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INDIANA PHARMACISTS ALLIANCE
CONTINUING PHARMACY EDUCATION (CPE)
2015 ARTICLE 5
HOME URINE PREGNANCY TESTS:
APPLICATION OF IMMUNOLOGY TO PATIENT CARE
Corresponding Author:
Jane E. Krause, MS, RPh
Clinical Associate Professor of Pharmacy
Practice
Purdue College of Pharmacy
[email protected]
2. Describe the immunological principles
Additional Authors:
E. Denise VanHyfte, PharmD
PGY-1 Pharmacy Practice Resident
Lutheran Health Network
[email protected]
4. Explain the possibility for false negative
Jenny N. Wulf, PharmD
PGY-1 Pharmacy Practice Resident
Union Hospital
[email protected]
Tony R. Hazbun, PhD
Associate Professor of Medicinal Chemistry and
Molecular Pharmacology
Purdue College of Pharmacy
[email protected]
ACPE no. 0120-0000-022-H04-P
1.5 Contact Hour (.15 CEU’s)
This is a knowledge based activity.
See the end of the article for CE
details. Target Audience: Pharmacists
Faculty Disclosure: Faculty have
no conflicts of interest to disclose
Goal:
To apply immunology principles when
educating and counseling patients regarding
home urine pregnancy tests.
Learning Objectives:
Upon completion of this article the learner
should be able to:
1. Summarize the history and use of urine
pregnancy tests.
underlying the use of antibody based urine
pregnancy tests.
3. Explain manufacturer claims of accuracy
and performance.
and false positive results.
5. Describe the procedure for patient use of
urine pregnancy tests and appropriate
counseling points.
Introduction
A “pregnancy test” is defined as a procedure
intended to reveal the presence or absence of a
pregnancy.1 Currently, there are over 60
different brands of US Food and Drug
Administration (FDA) cleared home pregnancy
tests on the market in the United States.2 Many
of the brands are produced by the same
manufacturers; according to a market research
report, there are 11 different manufacturers of
home pregnancy tests.3 In addition, products
may advertise early results, digital results, or
low price. With the numerous products
available, pharmacists have the opportunity to
provide valuable education to patients about
selecting a home pregnancy test along with the
proper way to perform the test and interpret the
results.
Evolution of Home Pregnancy Tests
Women have been attempting to predict
pregnancy for centuries.2,4,5 Prior to the 1920s,
there was no scientific means of determining
pregnancy in a woman. Ancient Egyptian
writings tell of women trying to determine
pregnancy (and gender of the baby) by using
urine to fertilize barley and wheat seeds. If the
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barley grew, it was interpreted as being pregnant
with a male baby, and if the wheat grew, it was
interpreted as being pregnant with a female
baby. No growth indicated the female was not
pregnant. This historical practice is now
considered the first theory noticing a unique
substance in the urine of pregnant women.
In the early 1900s, research focused on the
human reproduction system with the scientific
identification of hormones.4,5 During the 1920s,
researchers discovered the hormone “human
chorionic gonadotropin” (hCG), and the role it
plays in pregnancy. In 1927, two scientists,
Selmar Aschheim and Bernhard Zondek,
developed the “Aschheim-Zondek” test (A-Z
test), a bioassay (animal based test) which tested
for the presence of hCG by injecting the
woman’s urine into an immature female mouse.
Researchers determined that injection of hCG
into laboratory animals would cause ovulation
requiring fatal surgery to determine the test
result. In the presence of hCG, the ovaries of
the immature laboratory animal would enlarge
and show follicular maturation.
In the early 1930s, the “rabbit test”, or
"Friedman test", was developed by Maurice
Harold Friedman, a physician and physiology
researcher at the University of Pennsylvania, as
a modification of the A-Z test.6,7,8,9 The “rabbit
test” consisted of injecting the woman's urine
into an immature female rabbit and then
surgically examining the rabbit's ovaries 36 to
48 hours later to obtain results. The “rabbit test”
became a widely used bioassay to test for
pregnancy. Although this test was found to be
more accurate than the “A-Z test”, both
bioassays were expensive, required animal
sacrifice, and took several days in the laboratory
to complete.4,5 With the “rabbit test”, it was a
common misconception among the public that
the injected rabbit would die only if the woman
was pregnant.6 This led to the phrase "the rabbit
died" being used as an expression for a positive
pregnancy test.
In 1960, a hemagglutination inhibition test for
pregnancy was developed by Leif Wide and Carl
Axel Gemzell.5,10 Because it used cells, this test
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was an immunoassay (e.g., based on an
antibody-antigen reaction), rather than a
bioassay.4,5 In this test, a urine sample was
combined with antibodies against hCG. In a
positive test, red blood cells aggregated in a
particular pattern which laboratory personnel
interpreted as pregnant. These types of tests
became commercially available to healthcare
professionals and serve as the precursor to
current pregnancy tests.4 However, there were
still problems with these tests including crossreactivity with medications, and the inability of
the test to distinguish between hCG and the
luteinizing hormone (LH) causing false positive
results.11
In the early 1970s, scientists at the National
Institutes of Health (NIH) researched the
biochemistry of hCG and determined the
biological specificity of hCG was in the betasubunit of the hormone.5 hCG is one of a family
of glycoprotein hormones, the others being LH,
follicle stimulating hormone (FSH), and
thyrotropin-stimulating hormone (TSH). Each
of these consists of two subunits: an alphasubunit (which is very similar in all four
hormones) and a beta-subunit which is unique or
characteristic of each individual hormone. Both
subunits of the molecule are needed for
biological activity but the beta-subunit
determines the specificity of action.1 This
discovery proved significant and in 1972,
Vaitukaitis, Braunstein, and Ross developed an
hCG beta-subunit radioimmunoassay that
distinguished between hCG and LH, making it
potentially useful as an early test for
pregnancy.4,5 The ability to measure low levels
of hCG in the presence of LH explains the great
sensitivity of current pregnancy tests.1
Interestingly, the radioimmunoassay was also
found to be useful for healthcare professionals
by allowing them to follow patients being
treated for hCG–secreting tumors.5,12
By 1977, the assay process was simplified
enough to allow marketing to the general
population and the first home pregnancy test
became available.4,5 Warner-Chilcott introduced
e.p.t.TM (“early pregnancy test”), which had been
submitted to the FDA for clearance the previous
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year.4 This was truly a breakthrough in home
diagnostics, as Warner-Chilcott claimed. What
once was confirmed by merely waiting for
outward signs of pregnancy or a test in the
physician’s office, the pregnancy test became
available for a woman to use in the privacy of
her own home. In a 1972 “Dear Pharmacist”
letter from Warner-Chilcott, pharmacists were
informed that “the e.p.t.TM consumer campaign
has been designed to direct the consumer to their
drug store to purchase the test.” At the time, the
cost of the test was $10 -$11.4,5,13 In January
1979, 30 second commercials for e.p.t.TM began
airing on television nationwide.13 This marked
the first time commercials for pregnancy tests
had been televised. (Note: e.p.t.TM is currently a
registered trademark of INSIGHT
Pharmaceuticals, Corp.)
These first generation home pregnancy tests
looked like a mini-science lab and came with a
vial of purified water, a test tube containing
sheep red blood cells, a medicine dropper, and a
clear plastic support for the test tube with an
angled mirror at the bottom.6,7 The test took
two hours to complete and was sensitive to
vibrations.2,4,7 These tests used ring, or tube
agglutination tests; a ring at the bottom of the
tube indicated a positive result.6
During the 1980s, tests were created in one-step
formats in which the testing reagent could be
contained on a single strip and situated within a
hand-held applicator.11 Results would appear
within ten minutes and sensitivity improved,
thereby decreasing wait time to test. In 1988,
Unilever introduced the first “one-step” test,
Clearblue EasyTM, so named for the blue stripe
that developed to indicate pregnancy.7 In 2003,
the next generation of home pregnancy tests
appeared with the FDA clearance of Clearblue
Easy’sTM digital pregnancy test.5 In place of a
blue line, the indicator screen displayed either
“pregnant” or “not pregnant”. (Note:
ClearblueTM is currently a registered trademark
of SPD Swiss Precision Diagnostics GmbH.)
Urine pregnancy tests are the most common
diagnostic assays used by patients at home.14
According to the FDA, one-third of
2015 ARTICLE 5
reproductive-age women have used a home
pregnancy test.15 Throughout recent years, sales
of these tests have remained consistent. In 1999,
about 19 million home pregnancy tests were sold
in the United States, with sales of about $230
million.16 Similarly, sales of home pregnancy
tests in 2009 equaled $227 million.17
Immunological Principles of Home
Pregnancy Tests
The role of hCG: Home pregnancy tests rely
on the detection of hCG in the urine.14 The
hCG hormone is an ideal marker for the
detection of pregnancy due to the physiological
fluctuation of hCG levels following implantation
of the fertilized egg (or embryo) into the uterine
wall. Fertilization takes place in the fallopian
tube and once the egg is fertilized, the embryo
travels down the fallopian tube to the uterus.
Once in the uterus, the embryo implants in the
lining of the uterine wall. At this point, the
trophoblast cells of the developing placenta
begin to produce hCG, and levels continue to
increase throughout the early stages of
pregnancy. Implantation typically occurs 5-8
days after ovulation; this is the same time that
hCG first becomes present in both the plasma
and the urine.14,18 Twenty milli-international
units/milliliter (mIU/mL) is the level of hCG
that is typically present in the urine of pregnant
women at approximately seven to ten days post
ovulation. Throughout pregnancy, hCG levels
rise at an average rate of 50% per day and at
about 10 weeks peak at an average level of
100,000 (mIU/mL). After 10 weeks the levels
will start to decline and typically stabilize near
20,000 mIU/mL for the remainder of
pregnancy.18 In general, hCG levels less than 5
mIU/mL are considered negative for pregnancy,
while hCG levels greater or equal to 25 mIU/mL
are considered positive for pregnancy.19,20
Immunology and the mechanism of home
pregnancy tests: Some basic immunology
concepts are helpful in understanding the
mechanism of home pregnancy tests. Proteins
called antibodies are a very important
component of the body’s immune system.21
When the body is exposed to a foreign molecule,
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2015 ARTICLE 5
antibodies are produced that can bind to a
specific site on the molecule, and thereby tag it
for destruction. This helps to protect the body
from invading substances that may lead to
illness. The foreign molecule to which the
antibody binds is termed the antigen. The
specific region of the antigen that is recognized
by the antibody is called the epitope, and the
location where this binding takes place is the
antigen binding site.
How does this information relate to home
pregnancy tests? Home pregnancy tests are
designed using multiple antibodies.12 One set
of antibodies in a pregnancy test recognizes
hCG, which serves as an antigen. Different
brands of pregnancy tests utilize different
antibodies that typically recognize hCG.
However, the specific epitope of hCG that is
recognized varies from test to test based on the
antibody used. Therefore, the antigen binding
site on hCG varies from one pregnancy test to
another.
Antibodies are also called immunoglobulins (Ig)
and have a characteristic Y-shaped structure that
results from the assembly of light and heavy
polypeptide chains.22 The antibody structure
consists of two identical copies of a heavy chain
and two identical copies of a light chain (see
Figure 1)22, which are named in this manner
because of their relative molecular weights.
Each chain has a variable region that differs in
amino acid sequence between one antibody and
another, and the remaining region is constant in
sequence. The variable regions of the light and
heavy chain form two identical antigen-binding
sites. During a pregnancy test it is these variable
regions that will bind hCG. The constant region
of the heavy chain is an important factor in the
pregnancy test because a second antibody
recognizes the constant region of the anti-hCG
antibody. This second antibody-antibody
interaction leads to the formation of the control
line within pregnancy tests (see Figure 2).
In addition to their critical role in the immune
system, antibodies are important analytical tools
due to their ability to bind antigens with high
specificity.22 It is possible to generate an
antibody to any antigen of interest by simply
injecting the antigen into a host animal capable
of mounting an immune response. Typically,
the host animal is a mouse or a rabbit. Note that
one can generate an antibody that recognizes an
antibody from a different species. For example,
if a rabbit is injected with a mouse
immunoglobulin, then one can collect antibodies
from the rabbit that recognize and bind to mouse
immunoglobulin. Two different types of
antibodies, generated against two different
classes of antigens, are used in the home
pregnancy test: (i) antibodies that recognize
hCG, and (ii) antibodies that recognize
immunoglobulins from a different species. The
principles of operation of a home pregnancy test
are summarized as follows (see Figure 2)22:
1. A sample of urine is collected. For the
most accurate result, it is suggested to
use the first urine in the morning, when
hCG levels are most concentrated.
2. Two to three drops of the urine sample
are applied to the bottom of the test
strip. The strip has two sets of
antibodies that recognize and bind to
hCG. The first set of anti-hCG
antibodies (termed “mobile” antibodies)
are localized at the bottom part of the
strip or absorbent tip. These antibodies
are drawn up the strip by capillary
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action as soon as they come in contact
with the urine sample. The second set
of anti-hCG antibodies (termed
“immobilized antibodies”) are fixed to
the test region of the strip.
3. If the individual is pregnant, then hCG
will form a complex with the mobile
anti-HCG antibodies and flow up the
strip. Upon reaching the test region, the
mobile anti-hCG/hCG complex is
captured by the immobilized anti-hCG
antibodies. An enzyme on the first set
of anti-hCG antibodies produces a color
reaction in this region of the strip.
4. Mobile anti-hCG antibodies that are not
bound to hCG flow past the
immobilized anti-hCG antibodies to the
control region, where they are captured
by a third set of antibodies that
recognize the constant region of
immunoglobulins. An enzyme on the
first set of anti-hCG antibodies produces
a color change in this region of the strip.
This step verifies that the mobile antihCG antibodies have traveled up the
strip and, therefore, ensures that the test
has functioned properly.
5. The test is complete within 3-5 minutes.
A positive test occurs when hCG is
present at levels in excess of the test
sensitivity (e.g., 25 mIU/mL).
2015 ARTICLE 5
Manufacturer Claims
All marketed devices claim to be “over 99%
accurate” and able to “detect pregnancy by the
first day of the expected period”.23 Some
products claim to be able to detect pregnancy
even earlier (e.g., 4–5 days before the expected
period). Such claims by the manufacturers can
be misleading and confusing to patients, and
having an understanding of these claims is
helpful when counseling patients.24
Regulation of pregnancy tests: Pregnancy
tests are considered a medical device and must
be “cleared” (not “approved”25) by the FDA.12
Pregnancy tests are classified as an hCG test
system intended for the early detection of
pregnancy and the manufacturer is required to
submit a premarket notification, or 510(K). A
search of the FDA database with the term
“pregnancy” shows that 293 such submissions
occurred from 1977 to 2014.26 The 510(K)
submission requires, in part, clinical data
supporting what are termed “Performance
Characteristics/Laboratory Evaluation”. For
example, data must be provided for the
following12:

Method Comparison/Accuracy Study 12:
The new device is compared to a
predicate or already cleared device.
Testing is done by splitting samples
between both subjects (home users) as
well as professionals. The subjects are
to perform the test without guidance or
assistance, and the results between both
professionals and subjects are compared
to determine ease of use. The testing
involving the subjects and professionals
utilizes the new pregnancy test device
being studied as well as a predicate
device. Related to this, the statement
“over 99% accurate” means that when
compared to a predicate device already
on the market, the new test will produce
the same results as the predicate device
at least 99% of the time. The FDA
recommends expressing this data in
terms of percent accuracy, which should
never exceed >99%.
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
Sensitivity/Detection Limit 12: The
minimum urinary concentration of hCG
that can reliably produce positive results
is referred to as the detection limit, or
sensitivity, of the pregnancy test. The
concentrations of hCG at the claimed
sensitivity must produce positive results
in at least 95% of samples tested. Assay
sensitivity should be such that small
amounts of hCG will be detected while
false-positive results due to the presence
of LH will be minimized. Additionally,
it is expected that all positive results will
have reacted within the time frame
specified.

Expected Values 12: Expected values or
pregnancy detection rates may be
provided by the manufacturer when
testing is done before the day of the
expected period (i.e., the percentage rate
of detected pregnant results by day
relative to the day of the expected
period). The manufacturer may state the
test is capable of detecting pregnancy by
the first day of the expected period and
no sooner, unless validated by clinical
data.
Sensitivity and timing of use: Home pregnancy
tests vary in the level of hCG that they are able
to detect.23 A common detection limit for many
pregnancy tests is 25 mIU/mL; however the
limits range from 6.3 - 50 mIU/mL.2,23 In order
to prevent false negative results, a woman must
perform the pregnancy test at a time when her
urinary hCG concentrations are at or above the
detection limit of the test. One study evaluating
urinary hCG concentrations in 109 pregnant
women, found an average concentration of 137
mIU/mL on the first day of the expected
period.14 However, when adjusting for
variability among women in length of menstrual
cycles as well as differences in the time between
ovulation and next menstrual period, the 10th and
90th percentiles of the hCG concentrations on the
first day of the expected period were 15
mIU/mL and 853 mIU/mL respectively.14,27
It is important to note that regardless of test
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sensitivity, a pregnancy cannot be detected prior
to implantation.28 There can be significant
variability in the timing of implantation from
one woman to the next. In approximately 10%
of women, implantation does not occur until
after the first day of the expected menstrual
period. Because hCG production by trophoblast
cells does not begin until implantation, these
women will not have elevated levels of hCG by
the first day of the expected period.14,28 This
shows that there will be a portion of women
whose hCG levels will not rise above the
detection limit of the test until sometime after
the first day of the expected menstrual period.
For less sensitive tests, the day by which a
pregnancy can be detected will be even later.
By waiting until a week after the first day of the
expected menstrual cycle, over 97% of all
pregnant women should have hCG levels above
the detection limit of any pregnancy test, and
therefore produce a positive result.28
Inaccuracies
False positives: If a pregnancy test produces a
positive result, but the woman is not actually
pregnant, it is referred to as a false positive.
There are several factors that can lead to false
positive results in home pregnancy testing. Some
of the contributing factors toward a false
positive result include recent birth or
miscarriage, menopause, certain fertility
medications, as well as various disease states.29
After giving birth or experiencing a miscarriage,
hCG levels can remain elevated for
approximately eight weeks. If a woman were to
take a pregnancy test within this eight week time
frame, it could produce a false positive
result.14,29
A “biochemical pregnancy” is when a fertilized
embryo implants briefly in the womb (hCG is
produced for a short period of time), but the
pregnancy does not continue and is followed by
normal menstruation.30 In such a situation, a
positive pregnancy test result can happen and
several days later, negative results occur. A
biochemical pregnancy and resulting positive
test result is one of the emotional hazards of
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early-detection pregnancy tests. This is different
than a later-term miscarriage and women should
understand that biochemical pregnancies are not
uncommon and most women are not even aware
that they were pregnant as a biochemical
pregnancy often ends before physical pregnancy
symptoms manifest.
Failure to observe manufacturer directions on
test interpretation can also be associated with a
false positive result.30 A negative pregnancy test
result can appear faintly positive if interpreted
after the time interval specified by the
manufacturer (e.g., after 10 minutes).
Interpreting test results after the time interval
specified are considered invalid.
Another contributing factor to inaccuracies
caused by false positives is menopause.14
Women in peri-menopause or menopause may
have elevated levels of hCG leading to a positive
result on a pregnancy test. The only medications
known to produce false positive results are
certain fertility drugs that contain hCG (e.g.,
chorionic gonadotropin for injection). If a
woman is using one of these medications it is
very important that she is aware of the
interactions between pregnancy tests and the
fertility treatment. There are also some cancers
and medical conditions that can lead to elevated
levels of hCG and thereby produce a false
positive pregnancy test.14,29 Examples of these
include choriocarcinoma as well as neoplasms of
the ovaries.
The commonality between all of these factors
contributing to false positive results is that they
all lead to elevated levels of hCG.14,29 This
shows that is possible for hCG to be elevated
even in the absence of pregnancy. Because of
this, a positive home pregnancy test result
should always be followed up and confirmed by
a physician.
False negatives: If a pregnancy test produces a
negative result, but the woman is actually
pregnant it is referred to as a false negative.14
Just as with false positive tests, there are several
different factors that can contribute to false
negative results in home pregnancy tests. Some
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of the contributing factors include testing too
early in pregnancy, using urine that is too
diluted, or user error.
The concept of false negative results was
discussed briefly as it relates to manufacturer
claims of sensitivity. As mentioned previously,
the level of hCG must be at or above the
detection limit of a test in order to reliably
produce a positive result.23 The most common
cause of false negative results is women testing
too early in their cycle before the urinary
concentrations of hCG are high enough to be
detected by the pregnancy test.14 The timing for
when hCG starts to rise is different for every
woman due to variability in menstrual cycle
lengths as well as time to implantation.28,31 In
addition, many women do not have accurate
estimations of their menstrual cycle dates and
this can lead to their testing too early.14
Understanding this variability and the need to
follow-up a negative result with a subsequent
pregnancy test is important for the health and
safety of both mother and baby.
The other contributors to false negatives are
using urine that is too diluted and user error.14
Urinary concentrations of hCG are most
concentrated in the morning, therefore testing
with first morning urine can help to decrease the
risk of false negatives due to urine that is too
dilute.32 Too much fluid consumption can also
lead to urine that is too dilute. If a woman is not
able to utilize the first urine of the morning for
testing, then decreasing fluid consumption 4 to 6
hours prior to testing can help prevent false
negatives.
Finally, user error can also lead to false negative
results. In studies where pregnancy tests are
performed both by trained technicians and
volunteers, there have been significantly more
false negative results in the volunteer group
versus the trained technician group.24 These
false negatives have been attributed to user error
by volunteers not fully understanding product
instructions. To prevent this form of false
negative results, proper counseling on device use
as well as encouraging thorough reading of
package instructions prior to testing should
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always be done.32
Interference: With the development of
monoclonal antibodies with a greater degree of
specificity to hCG, the rate of interference in
home pregnancy testing has declined.12 As
described in the “Evolution of Home Pregnancy
Tests” section of this article, by utilizing the
differences in the structures of the beta-subunits,
manufacturers have been able to develop
monoclonal antibodies extremely specific to
hCG, thereby minimizing interference involving
LH, FSH, or TSH.
In order for a pregnancy test to be cleared by the
FDA, it must undergo interference studies.12 A
portion of the interference studies include testing
the pregnancy kits with urine samples spiked
with LH, FSH, and TSH. The hCG antibodies
used in the pregnancy tests should be specific
enough to not significantly cross-react with
these substances. Another aspect of interference
studies involves testing the pregnancy kit in the
presence of various chemical and biological
analytes to determine the effect on functionality
and accuracy of the pregnancy test. Examples of
these analytes include caffeine, ascorbic acid,
hemoglobin, glucose, and albumin. In addition
to these studies, pregnancy tests are also
evaluated for interference based upon pH level.
The home pregnancy tests must pass these
interference studies in order to make it to
market.
Patient Use of Home Pregnancy Tests
It is suggested that a pharmacist offer assistance
when he/she notices a patient looking at or
purchasing a pregnancy test.30 Examples of
open-ended questions to ask the patient during
this conversation are included in Table 133.
When recommending a home pregnancy test,
consideration should be given to the following:
test sensitivity [or the hCG threshold at which a
positive result is indicated (the lower the number
- the more sensitive the test)], ease of use and
interpretation of results, and price. In addition
to single packs, tests are also sold in twin and
triplet packs, which offers a more economical
option if testing is done more than once.14
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Generic tests (as compared to brand-name tests)
are FDA cleared, affordable, and equally
reliable. Digital tests tend to be the most
expensive. Sensitivity information may be
found on the box, package insert, or may be
obtained by contacting the manufacturer (phone
number given on box and/or package insert). As
will be noted on the box, many products give
instructions for use in both English and Spanish
languages. Steps to follow when using a home
pregnancy test are summarized in Table
212,30,34,35. Reviewing this information with
patients will help to ensure the correct use of the
product and interpretation of the results.
Table 1. Conversation Starter with Patient:
Examples of Open-Ended Questions
How can I help you?
Who is the product for? (Is this product for
you?)
How late is your period? (What day do/did
you expect your period to begin?)
Are your periods usually regular?
What medications do you currently take?
Do you regularly see a physician for any
medical conditions?
Have you used a pregnancy test before?
What preference, if any, do you have between
products? (e.g., test stick versus test cassette)
It is interesting to note a few recent studies
involving patient use of pregnancy tests. For
example, a recent study of over 100 women
showed that greater than 95% preferred holding
a “test stick” in their stream of urine as
compared to collecting urine in a cup for use
with the “test cassette” or immersion of the “test
stick”.14 This group of women preferred this
format because it was the most efficient
(involved the fewest steps). Another study
showed that one in four women misread results
with line-based pregnancy tests where the
appearance of colored lines has to be evaluated
by the user to determine the result.36 This
illustrates the importance of patient education
with interpreting test results. In addition, the
digital display of results (“pregnant, “not
pregnant”) with some tests has removed the need
for users to interpret line-based results.
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Table 2. Patient Education: Steps in Using Home Pregnancy Tests
Before Using the Test:
 Check the expiration date on the package. Do not use a test after its expiration date.
 Store the test in a cool, dry place. Avoid exposure to heat and sunlight. Do not freeze.
 Carefully read the instructions on box and product insert as directions vary between products.
Using the Test:
 Follow the directions exactly. If a step requires timing, use a clock with a second hand or a stopwatch.
 Most products recommend the use of the first morning urine. If testing before the period is expected,
use first morning urine to maximize the chances of picking up the lower level of hCG. If testing on or
after the day when period is expected, in general, she may test urine any time during the day.
 Test Stick (Figure 3): Remove test stick from wrapper and remove cap from adsorbent tip; hold the test
stick by the thumb grip with the tip pointing downward and result window facing away from body; place
the tip in urine stream for length of time recommended by manufacturer OR collect urine in a cup and
immerse the entire tip in the urine for the recommended time. Replace the cap while holding the tip
downward; lay the stick on a flat, dry surface with the result window facing up.
 Test Cassette (Figure 3): Remove the test cassette from wrapper and place on a flat, dry surface;
collect urine in a cup (clean and dry); apply indicated number of drops of urine (dropper provided with
product) into the round well.
 Test the urine sample immediately if collecting in a cup. If urine must be tested later, keep the sample
refrigerated.
 Do not dip the pregnancy test into the toilet bowl as the urine is too dilute.
 Tests are not to be taken internally and are single-use. Do not reuse a test.
Test Reaction Time:
 It is important to adhere to the test reaction time. Every test has a time interval that must be respected,
typically between five and ten minutes.
 Results determined after the reaction time are considered invalid. A negative test may appear positive
after the given reaction time of the test as the chemical composition of urine can cause a “ghost line” or
“evaporation line” in the result window in the location of the antibody line.
Interpreting Results:
 Although products vary, a positive test (pregnant) will display a “control line” and a “test line”. “Test
lines” can appear as a simple color band, a plus sign, or in the case of digital pregnancy test, actual
words appear. A faint result/test line is indicative of a positive result as long as the test was interpreted
within the given reaction time as specified in the directions.
 The control line indicates that the test has been performed correctly and is functioning properly. If the
control line does not appear, the test is considered invalid.
Follow-Up:
 Record the results of the test.
 If the test result is positive (pregnant), it is important for the patient to see a physician to confirm and
discuss the results. Most healthcare providers also use a urine pregnancy test to confirm. However, a
blood test may be used by the healthcare professional to determine the exact level of hCG.
 If the test is negative (not pregnant), repeat the test in 3-7 days if menses has not started. If the repeat
test is negative, it is most likely that the patient is not pregnant. If menses does not start within one
week and repeat testing is still negative, a woman should call her physician.
Questions:
 Questions about the test can be directed to a pharmacist, physician, or to the manufacturer (phone
number on the box and/or package insert).
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2015 ARTICLE 5
As previously mentioned, package labeling can
be confusing. Care must be taken when
interpreting “over 99% accurate”.34 This
accuracy claim is often not what potential users
expect as it may refer to the “Method
Comparison/Accuracy Study” described earlier
which means that when compared to a predicate
device already on the market, this test will
produce the same results as the predicate device
at least 99% of the time.12 Or, it may refer to a
claim of “over 99% accurate” in detecting an
existing pregnancy as described earlier in the
“Expected Values”(or pregnancy detection rate).
In this case, the claim would likely be
accompanied by additional qualifying
information such as “over 99% accurate at
detecting typical pregnancy hormone levels from
the day of the expected period”.37,38 In addition,
percentage rate of detected pregnant results on
days prior to the expected period may be
presented.12
may state “over 99% accurate” and “results five
days sooner” leading the patient to believe that
the product is “over 99% accurate in providing
results five days sooner”.34 On closer
inspection, information on the side of the box
(or, it may be included in the package insert)
explains that the product is “over 99% accurate
from the day of the expected period”. It goes
onto say that testing one day before the expected
period will give a positive result in 95% of
pregnant users, testing two days before will give
a 90% rate, three days sooner gives a 82%
positive rate, and testing 4 days before gives
only a 51% positive rate. It is also
recommended on the package to test on the first
day of the expected period. Clarifying such
package information to the patient and
distinguishing between percentages related to
“Method Comparison/Accuracy Study” and
“Expected Values” (pregnancy detection rates)
is essential.
According to Grenache, patients often lack an
appreciation of the strengths and limitations of
pregnancy tests and rely heavily on marketing
claims to guide their purchasing decisions.24 For
example, the labeling on the front of the box
FUTURE TRENDS
A recent innovation in pregnancy testing
involves a point-of-care device relying on a
blood sample, which is important when a patient
INDIANA PHARMACISTS ALLIANCE
CONTINUING PHARMACY EDUCATION (CPE)
is unable to produce urine (e.g., due to
dehydration, or if the patient is unconscious).
The blood based test was cleared by the FDA in
2015 and can detect low levels of hCG with
several droplets of blood in 10 minutes and can
aid physicians in identifying if a woman is
pregnant in an emergency situation.39
An additional trend in pregnancy testing is the
desire for more information by the patient and
this has become evident with a recently cleared
pregnancy test which estimates the number of
weeks that have occurred since ovulation.40
Future tests will likely follow this trend of
providing additional information and semiquantitative measurements.
CONCLUSION
Pregnancy testing has evolved dramatically over
the centuries. Today, home urine pregnancy
tests provide privacy, convenience, reliable
results, and have helped to empower women to
maintain control over a portion of their
reproductive lives.2 Pharmacists play a vital role
in guiding patients through the vast amount of
information available regarding pregnancy tests
including the principles of operation and
clarification of product labeling.41 Opportunities
for counseling exist during the recommendation
for a product, interpretation of test results, and
referral of the patient for confirmation or further
investigation.
2015 ARTICLE 5
The Pharmacists Education
Foundation (PEF) is accredited by
the Accreditation Council for Pharmacy
Education (ACPE) as a provider of
continuing pharmacy education. To receive
continuing pharmacy education (CPE)
pharmacists MUST COMPLETE THE
ONLINE QUIZ AND EVALUATION
FORM. A score of 70% or above is required
to receive CPE credit. The link to the quiz
can be accessed from the home study section
in the CE Portal of the IPA website,
www.indianapharmacists.org. This is a free
service of IPA members in 2015. Initial
release date: 12/16/2015. Expiration Date:
12/16/2018. Questions: Call IPA office at
317-634-4968.
INDIANA PHARMACISTS ALLIANCE
CONTINUING PHARMACY EDUCATION (CPE)
2015 ARTICLE 5
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