chroniclces of chemical tests in determining whether or not there is occurrence of pregnancy

1. AMOO ABDULAKEEM ENIOLA
(BMLS, AMLSCN)
Intern medical laboratory scientist,
Department of chemical pathology,
LTH Osogbo.
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2.  INTRODUCTION
 HISTORY
 hCG
 SAMPLE OF CHOICE
 RECOMMENDATION
 CONCLUSION
 REFERENCES
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3.  Pregnancy in simple terms is the period from
conception to birth. This period is usually 40weeks
after the Last Menstrual Period (LMP)
 Pregnancy Test (PT) attempts to determine whether a
woman is pregnant.
 Different methods had been designed since the early
times for the detection of pregnancy
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4.  In 1200 AD, it was believed that milk would
float on top of urine from a pregnant woman
 Earliest known pregnancy testing dates back to
1350 BC
 First scientifically based pregnancy test (PT) was
based on the presnce of Human Chorionic
Gonadotropin (hCG) in pregnant women urine.
(Loraine,2000)
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5.  The urine is introduced in animals (e.g
frogs toads etc)which are subsequently
observed for ovulation.
 In presence of ovulation, a pregnancy test
is said to be ‘positive’
 hCG detection and quantification had
since been in existence till now
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6.  Human Chorionic Gonadotropin (hCG) is an
oddball molecule, which forms one of the
four major glycoprotein hormones .
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7. BIOCHEMISTRY
 its composed of an alpha and beta sub
units
 The alpha subunit is 92 amino acid long
and weighs 14,500 daltons. The beta
subunit which is non covalently linked to
the alpha subunit is 145amino acids in
length, weighs 22,000 daltons
 Alpha and beta subunits has two N-
linked oligosaccharride and 4 O-linked
oligosaccharride respectively
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10.  Production
synthesized by trophoblastic tissue at time of
blastocyst implantation
 pituitary gland
 benign gastrointestinal neoplasm
 other neoplasm
 6-9days after conception
Function
hCG takes over the function of LH. Also:
stimulates progesterone production by corpus luteum
stimulates fetal gonads
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11. variants
 hCG
 hCG-H
 hCG
 hCGB-H
 hCG-S
 Total hCG is usually measured during
quantitatitve assays
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12.  The commonest and modern method for the
detection of pregnancy using hCG is based on
immunochromatographic principle (Matsuura
et al 2000)
 It could detect the whole molecule of hCG ,its
variants and products in urine and
serum/plasma sample
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15. Source: www.biochemistry.org
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17. MERITS -URINE
 Represents all hCG forms
produced
 hCG is degraded rapidly during
clearance of hCG, following
parturition, abortion of pregnancy,
evacuation of hydatidi-form mole
or ectopic pregnancy etc
 residual hCG or the last remnants
of hCG clearance may only be in
urine
 in a person having heterophilic
antibodies interfering with Serum
measurements, only a urine hCG
assay is meaningful.
LIMITATION - URINE
 The kidney can cleave hCG
forms.
 hCG-free β that is missing
the β C-terminal peptide,
for instance, can be cleaved
further by exopeptidases
and glycosidases in the
kidney, such that only βcore
fragment is present in the
urine.
 As reported by Stenman
and Alfhan (58), urine hCG
can change greatly with
freezing at –20°C and
thawing.
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18.  SERUM –MERITS
 Is a stable and
constant fluid medium
and does not vary with
liquid intake
 Serum has constant pH
and serum containing
hCG can be frozen and
thawed without
significant loss of hCG.
 SERUM-DEMERITS
 hCG and its variants
and degradation
intermediates are all
cleared from serum at
dramatically different
clearance rates.
 2-Heterophillic
antibodies interfere
with serum
measurements.
(Laurence 2011)
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20.  A comprehensive study of 27 brands of pregnancy
tests sold in France in 1989 showed that only 11 of
27 brands had 100% sensitivity and specificity
 Modern qualitative test kits have been confirmed to
be 100% sensitive ,precise and reproducible
 Despite this , there are recent report of interfering
substances and conditions that relate to their false
positivities and negativities
 False positive (FP) and negative (FN) results have
been largely related to the age ,activity and
educational level of the individual performing the
tests (Daviaud et al 1993)
 Poor comprehension of instructions, particularly
understanding how to visually interprete the test
amounts to false negative result often(Daviaud et al
1993)
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21.  Sensitive kits may give FP results in case of
exogenous hCG administration
 Other interfering substances and conditions
◦ Serum human anti-mouse antibodies
◦ -neoplasm autocrine hCG secretion
◦ -nephrotic range proteinuria
◦ -tubo ovarian abscess
◦ -Hsiu-fen et al recently reported WBC concentration
in urine could yield false positive result
 - Drugs
◦ -chlorpromazine
◦ -phenothiazine
◦ -methadone
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22.  Inexperienced end user
 Inadequate adherence to manufacturer
instruction of use
 HOOK EFFECT- a problem characterised
by masking of the binding site of the
analyte of interest by other molecules of
similar homology
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23.  EARLY PREGNANCY FACTOR (EPF) Test for
pregnancy which can give the quickest result
after fertilization is a ROSSETTE INHIBITION
ASSAY for (Fan and Zheng,1997)
◦ EPF can be detected in blood within 48 hours of
fertilization
 limitation
 Expensive
 Time-consuming
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24.  Its expected that hCG should rise exponentially
as the gestational period procedes up till 10th
week
a stray from this norm is usually indicative of
some disease
◦ -Ectopic pregnancy
◦ -Threatened abortion etc
 Measures hCG levels as low as 1mIU/ml
 Comonly measures hCG as total hCG
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25.  IT is interesting to know that only two of the five
variants of hCG reflects pregnancy;
 hormone hCG and autocrine hCG
 The common over the counter and point of care
tests kits can pick these secretions
 If other variants are to be measured by scientists,
 Quantitative determination of hCG is
recommended,and if its to be detected urine PT is
recommended
 -To avoid avoid FN and FP results
◦ -cloudy urine shuld be centrifuged before usage
◦ -serum PT should be correlated with the current
diagnosis
◦ -clinicians are advised to always state the correct
diagnosis when requesting for serum PT
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26.  hCG still remain the chief biomarker in
detection of pregnacy till date. keeping
abreast of upto date information in this age
of technological advancement, becomes
irresistibly germane to medical laboratory
scientists and other laboratorians
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27. THANKS
FOR
LISTENING
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28.  Daviaud J, Fournet D, Ballongue C, et al Reliability and feasibilityof
pregnancy home-use tests: laboratory validation and
diagnosticevaluation by 638 volunteers. Clin Chem1993;39:5359.
 Joel R. L. Ehrenkranz .Home and Point-of-Care Pregnancy Tests: A
Review of the TechnologyEPIDEMIOLOGY May 2002, Vol. 13 No. 3S
 Loraine JA. The Clinical Application of Hormone Assay Edinburgh: E and
S Livingston,2000.
 Matsuura S, Chen HC, Hodgen GD. Antibodies to the carboxyl- terminal
fragment of human chorionic gonadotropin beta-subunit:
characterization of antibody recognition sites using synthetic peptide
analogues.Biochemistry2000;17:575 580.
 Nisula BC, Wehmann RE. Distribution, metabolism, and excretion of
human chorionic gonadotropin and its subunits in man. Segal ST
1980;231 252, Plenum Press,Chorionic gonadotropin New York.
 www.biochemistry.org

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39.  Most pregnancy tests used today, whether a
home urine test, a physician's office urine
or blood test, or a clinical laboratory blood
test are "sandwich assays".
 Sandwich assays use two or more animal
antibodies raised against different sites on
Human Chorionic Gonadotropin (hCG).
 Usually a mouse monoclonal antibody
against one site on the hCG molecule, and a
mouse monoclonal, or a sheep, rabbit or a
goat polyclonal antibody against a second
distant site on the hCG molecule.
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41. But understanding the concepts of the
principle may be challenging.
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42.  Human chorionic gonadotropin (hCG) is a
glycopeptide hormone produced by the
placenta during pregnancy.
 The appearance and rapid rise in the
concentration of hCG in the woman's urine
makes it a good pregnancy marker.
 Usually, concentration of hCG in urine is at
least 25 mIU/ml as early as seven to ten
days after conception.
 The concentration increases steadily and
reaches its maximum between the eighth
and eleventh weeks of pregnancy.
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44.  The pregnancy testing device contains a unique set of dye-
conjugated and immobilized antibodies used to produce a
distinctive visual pattern indicating elevated concentration of hCG
(=25 mIU/ml) in the test sample.
 One antibody, the capture antibody, is in a solid phase permanently
attached to a tube, plate, membrane, or bead.
 Conjugate pad contains the label reagent, i.e. antibody labeled with
either red, gold or blue latex particles.
 Sample is applied and dissolves the label mixture and migrates to
the zone of immobilized antibody lines. If hCG is present, labeled
antibody-dye conjugate binds it, forming an antibody-antigen
complex.
 Positive, that is hCG containing, sample causes the formation of a
colored test line, which indicates a positive test result.
 As the reaction mixture continues to flow along the test membrane,
the complex binds to the anti-hCG antibody in the test zone of the
membrane, and produces a color band.
 Unbound conjugate binds to the reagents immobilized in the control
zone producing a color band, demonstrating proper performance of
the test.
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45.  Device with solid phase capture antibody to one
site on hCG, and liquid phase tracer antibody (label
shown by red star) to second or distant site on
hCG. In this way the label becomes immobilized.
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46.  Serum or urine containing hCG (shown as ab) added to device. The hCG
forms a sandwich or bridge between capture and tracer antibody. After a
short incubation period the hCG binds both the solid phase and liquid phase
antibodies linking them.
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47.  Excess tracer antibody is washed away. Amount of label or tracer (red star) is measured.
This is proportional to amount of hCG.
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49.  NOTE: Bring test components and specimens to
room temperature prior to testing.
 Remove a Testing Device from the foil pouch by
tearing at the "notch" and place it on a level
surface.
 Holding a Sample Dropper vertically, add exactly
four drops of the urine specimen to the sample
well. NOTE: Picture shows incorrect orientation of
dropper to test area, must be completely vertical to
ensure adequate sample.
 Read results at time indicated in procedure.
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50.  If two color bands are visible the test is
positive.
 The presence of a Control Band only indicates
a negative test.
 If two color bands are visible the test is
positive.
 The presence of a Control Band only indicates
a negative test.
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52.  The Control Band is used as a reference and
built in quality control check.
 If the Test Band is darker or similar to the
Control band, the test result is considered
positive.
 The Control Band is used for procedural
control to check whether the test reagents
are working properly and that a sufficient
amount of urine sample has been applied to
the test area.
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53.  If, after performing the test, no purple color
band is visible anywhere within the Results
Window, the result is considered invalid.
 If a color appears in the test area but NO
color appears in the control area, the test is
invalid.

 If, after performing the test, no purple color
band is visible anywhere within the Results
Window, the result is considered invalid.
 If a color appears in the test area but NO
color appears in the control area, the test is1/20/2018 53

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56.  The directions may not have been followed
correctly.
 Inadequate amount of sample has been
exposed to the test system.
 The test may have deteriorated.
 The directions may not have been followed
correctly.
 Inadequate amount of sample has been
exposed to the test system.
 The test may have deteriorated.
 The directions may not have been followed
correctly.1/20/2018 56

57.  Do not use test kit components after the
expiration dates.
 Dispose of all used test components in a
proper biohazard container.
 If specimens or test components have been
stored in a refrigerator, allow them to warm
to room temperature before performing the
test.
 Human specimens should be handled as if
capable of transmitting infectious agents.
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58.  Besides pregnancy, elevated concentrations of hCG may be found in
patients with both gestational and non-gestational trophoblastic
diseases. These conditions should be ruled out in the interpretation
of hCG levels to establish a diagnosis of pregnancy.
 A low incidence of false results can occur. Consult with a physician if
unexpected or inconsistent results.
 A normal pregnancy cannot be distinguished from an ectopic
pregnancy based on hCG levels alone.
 A spontaneous miscarriage may cause confusion in interpreting the
test results.
 A definitive diagnosis should not be based on the results of a single
test, but should only be made by the physician after all clinical and
laboratory findings have been evaluated.
 A negative result from a specimen collected from a woman in very
early pregnancy may be due to an unusually low concentration of
hCG. In such cases, the test should be repeated on a fresh specimen
obtained approximately two days later.
 A urine sample may be too diluted and thus may not contain a
representative concentration of hCG. If a negative result is obtained
with a urine specimen and pregnancy is still suspected, obtain a first
morning urine specimen and re-test.
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59.  This unfortunate patient came in with a late cycle. The pregnancy test was
positive but we couldn't see a pregnancy on her ultrasound scan. We
suspected an ectopic and this is what we found during laparoscopy.
 The pregnancy was lodged in the middle of her left tube. A small slit was
made over this and the pregnancy was removed without removing her tube.
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60.  Choriocarcinoma is a highly malignant germ cell
tumour which usually follows an abnormal
pregnancy with a hydatidiform mole. It may also
occur after a spontaneous abortion, and rarely,
may follow a normal pregnancy. The tumour
metastasizes early, by means of vascular invasion
and blood spread. Picture of liver.
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62.  Hydatidiform mole is a tumor of the placenta that is usually
benign. It develops from placental tissue during an early
pregnancy in which the embryo fails to develop normally. The
tumor consists of many small vesicles (sacs) and resembles a
large cluster of grapes. Although the condition is fairly rare in
the USA, it is common in the Orient and other parts of the
world. In the United States, molar pregnancy occurs in 1 of
every 1,000-1,200 pregnancies.
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63.  50% of all testicular tumors.
 Human chorionic gonadotropin-beta is a
better marker than hCG. For earlier
detection of recurrence, both markers
should be examined.
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64.  First morning urine usually contains the highest
concentration of hCG and is therefore the best
sample when performing the urine test. However,
randomly collected urine specimens may be used.
 Collect a urine specimen in a clean glass, plastic, or
wax coated container. Do not use preservatives.
 If the test is not run immediately following
collection of the sample specimen, but is to be run
within 48 hours following collection, the specimen
should be refrigerated (2-8°C), and brought back to
room temperature (15-28°C) before testing.
 If testing is delayed more than forty-eight hours,
the specimen should be frozen. A frozen specimen
should not be used if stored more than two weeks.
 Prior to testing, the frozen specimen must be
completely thawed, thoroughly mixed, and brought
to room temperature.
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65.  The use of controls is recommended to
verify proper kit performance.
 Quality control reagents (positive and
negative) should be tested according to
quality control requirements established by
the testing laboratory.
 Use controls in the same procedure as
specimens.
 This may be required with each test or only
when a new lot number is being put into
use.
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66.  http://www.saveontests.com/pregnancy_test_strip_instructions.htm
 http://www.ovulation-calculator.com/pregnancy-tests/pregnancy-
tests.htm
 http://sunflower.bio.indiana.edu/~aberndts/Z566/lab1image
s.html - fertilization
 http://www.healthsentinel.co.uk/professional/professional-pregnancy-test-
info.htm
 http://www.docsun.com/default.asp?id=26 – tubal pregnancy
 http://www.moondragon.org/obgyn/pregnancy/molar.html
 http://www.nlm.nih.gov/medlineplus/ency/article/001288.htm
seminoma
 http://www-
medlib.med.utah.edu/WebPath/TUTORIAL/PRENATAL/PRENATAL.html Some
pictures very graphic
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