HOME URINE PREGNANCY TESTS: APPLICATION OF IMMUNOLOGY … · a modification of the A-Z test.6,7,8,9 The “rabbit test” consisted of injecting the woman's urine into an immature

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]

Additional Authors:

E. Denise VanHyfte, PharmD

PGY-1 Pharmacy Practice Resident

Lutheran Health Network

[email protected]

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]

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.

2. Describe the immunological principles

underlying the use of antibody based urine

pregnancy tests.

3. Explain manufacturer claims of accuracy

and performance.

4. Explain the possibility for false negative

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

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

mailto:[email protected]

mailto:[email protected]


2015 ARTICLE 5

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

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 cross-

reactivity 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 beta-

subunit 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 alpha-

subunit (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


2015 ARTICLE 5

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

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,


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


2015 ARTICLE 5

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 anti-

hCG 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).

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%.


2015 ARTICLE 5

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

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


2015 ARTICLE 5

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

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


2015 ARTICLE 5

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

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).


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

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

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.

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


2015 ARTICLE 5

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 semi-

quantitative 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.

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.


2015 ARTICLE 5

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2015 ARTICLE 5

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Documents

HOME URINE PREGNANCY TESTS: APPLICATION OF IMMUNOLOGY … · a modification of the A-Z test.6,7,8,9 The “rabbit test” consisted of injecting the woman's urine into an immature