Pregnancy Testing
Terry Kotrla, MS, MT(ASCP)BB
Austin Community College
Introduction
• Most pregnancy tests used today, whether a
home urine test, a physician's office urine or
blood test, or a clinical laboratory blood test are
"sandwich assays".
• Sandwich assays use two or more animal
antibodies raised against different sites on
Human Chorionic Gonadotropin (hCG).
• Usually a mouse monoclonal antibody against
one site on the hCG molecule, and a mouse
monoclonal, or a sheep, rabbit or a goat
polyclonal antibody against a second distant site
on the hCG molecule.
Simple to Perform
But understanding the concepts of the principle
may be challenging.
SUMMARY AND EXPLANATION OF THE TEST
• Human chorionic gonadotropin (hCG) is a
glycopeptide hormone produced by the placenta
during pregnancy.
• The appearance and rapid rise in the
concentration of hCG in the woman's urine
makes it a good pregnancy marker.
• Usually, concentration of hCG in urine is at least
25 mIU/ml as early as seven to ten days after
conception.
• The concentration increases steadily and
reaches its maximum between the eighth and
eleventh weeks of pregnancy.
Fertilization
PRINCIPLE OF THE TEST
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The pregnancy testing device contains a unique set of dye-conjugated and
immobilized antibodies used to produce a distinctive visual pattern
indicating elevated concentration of hCG (=25 mIU/ml) in the test sample.
One antibody, the capture antibody, is in a solid phase permanently
attached to a tube, plate, membrane, or bead.
Conjugate pad contains the label reagent, i.e. antibody labeled with either
red, gold or blue latex particles.
Sample is applied and dissolves the label mixture and migrates to the zone
of immobilized antibody lines. If hCG is present, labeled antibody-dye
conjugate binds it, forming an antibody-antigen complex.
Positive, that is hCG containing, sample causes the formation of a colored
test line, which indicates a positive test result.
As the reaction mixture continues to flow along the test membrane, the
complex binds to the anti-hCG antibody in the test zone of the membrane,
and produces a color band.
Unbound conjugate binds to the reagents immobilized in the control zone
producing a color band, demonstrating proper performance of the test.
Figure 1
• Device with solid phase capture antibody to one site on
hCG, and liquid phase tracer antibody (label shown by
red star) to second or distant site on hCG. In this way the
label becomes immobilized.
Figure 2
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Serum or urine containing hCG (shown as ab) added to device. The hCG forms a
sandwich or bridge between capture and tracer antibody. After a short incubation
period the hCG binds both the solid phase and liquid phase antibodies linking them.
Figure 3
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Excess tracer antibody is washed away. Amount of label or tracer (red star) is measured. This is
proportional to amount of hCG.
Illustration of Principle
TEST PROCEDURE
• NOTE: Bring test components and specimens to room
temperature prior to testing.
• Remove a Testing Device from the foil pouch by tearing
at the "notch" and place it on a level surface.
• Holding a Sample Dropper vertically, add exactly four
drops of the urine specimen to the sample well. NOTE:
Picture shows incorrect orientation of dropper to test
area, must be completely vertical to ensure adequate
sample.
• Read results at time indicated in procedure.
Interpretation
• If two color bands are visible the test is
positive.
• The presence of a Control Band only
indicates a negative test.
Results
Function of the Control Band
• The Control Band is used as a reference and
built in quality control check.
• If the Test Band is darker or similar to the
Control band, the test result is considered
positive.
• The Control Band is used for procedural control
to check whether the test reagents are working
properly and that a sufficient amount of urine
sample has been applied to the test area.
Invalid Tests
• If, after performing the test, no purple color
band is visible anywhere within the
Results Window, the result is considered
invalid.
• If a color appears in the test area but NO
color appears in the control area, the test
is invalid.
Ovulation Test
Another Example of Results
Causes of Invalid Results
• The directions may not have been
followed correctly.
• Inadequate amount of sample has been
exposed to the test system.
• The test may have deteriorated.
PRECAUTIONS
• Do not use test kit components after the
expiration dates.
• Dispose of all used test components in a proper
biohazard container.
• If specimens or test components have been
stored in a refrigerator, allow them to warm to
room temperature before performing the test.
• Human specimens should be handled as if
capable of transmitting infectious agents.
Limitations of the Procedure
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Besides pregnancy, elevated concentrations of hCG may be found in
patients with both gestational and non-gestational trophoblastic diseases.
These conditions should be ruled out in the interpretation of hCG levels to
establish a diagnosis of pregnancy.
A low incidence of false results can occur. Consult with a physician if
unexpected or inconsistent results.
A normal pregnancy cannot be distinguished from an ectopic pregnancy
based on hCG levels alone.
A spontaneous miscarriage may cause confusion in interpreting the test
results.
A definitive diagnosis should not be based on the results of a single test, but
should only be made by the physician after all clinical and laboratory
findings have been evaluated.
A negative result from a specimen collected from a woman in very early
pregnancy may be due to an unusually low concentration of hCG. In such
cases, the test should be repeated on a fresh specimen obtained
approximately two days later.
A urine sample may be too diluted and thus may not contain a
representative concentration of hCG. If a negative result is obtained with a
urine specimen and pregnancy is still suspected, obtain a first morning urine
specimen and re-test.
Ectopic Pregnancy
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This unfortunate patient came in with a late cycle. The pregnancy test was positive
but we couldn't see a pregnancy on her ultrasound scan. We suspected an ectopic
and this is what we found during laparoscopy.
The pregnancy was lodged in the middle of her left tube. A small slit was made over
this and the pregnancy was removed without removing her tube.
Choriocarcinoma
• Choriocarcinoma is a highly malignant germ cell tumour
which usually follows an abnormal pregnancy with a
hydatidiform mole. It may also occur after a spontaneous
abortion, and rarely, may follow a normal pregnancy. The
tumour metastasizes early, by means of vascular
invasion and blood spread. Picture of liver.
Choriocarcinoma of Testes
Hydatiform Mole
• Hydatidiform mole is a tumor of the placenta that is usually
benign. It develops from placental tissue during an early
pregnancy in which the embryo fails to develop normally. The
tumor consists of many small vesicles (sacs) and resembles a
large cluster of grapes. Although the condition is fairly rare in
the USA, it is common in the Orient and other parts of the
world. In the United States, molar pregnancy occurs in 1 of
every 1,000-1,200 pregnancies.
Seminoma
• 50% of all testicular tumors.
• Human chorionic gonadotropin-beta is a better
marker than hCG. For earlier detection of
recurrence, both markers should be examined.
SPECIMEN COLLECTION AND STORAGE
• First morning urine usually contains the highest
concentration of hCG and is therefore the best sample
when performing the urine test. However, randomly
collected urine specimens may be used.
• Collect a urine specimen in a clean glass, plastic, or wax
coated container. Do not use preservatives.
• If the test is not run immediately following collection of
the sample specimen, but is to be run within 48 hours
following collection, the specimen should be refrigerated
(2-8°C), and brought back to room temperature (1528°C) before testing.
• If testing is delayed more than forty-eight hours, the
specimen should be frozen. A frozen specimen should
not be used if stored more than two weeks.
• Prior to testing, the frozen specimen must be completely
thawed, thoroughly mixed, and brought to room
temperature.
QUALITY CONTROL
• The use of controls is recommended to verify
proper kit performance.
• Quality control reagents (positive and negative)
should be tested according to quality control
requirements established by the testing
laboratory.
• Use controls in the same procedure as
specimens.
• This may be required with each test or only
when a new lot number is being put into use.
Reference
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http://www.saveontests.com/pregnancy_test_strip_instructions.htm
http://www.ovulation-calculator.com/pregnancy-tests/pregnancy-tests.htm
• http://sunflower.bio.indiana.edu/~aberndts/Z566/lab1images.html fertilization
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http://www.healthsentinel.co.uk/professional/professional-pregnancy-test-info.htm
• http://www.docsun.com/default.asp?id=26 – tubal pregnancy
• http://www.moondragon.org/obgyn/pregnancy/molar.html
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http://www.nlm.nih.gov/medlineplus/ency/article/001288.htm seminoma
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http://www-medlib.med.utah.edu/WebPath/TUTORIAL/PRENATAL/PRENATAL.html
Some pictures very graphic