PREGNANCY TESTS

shaibana
INTRODUCTION
• 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.
• 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
 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)
HISTORY
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
HUMAN CHORIONIC GONADOTROPIN
• 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
• 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
SPECIMEN
• 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.
METHODS FOR MEASURING β-Hcg LEVEL
1. Bio-assays
2. Immuno-assays
3. Radio immuno-assays
4. Radio receptor assays
5. Immuno metric assays
Bio assays
• 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.
 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.
• The ovaries are examined
for the presence of
corpus leuteum and
haemorrhage which
indicates ovulation.
 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.
 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
 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.
 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.
Disadvantages of Biological Tests
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
Immuno assay
• Simple, expensive and rapid test
• Not very sensitive, therefore more often used for
qualitative or semi quantitative detection of
hCG
 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.
• The test performed on a slide and requires only a few
minutes.
• Sensitivity :- 500-4000mIU/ml
 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.
 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.
Radio immuno assay
• 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 receptor assay
• 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
Immunometric assay
• 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.
ADVANTAGES OF IMMUNOLOGICAL
TESTS FOR PREGNANCY
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
 DIAGNOSIS OF NORMAL PREGNANCY
• 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.
• Results depends on the sensitivity of test used.
• Serum hCG values ranges from 100-100000mIU/ml.
DIAGNOSIS OF ECTOPIC PREGNANCY
• 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%.
• In trophobastic disease very high values of hCG is
seen( 3-100 times higher than normal pregnancy.
 Gravindex method
 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.
 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.
 Requirements
 Granvindex test kit
contains anti hCG serum
hCG latex Ag
test slide
dispossible
applicator
stick
 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.
 Observation
Patient urine Latex particle Negative
agglutination with in 2
min
Patient urine Homogenous suspension, Positive
no agglutination.
Negative Latex particle negative
control urine agglutination within 2 min
  Direct test
Urine drop Latex reagent 1 drop agglutination
which contains hCG Ab
coated particles.
Presence of hCG Ag cambines with Ab positive
hCG Ag

Urine drop Latex reagent 1 drop No

which contains hCG Ab agglutination
coated particles.
hCG Ag No hCG Ag to combine negative
absent with Ab
  Indirect test
Urine drop Antiserum 1 Latex reagent 2 No
drop which drops which contain agglutination
contain hCG Ab hCG Ag coated
particle

Presence of hCG Ag No hCG Ab to positive

hCG Ag combines with combine with latex
hCG Ab particles

Urine drop Antiserum 1 Latex reagent 2 agglutination

drop which drops which contain
contain hCG Ab hCG Ag coated
particle

Absence of hCG Ab remains hCG Ab combines negative

hCG Ag free with latex particles
Visipreg strip and card method
 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.
 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
 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

MODERN TESTS
• 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.
Thank you……