What is Truth of Home Pregnancy Test Kit?

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

2. 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.

3. Contents
• Structure of hCG (free subnuits of hCG)
• Home pregnancy test kit
• False negative of HPT kit
• False positive of HPT kit
• Summaries

4. Human Chorionic
Gonadotropin

5. Human Chorionic Gonadotropin (hCG) (1)
• 237 amino acid glycoprotein
hormone composed of two dissimilar
α-Subunit
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 β-Subunit
pregnancy and trophoblast disease,
and in small amounts by poorly
differentiated cancer (testicular ca.,
ovarian ca., bladder ca)
Cole 1996, Javadpour 1977
Williams Obstetrics 23rd Ed

6. Human Chorionic Gonadotropin (hCG) (2)
α-subunit of hCG
• Identical to those of lutenizing
Same as that α-
hormone (LH), follicle – subunit of hCG
stimulating hormone (FSH),
and thyroid- stimulating
hormone (TSH) •Hyperglycosylated,
• Composed of 92 aminoacids with larger, more-
complex N-linked
linked by five disulfied bridges oligosaccharides
• Two forms of free α-subunit
•Majority of free α
molecules in pregnant
are present in serum and urine
urine.
Prevent combination of large free α with ß-subunit

7. Human Chorionic Gonadotropin (hCG) (3)
ß-subunit of hCG
• 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

8. Human Chorionic Gonadotropin (hCG) (4)
Nicked hCG
• 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.
Cole 1993, Kardana 1997

9. Human Chorionic Gonadotropin (hCG) (5)
Free ß-hCG
• 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
Cole 1993

10. Human Chorionic Gonadotropin (hCG) (6)
ß-core fragment
• 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

11. 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. Mean concentration of hCG in serum of women throughout
normal pregnancy.
• Doubling time of plasma hCG
conc. is 1.4 to 2.0 days.
• Serum hCG levels reach peak
levels at 60 to 70 days.

12. Boxplot comparison of the first day of hCG detection
postgonadotropin surge for cycles
Serum
• Serum hCG detectable 9-11
days after conception (1-3
days before expected menses)
• Urine hCG usually
Urine detectable 1-2 days later
(highly variable)
Early pregnancy loss (EPL), clinical spontaneous abortion (CAB), or successful pregnancy (SP)
Lohstroh. Secretion and excretion of hCG during early pregnancy. Fertil Steril 2005.

13. Human Chorionic Gonadotropin (hCG) (8)
• Multifetal pregnancy
Higher maternal • Erythroblastosis fetalis with fetal hemolytic anemia
plasma hCG • Gestational trophoblastic dz
level • Down SD
• Early pregnancy wastage
Lower maternal
• Ectopic pregnancy
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

14. Home Pregnancy Test

15. 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
Why do women choose to use HPT?
professional health care.
• Speed of obtaining results
• Convenience of testing at home
• Women is the first person to know that she is pregnant.

16. Home Pregnancy Test

17. Home Pregnancy Test
Negative test

18. Home Pregnancy Test
Positive test

19. 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 .

20. 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.
Actually based on tests run on samples collected
after 15 days or more beyond the missed menses.
Maoi 1979
• 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

21. Sensitivity vs Accuracy
• TP/(TP + FN)
Sensitivity • (Number of true positive assessment)/
(Number of all positive assessment)
• (TN + TP)/(TN + TP + FN + FP)
Accuracy • (Number of correct assessment)/
(Number of all assessment)
TP: True positive, TN: True negative, FP: False positive, FN: False negative

22. Home Pregnancy Test
Combined and Brand Comparison of In-Home Pregnancy Test for Sensitivity, Specificity, and Predictive Value
(expressed as per cent)
 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 Mary 1986
 Multistix® SG Reagent Strips

23. Home Pregnancy Test
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
Mary 1986

24. 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

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

26. Home Pregnancy Test
Accuracy of home pregnancy tests at the time of missed menses (2)
Most sensitive test kit
Cole et al 2004

27. Home Pregnancy Test
Relationship between Gestational Age, Quantitative β-Human Chorionic Gonadotropin Levels, and
Ultrasound Findings
Time Elapsed from 1st Day of Last Normal Quantitative β-HCG Level (mIU/mL) Using
Ultrasound Findings
Menstrual Period the IRP
<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-hC
G > 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
Roberts: Clinical Procedures in Emergency Medicine, 5th ed.

28. False Negative of HPT kit

29. Home Pregnancy Test
Causes of False-Negative
• 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

30. Home Pregnancy Test
Failure to read or follow instructions
• 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

31. Home Pregnancy Test
Dehydration and Decreased urinary pH
If the pH of the urine is
below 5, then the test might
Concentration
Acidic urine theoretically affect the
of urine
result.
This kit is tested for the
Dehydration
effect of urine pH from 5-9
with hCG concentrations at
False Negative 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.

32. Home Pregnancy Test
Hepatitis and Increase renal excretion
High
All of the
dose
serum hCG
hook
might be
effect
metabolising
in kidney,
If the and excreted
metabolism of in the urine
hCG is affected
in hepatitis.

33. Home Pregnancy Test
Normal Sandwich Assay
 Antigen is sandwiched between the antibody coated on the
plate and the conjugate
C C C C
C C
Positive

34. Home Pregnancy Test
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
C C C C
C C
C C
C C C C
C C C C C C
False negative

35. False Positive of HPT kit

36. Home Pregnancy Test
False Positive
False Positive
 Rare occurrence (estimated at 1 in
10,000 to 1 in 100,000 tests)
Abeloff’s Clinical Oncology 2008
 False-positive results have been Signaling Ab
attributed to ectopic sources of hCG or
elevated levels of circulating luteinizing Interfering Ab
hormone
Capture Ab
 Ectopic hCG production: small cell ca
of the lung
 High level of lutenizing hormone:
postmenopausal women, women just
before ovulation Bastian et al 1998

37. 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.

38. Summary

39. 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.

40. Acknowledgment
• Dr. Irena Nulman
• Dr. Shahnaz Akhtar Chaudry

41. Thank you for your attention!!