time-consuming.
MODERN TESTS

51. Thank you……