What is Truth of Home Pregnancy Test Kit? Research Fellow of Motherisk Korean Motherisk Program Div. of Maternal-Fetal Medicine, Dept. of OB & GYN, Cheil General Hospital & Women’s Healthcare Center, School of Medicine, Kwan-dong University, Seoul, Korea

June Seek Choi, M.D., PhD

Case

• A 44 year old woman in her second pregnancy was treated for

acne with 40 mg isotretinoin daily.

• Before starting isotretinoin, she took a negative pregnancy test

which revealed she was not pregnant. For contraception, she used

only condoms after starting of medication. She did not know the

exact date of last menstrual period and did not take ultrasound.

• After medication, her next periods were delayed and she had

taken another urine hCG. The result was a positive urine

pregnancy test.

Contents • Structure of hCG (free subnuits of hCG)

• Home pregnancy test kit

• False negative of HPT kit

• False positive of HPT kit

• Summaries

Human Chorionic Gonadotropin

Human Chorionic Gonadotropin (hCG) (1) • 237 amino acid glycoprotein

hormone composed of two dissimilar

subunits, α and ß, joined

noncovalently.

• Molecular weight: 36,000~ 40,000 Da

• Plasma half life of intact hCG: 36 hrs

• Produced by trophoblast tissue in

pregnancy and trophoblast disease,

and in small amounts by poorly

differentiated cancer (testicular ca.,

ovarian ca., bladder ca)

Cole 1996, Javadpour 1977

α-Subunit

β-Subunit

Williams Obstetrics 23rd Ed

Human Chorionic Gonadotropin (hCG) (2)

• Identical to those of lutenizing

hormone (LH), follicle –

stimulating hormone (FSH),

and thyroid- stimulating

hormone (TSH)

• Composed of 92 aminoacids

linked by five disulfied bridges

• Two forms of free α-subunit

are present in serum and

urine.

α-subunit of hCG

Same as that α-subunit of hCG

•Hyperglycosylated,

with larger, more-

complex N-linked

oligosaccharides

•Majority of free α

molecules in pregnant

urine

Prevent combination of large free α with ß-subunit

Human Chorionic Gonadotropin (hCG) (3)

• Unique, distinguishes hCG form

the other glycoprotein hormones

• Composed of 145 amino acids

linked by six disulfide bridges

• Contains two N-linked

oligosaccharide side chains

• 4 O-linked oligosaccharide units

ß-subunit of hCG

Human Chorionic Gonadotropin (hCG) (4)

• Single cleavage in the ß-subunit

peptide, between residues 47 and

48

• Nicked hCG concentrations peak

at the same time as nonnicked

hCG concentrations, at around 10

weeks of gestation.

• Nicked hCG is unstable, rapidly

breaking up into nicked free ß and

free α in serum.

Nicked hCG

Cole 1993, Kardana 1997

Human Chorionic Gonadotropin (hCG) (5)

• Present in serum and urine

sample

• Free ß concentrations peak at

around the 10th week of gestation

• Total serum free ß concentration

is very low, 0.9% of the hCG

concentration in the 2nd month of

gestation, declining to 0.5% of the

hCG concentration in the 9th

month of pregnancy

Free ß-hCG

Cole 1993

Human Chorionic Gonadotropin (hCG) (6)

• Terminal degradation product of hCG

• Principal hCG ß-subunit-related molecule

in pregnancy urine sample (undetectable

in pregnancy serum)

• At 5 weeks of gestation, ß-core fragment

concentrations start to increase sharply

• ß-core fragment concentrations are 58%

of urine hCG concentration in the 2nd

month of pregnancy, rising 305% of hCG

concentrations in the final month of

gestation

ß-core fragment

Human Chorionic Gonadotropin (hCG) (7) • Trophoblast cells produce hCG in

amounts that increase

exponentially following

implantation.

• With a sensitive test, hCG can be

detected in maternal plasma or

urine by 8 to 9 days after

ovulation.

• Doubling time of plasma hCG

conc. is 1.4 to 2.0 days.

• Serum hCG levels reach peak

levels at 60 to 70 days.

Mean concentration of hCG in serum of women throughout normal pregnancy.

Boxplot comparison of the first day of hCG detection postgonadotropin surge for cycles

Lohstroh. Secretion and excretion of hCG during early pregnancy. Fertil Steril 2005. Early pregnancy loss (EPL), clinical spontaneous abortion (CAB), or successful pregnancy (SP)

Serum

Urine

• Serum hCG detectable 9-11

days after conception (1-3

days before expected menses)

• Urine hCG usually

detectable 1-2 days later

(highly variable)

Human Chorionic Gonadotropin (hCG) (8) • Multifetal pregnancy • Erythroblastosis fetalis with fetal hemolytic anemia • Gestational trophoblastic dz • Down SD

Higher maternal plasma hCG level

• Early pregnancy wastage • Ectopic pregnancy

Lower maternal plasma hCG level

Metabolic clearance of hCG

• Renal clearance of hCG accounts for 30% of its metabolic

clearance.

• 70% of hCG is cleared by liver.

• Clearance of ß- and α-subunit are about 10-fold and 30-fold

greater than that of intact hCG. Williams Obstetrics 23rd Ed

Home Pregnancy Test

Home Pregnancy Test • The first in-home test (e.p.t. ®) was released prior to May 28, 1976

without FDA approval.

• There are > 100 commercial assays available for measuring hCG

concentrations in serum and urine samples.

• In 1999, 19 million OCT pregnancy test kits were sold in the USA

with sales of almost $230 million Wilcox et al 2001

In US, about 33% of women have used a HPT kit to

determine their pregnancy status before seeking

professional health care. Why do women choose to use HPT?

• Speed of obtaining results • Convenience of testing at home • Women is the first person to know that she is pregnant.

Home Pregnancy Test

Home Pregnancy Test Negative test

Home Pregnancy Test Positive test

Home Pregnancy Test Sensitivities checked with manufacturers in July 2008

Test Name Lowest hCG detected BabyHopes 10 mIU Generic Pregnancy Test Strip 10 mIU

AimStrip (aka AimStick) Pregnancy Test Strip 20 mIU BabyHopes Generic Pregnancy Test Strip 20 mIU

Accuclear Pregnancy Test 25 mIU Answer Early Result Pregnancy Test 25 mIU

Clearblue Easy +/- 25 mIU Clearblue Easy Digital (1 piece, blue cap) 25 mIU

Clear Choice w/ Cup 25 mIU Confirm 1-Step Pregnancy Test 25 mIU

CVS Early Result Pregnancy Test 25 mIU Dollar Store Brand (mini-strip, cassette, & midstream) 25 mIU

Early Detect 25 mIU e.p.t. +/- Test 25 mIU

e.p.t. 1-Step Digital Test 25 mIU e.p.t. Certainty Digital Test 25 mIU

Equate +/- (Wal-mart) 25 mIU Equate Pregnancy Test (Wal-mart) 25 mIU

Fact Plus Midstream Pregnancy Test 25 mIU First Response Early Result Pregnancy Test 25 mIU

One Step Be Sure Pregnancy Test 25 mIU Right Aid Pregnancy Test 25 mIU

Target Brand Pregnancy Test 25 mIU Walgreen Midstream Pregnancy Test 25 mIU

Walgreen Digital 25 mIU Fact Plus Cassette 40 mIU Clearblue Digital 50 mIU

CVS "Early Result" Cassette Pregnancy Test 50 mIU Drug Emporium Brand Pregnancy Test 50 mIU

early Pregnancy test 50 mIU Walgreens Cassette Pregnancy Test 50 mIU

Copyright © 2000-2012 BabyHopes.com.

Home Pregnancy Test • Companies claim their products to be up to 99% accurate when

used to detect pregnancy as early as 6 to 9 days after the missed

period.

• When urine samples were obtained for testing prior to 13 days

past the missed period, accuracy was only 69%. collected

beyond 13 days improved accuracy to 96.2%

Driscoll 1971

Actually based on tests run on samples collected

after 15 days or more beyond the missed menses. Maoi 1979

Sensitivity vs Accuracy

• TP/(TP + FN) • (Number of true positive assessment)/

(Number of all positive assessment) Sensitivity

• (TN + TP)/(TN + TP + FN + FP) • (Number of correct assessment)/

(Number of all assessment) Accuracy

TP: True positive, TN: True negative, FP: False positive, FN: False negative

Home Pregnancy Test

36 Daisy 2 TM Home Pregnancy Test Kit,

72 e.p.t.® In-Home Early Pregnancy Test Kits

74 Answer® At-Home Early Pregnancy Test Kits

Sensi-Tex TM

Multistix® SG Reagent Strips Mary 1986

Combined and Brand Comparison of In-Home Pregnancy Test for Sensitivity, Specificity, and Predictive Value (expressed as per cent)

Home Pregnancy Test

Mary 1986

Combined Accuracy and Brand Accuracy of In-Horne PregnancyTests for Early Detection Compared to Manufacturer Claims

FDA average accuracy claim for all branch in per cent

Home Pregnancy Test Diagnostic Efficiency of Home Pregnancy Test Kits: Meta-analysis

• An effectiveness score of 1.0 means that 27% of pregnant women who have tests results equivalent to those of women who are not pregnant. • An effective score of 3.0 would yield results for pregnant women that are 3 SD away from those of a population of non-pregnant women.

Bastian 1998

Accuracy of home pregnancy tests at the time of missed menses (1) Home Pregnancy Test

Urine from non-pregnant women (5) + Recombinant hCG

0 mIU/ml 12.5 mIU/ml 25 mIU/ ml 50 mIU/ml 100mIU/ml

18 HPT brands were tested with 5 different urine hCG

concentration by 3 technicians

Cole et al 2004

Home Pregnancy Test Accuracy of home pregnancy tests at the time of missed menses (2)

Most sensitive test kit

Cole et al 2004

Time Elapsed from 1st Day of Last Normal Menstrual Period

Quantitative β-HCG Level (mIU/mL) Using the IRP Ultrasound Findings

<28 days 5–50

4–5 wk 50–500

From about 4.5 wk and Q β-HCG 1000–1500.

EVU can show signs of IUP

5–6 wk 100–10,000

Definitely abnormal if no DDS seen by TVU with Q β-hCG > 2000 or by TAU with Q β-hCG > 6500

Abnormal if no YS with MSD > 10 mm

6–7 wk 1000–30,000

FP, cardiac activity 5.5–7 wk, Qβ-hCG > 10,000

Abnormal if no FP with MSD > 18 mm

7–8 w 3500–115,000

8–14 wk 12,000–270,000

>10 wk 270,000–15,000

Relationship between Gestational Age, Quantitative β-Human Chorionic Gonadotropin Levels, and Ultrasound Findings

Roberts: Clinical Procedures in Emergency Medicine, 5th ed.

Home Pregnancy Test

False Negative of HPT kit

• Women may be obtaining their samples before the recommended

number of days after their first missed menstrual period (usually 9

days).

• Operation errors result from failure to read or follow instructions.

Only 32% of users complied with all test kit instructions. The

incidence of false-negative results was 24.3%. Valvanis 1982

• Dehydration, decreased urinary pH, bilirubin, hepatitis Yunus 2007

• Ectopic pregnancy, triplet pregnancy, cancer, gestational

trophoblastic diseases American Association for Clinical chemistry 2001-2012

Home Pregnancy Test Causes of False-Negative

• The person may have erroneously calculated the time of onset of

her missed period, she may have a delayed menses due to an early

pregnancy loss or delayed ovulation, or implantation may

have prolonged the onset of significant hCG production.

• Although the test instruction sheets were easy to understand, an

erroneously performed test or faintly discernible result may also

explain a falsely negative result.

Wilcox et al 2001, O’Connor et al 1998, Bjercke et al 1999

Home Pregnancy Test Failure to read or follow instructions

Home Pregnancy Test

Dehydration

Concentration of urine Acidic urine

Dehydration and Decreased urinary pH

If the pH of the urine is

below 5, then the test might

theoretically affect the

result.

This kit is tested for the

effect of urine pH from 5-9

with hCG concentrations at

20 mIU/mL and that did not

affect the outcome of the

result.

Solubility of hCG is enhanced in acidic urine.

Acidic condition could have made hCG more

soluble in urine.

False Negative

Home Pregnancy Test

If the metabolism of hCG is affected in hepatitis.

All of the serum hCG might be metabolising in kidney, and excreted in the urine

High dose hook effect

Hepatitis and Increase renal excretion

Home Pregnancy Test

C

C

C C

C

C

Positive

Antigen is sandwiched between the antibody coated on the

plate and the conjugate

Normal Sandwich Assay

C

C

C

C

C C

C

C C

C

C

C

C

C C

C

C C

False negative

Hook Effect in ELISA

Well-documented interference that occurs when extremely high concentrations

of an analyte occupy all the sites on both the capture and detection antibodies and

prevent the formation of a so-called sandwich in 2-site immunoassays.

Few or no tracer-Ab + Ag + capture Ab complex are formed false negative

Home Pregnancy Test

False Positive of HPT kit

Signaling Ab

Capture Ab

Interfering Ab

False Positive Home Pregnancy Test False Positive

Rare occurrence (estimated at 1 in

10,000 to 1 in 100,000 tests)

False-positive results have been

attributed to ectopic sources of hCG or

elevated levels of circulating luteinizing

hormone

Ectopic hCG production: small cell ca

of the lung

High level of lutenizing hormone:

postmenopausal women, women just

before ovulation Bastian et al 1998

Abeloff’s Clinical Oncology 2008

Back to Our Case

Even though we did not have any information what her

the gestational weeks of last exposure, her first pregnancy

test may be taken so early.

Summary

Summaries • HPT kit instructions should be reviewed to make sure women

understand them.

• Encourage women to wait at least 2 weeks after a missed period

before performing the test

• Notify women of the potential for false-negative results

• Negative or inconclusive results in patients suspected pregnancy

should be further evaluated by serum quantification of hCG and

ultrasound.

• Another option to purchase a kit with two tests would be resonable

if the patient understands in advance that testing at the day of the

missed period or earlier can yield negative or only faintly positive

results.

Acknowledgment

•Dr. Irena Nulman •Dr. Shahnaz Akhtar Chaudry

Thank you for your attention!!