MLAB 1335 Immunology
Laboratory Exercise 3
Urine Pregnancy Enzyme Immunoassay Test
Objectives
1. Define human chorionic gonadotropin (hCG) and state its function.
2. State the principle of the enzyme immunoassay procedure for the detection of hCG.
3. Differentiate between a qualitative and quantitative assay.
4. State when the urine sample for pregnancy testing should be collected.
5. State the time and temperature for an hCG sample storage.
6. Perform urine pregnancy testing on controls and patient specimens with 100% accuracy.
7. Evaluate reagent package inserts to determine the significance of abnormal results, limitations of the procedure,
and troubleshooting procedures when control results are unacceptable.
8. Appropriately record and report results as instructed.
9. State whether the results can be reported to the physician.
10. List 8 conditions, other than pregnancy, for which hCG levels may be ordered.
Introduction
Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by the developing placenta shortly after
fertilization. The role of hCG is to prevent the disintegration of the corpus luteum. In normal pregnancy, the hCG
hormone can be found in detectable amounts in serum and urine of pregnant women 7-10 days following fertilization.
The concentration of hCG doubles every 36 - 48 hours during the early weeks, frequently exceeding 100 mlU/ml by the
first missed menstrual period.
Human chorionic gonadotropin peaks in the range of 30,000- 200,000 mlU/ml by 10-12 weeks of pregnancy (end of first
trimester). During the second and third trimesters, the hCG levels fall to plateau at approximately 1/6 of the peak level.
Following delivery, the hCG hormone is rapidly cleared from the blood and pregnancy tests become negative in three to
four days.
The appearance of hCG in urine soon after conception and its subsequent rise in concentration during early gestational
growth provides an excellent marker for the early detection of pregnancy.
Principle
Modern pregnancy tests use monoclonal antibodies selective for the beta area of the hCG molecule. Most of the urine
test systems incorporate monoclonal anti-hCG antibody which after binding with the hCG migrate along a membrane to
an area where the molecules become bound to a “fixed” anti-hCG. The fixed anti-hCG is complexed with an enzyme or
other color producing molecule. The result will be the production of a visible marker.
This type of test is considered a “qualitative” test. The result is either positive or negative. There can be no
determination of the amount of hCG present with a qualitative test; it simply indicates the presence or absence of hCG in
the sample being tested. There are times when a physician wants a “quantitative” result which is the actual amount of
hCG present. This requires a serum sample and an instrument set up to determine the actual concentration of hCG.
Quantitative hCG levels assist the physician in determining the actual gestational age of the fetus or may also indicate that
this is a multiple pregnancy, i.e., twins, triplets, etc.
Specimen Requirements
Specimen requirements may be and/or serum and urine. Read the instructions carefully to determine acceptable sample
types. All samples must reach room temperature prior to testing.
Random urine specimen is appropriate for hCG testing, but the first morning urine is optimal because it generally contains
the highest concentration of hCG. Urine specimens should be collected in any clean, dry, plastic container.
Refer to the package insert for specific sample storage requirements. When performing a procedure for the first time
carefully review the sample storage requirements, length of time for storage, and other manufacturer’s allowable storage
requirements.
Exercise 3 Pregnancy Test Enzyme Immunoassay
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Quality Control
Serum and urine qualitative immunoassay tests have built in controls.
 The “C” is the internal procedural control and must produce a colored line. It confirms sufficient specimen volume
and correct procedural technique.
 The background of the cassette serves as the negative background control and must remain colorless.
If the control line does NOT produce a colored line OR if the white background produces color the test is invalid.
When invalid results are obtained the first course of action should be to repeat the test. If another kit is available repeat
patient testing on the new kit. If the same invalid result is obtained, discuss with the supervisor. The manufacturer may
need to be contacted.
It is recommended that external quality control (QC) solutions known to be positive and negative for hCG be evaluated to
verify proper test performance. This is a requirement at most facilities which perform pregnancy tests. When kits with
new lot numbers are received two devices are used to test with the positive and negative external controls to validate the
kit.
Facilities which perform large numbers of pregnancy tests will order large volumes of kits with the same lot numbers as
this type of quality control only needs to be performed once per lot number. It is crucial that when a new kit is opened
that the individual performing the test verify that the QC has been performed. If it has not then the external QC solutions
must be tested prior to performing testing on patient samples.
Results
A positive result usually indicates that a woman is pregnant, but may have had a miscarriage or therapeutic abortion. A
confirmed diagnosis of pregnancy should be made by a physician after evaluating all clinical and laboratory findings.
False positive and false negative results can occur.
A “false positive” is a test result which is positive but the woman is not or has not been recently pregnant. Certain types
of tumors or malignancies can produce hCG such as choriocarcinoma, hydatidiform mole, testicular and prostate tumors.
The following medications contain hCG and may cause a false positive: Pregnyl, Profasi, Pergonal and APL. These are
used in the treatment of infertility.
A “false negative” is a test result which is negative but the woman is pregnant. The most common reason for a false
negative is that a first morning sample was not used and the amount of hCG is too small to be detected by the test.
Technical errors can lead to erroneous results and include:
 Insufficient sample used
 Incorrect timing
 Misinterpretation of the results
Clinical Indications
While confirmation to diagnose pregnancy is the most common reason a pregnancy test is ordered there are many other
conditions which may warrant this test:
1. Suspected choriocarcinoma
2. Suspected hydatidiform mole
3. Suspected molar pregnancy.
4. Testicular tumors
5. Prostatic cancer
6. Breast cancer
7. Lung cancer
8. Pre-surgical procedures
9. Pre-X-ray and radioisotopic procedures
Exercise 3 Pregnancy Test Enzyme Immunoassay
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All gestational trophoblastic tumors produce hCG. This hormone is a valuable marker for these tumors. The level
correlates with tumor mass and has prognostic value. hCG is extremely sensitive, being elevated in women with minute
amounts of tumor. The patient is followed weekly during treatment. At the completion of treatment indefinite follow up
is advised to detect recurrence.
Quantitative β-hCG levels are used for male patients, along with other tests, to diagnose certain cancers, determine the
stage of the cancer, evaluate response to therapy and monitor early detection of relapse.
Reference levels in normal pregnancy for a quantitative β-hCG
The following is a list of serum β-hCG levels. (LMP is the last menstrual period dated from the first day of the last period.)
The levels grow exponentially after conception and implantation.
Weeks since LMP
mIU/mL
3
5 – 50
4
5 – 426
5
18 – 7,340
6
1,080 – 56,500
7–8
7,650 – 229,000
9 – 12
25,700 – 288,000
13 – 16
13,300 – 254,000
17 – 24
4,060 – 165,400
25 – 40
3,640 – 117,000
Non-pregnant females
<5.0
Postmenopausal females <9.5
Exercise 3 Pregnancy Test Enzyme Immunoassay
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Lab (30)
SQ (15)
Total (45)
MLAB 1335 Immunology
Laboratory Exercise 3
Urine Pregnancy Enzyme Immunoassay Test
Name
Date ____________________
Procedure Points ___________/30
Kit Information – USE KIT OR PACKAGE INSERT
Test Kit Name
Manufacturer
Lot Number
Expiration Date
Storage Temperature
Points
1
1
1
1
1
Sample Information
Points
1
2
2
Type(s) of samples which may be used.
Storage Temperature (list each one)
Length of time of storage
Refer to the package insert as to the terminology used to report out the results and to answer the following questions.
Each of the following items in the chart is worth 2 points (16 total). Points will be deducted for unacceptable correction of
errors.
Patient Name
ID Number
Patient
Result
Positive Control Line
Result
Background
Result
1.
2.
Validity of Results
Can these results be reported out? Check ONE
If “No”, explain:
Test Completion Information
Initials of individual performing test (Tech)
Date of completion
Time of completion
Exercise 3 Pregnancy Test Enzyme Immunoassay
Yes
No
Point
1
Points
1
1
1
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MLAB 1335 IMMUNOLOGY/SEROLOGY
EXERCISE 3: Urine Pregnancy Enzyme Immunoassay Test
Study Questions
Name
__________________
Date ________
Points _______/15
Refer to the course laboratory materials to answer the following questions.
1.
What substance is being detected in the pregnancy test? No abbreviations. (1 point)
2.
State the principle of pregnancy tests which use monoclonal antibodies. (2 points)
3.
The immunoassay pregnancy test has built in controls. (2 points)
a. State the purpose of the “C” area and the expected result.
b.
4.
State the purpose of the background and the expected result.
List and describe two limitations of the procedure. (2 points)
a.
b.
5.
If a refrigerated sample is used, what must be done first prior to performing the test? (2 points)
6.
Define “false positive” and give one example. (2 point)
7.
Define “false negative” and give one example. (2 point)
8.
List 4 conditions other than pregnancy or pregnancy related conditions in which hCG may be detected. (2 points)
a.
b.
c.
d.
Exercise 3 Pregnancy Test Enzyme Immunoassay
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