“PREGNANCY TEST”

• Misnomer
- do not actually determine pregnancy,
but human chorionic gonadotrophin (hCG)

- used to diagnose conditions other than

pregnancy
 HUMAN CHORIONIC
GONADOTROPHIN

Alpha polypeptide subunit MW=

18,000
Beta polypeptide subunit MW=
 Bioassay
• 1928 - Aschheim-Zondek: Also known as the A-
Z Test, this very first scientific pregnancy test was
developed in Germany. It involved several
subcutaneous injections of varying amounts of a
woman's urine into the backs of immature female
mice weighing between five and eight grams. One
hundred hours after the first injection, the mice were
killed and the ovaries were examined. The A-Z test is
named for German researchers Selmar Aschheim
(1878-1965) and Bernhard Zondek (1891-1966).
Zondek was the first person to describe the ovarian
hormone and earned worldwide renown for
discovering gonadotropins.
• 1931 - Friedman: A refined version of the Aschheim-
Zondek test, the Friedman test used rabbits instead
of mice. Its advantages were the availability of the
animals, a reduced error rate, and the reduced time
required for completion of the test. The subject's
urine was injected into the ear vein of a female
rabbit. The test, named for Maurice H. Friedman
(1903-1991), could be read within 36 to 48 hours
after injecting the urine.
•1939 - Hogben (Xenopus): In the Hogben test,
a female African clawed toad (Xenopus laevis) is injected
with urine (or an extract) into the dorsal lymph sac. The
presence of five, six, or more eggs within four to twelve
hours indicates pregnancy. A similar test was done using
male frogs or male toads. A woman's urine or serum is
injected into the dorsal lymph sac of two male frogs
(Rana pipiens) or male toads (Bufo marinus). The
presence of spermatozoa in the cloacal fluid of both
animals is positive; in one animal, inconclusive; in
neither animal, negative. This test is named for the
British biologist Lancelot Hogben (1895-1975).
Although the Hogben pregnancy test had the advantage
of not killing the test animals, it was replaced by
 Limitations:
• Interference from LH
• Technical difficulty
• Standardization
• High Cost
• Maintain animal colonies
 Immunoassay
• Started in 1960’s
• Expressed in International Units
• 1 IU = 83.3 mg/dl
 Immunoassay
Hemagglutination Inhibition (Urine)
• Uses Anti-HCG
• If HCG is present, Neutralization would
occur
• HCG coated RBC indicator cells would
sink to the bottom of test tube and form a
ring
• Took 1 to 2 hours
 Immunoassay
Hemagglutination Inhibition
(Urine)
 Immunoassay
Latex Particle Agglutination
Inhibition
 Immunoassay
Direct Latex Particle
Agglutination
• Tube tests HAI and LAI 1-2 IU (5-7 days
after first missed menses
• Slide tests 2-5 IU (14-21 days after
missed)
 Immunoassay

Radioimmunoass
ay
•Uses radioisotopes
•More sensitive and could use serum
•Not affected by LH as much
•Need special equipment to read
•Usually was done when early dx was
important
Immunoassay
Enzyme
Immunoassay

ELISA Kit
 Immunoassay

Enzyme
Immunoassay
•Monoclonal Beta - HCG Enzyme Tests
•Very sensitive
•Can pick up .05 IU (1 week after
implantation)
• specificity is high because cross reaction
with
other hormones besides hCG is not a
 Quantitative Pregnancy Testing
• Ectopic pregnancies low levels
• low levels in 1 st trimester = bad prognosis
• Trophoblastic Tumors
- Hydatiform Mole - benign grape-like mass
- Choriocarcinoma - may follow usual childbirth
or incomplete removal of hydatiform mole,
malignant
*Increased levels of up to 6,000,000 IU vs.
100,000 in normal pregnancy
• Increased HCG
-Seen in multiple pregnancies and
Eclampsia
-High levels HCG seen in males that have
testicular tumors
Home pregnancy test
 Home pregnancy test

• Wait at least 1 week after the date of the expected period

• Use first morning urine
• Try to test the urine sample immediately after collection
• If testing occurs at other times of day, restrict fluid intake for 4-6 hours
• Waiting 10 minutes to read results improve results
• If test is negative, test again in 1 week if menstruation has not started
 Human Placental Lactogen (HPL)
• Synthesized in large quantities by placenta during last two trimesters
• Structurally similar to both prolactin and growth hormone
• Insulin antagonist
• Exact role is unclear

Alpha Feto Protein (AFP)

•Detection of Neural Tube Defects (NTDs)

Neural tube defects include both spina bifida(improper closure of the fetal
spine) and anencephaly(improper closure of the fetal skull). Anencephaly is a

lethal condition.
Pap Smear Collection
Technique
 Who should have a Pap smear?
• The American Cancer Society recommends that you
have your first Pap smear about three years after first
having sexual relations or at age 21. After age 21, the
guidelines are as follows:
 Prior to Pap Smear:
Try to schedule your Pap smear when you are not menstruating. If
your cycle is unpredictable, and you start your period when it will
coincide with your appointment, call your doctor's office as soon as
possible to see if the doctor may recommend rescheduling.
To ensure that you get the most accurate results, it is recommended
that 48 hours before having a Pap smear you should not:
• have sex
• douche
• use tampons
• use spermicidal foams, creams, or inserts
• use vaginal creams or suppositories

All of these things can wash away or hide abnormal cells.

 Positioning
• Privacy
• Buttocks just off table
• Good Lighting
• Drape

PARENTAL
GUIDANCE
Padded Stirrups
• Soft, padded stirrups
• Oven mitts
• Socks
 Inspect
• Spread labia
• Discharge
• Ulcers
• Growths

PARENTAL
GUIDANCE
 Anatomy

PARENTAL
GUIDANCE
Vaginal Speculum
Warm Speculum
• Warm water
• Not too hot
• Lubricates speculum
• Don’t use K-Y,
Surgilube or Vaseline
to lubricate speculum
 Insert Speculum
• Spread labia
• Keep labia apart
• Blades remain
closed until fully
inserted

PARENTAL
GUIDANCE
Squamo-Columnar Junction

• Junction of pink cervical

skin and red endocervical
canal
• Key portion of the cervix to
sample
• Most likely site of
dysplasia
Ayers Spatula

• Concave end to fit the

cervix
• Convex end for vaginal
wall and vaginal pool
scrapings
Sample Cervix

• Use concave end

• Rotate 360 degrees
• Don’t use too much
force (bleeding, pain)
• Don’t use too little
force (inadequate
sample)
Cytobrush

• Insert ~ 2 cm (until
brush is fully inside
canal)
• Rotate only 180
degrees (otherwise will
cause bleeding)
Make Pap Smear
• As thin as possible
• Properly labeled
Spray with Fixative

• Within 10-15
seconds
• Allow to fully dry
before packaging
• Cytologic Fixative
(hairspray works
acceptably also)
Factors That Diminish the Accuracy of Pap Smears

• Clinician Related Factors

- Contamination with blood or lubricants
- Mislabeled or unlabeled slides
- Inadequate clinical history
- Inadequate sampling of the transformation zone
- Slide material too thick or insufficient
- Performing pap in spite of obvious infection

• Laboratory Related Factors

- Confusing smears or names
- Failure to identify dysplastic cells
- Misinterpretation of diagnostic cells
- Poorly controlled technical process
 Background
• George Papanicolaou, MD, PhD, discovered that
tumor cells could be found in vaginal fluid of
women with cervical cancer
• 1928 - presented his paper entitled "New Cancer
Diagnosis" at the Third Race Betterment
Conference in Battle Creek, Michigan,
• 1943 - the concepts of early cancer and
carcinoma in situ were widely understood, and
the potential of the "Pap smear" for cancer
prevention was finally appreciated
The Pap Smear
• It was a Canadian physician, J. Ernest
Ayre, described the method we know
today as the Pap smear
• 1940s, cytology laboratories were
opening, and by the 1950s, Pap smear
screening was widespread, even before
clinical trials could be performed
 UNDERLYING PRINCIPLE OF CYTOLOGY

EXFOLIATIVE CYTOLOGY

DIFFERENT TYPES OF CELLS ARE PERIODICALLY DESQUAMATED FROM

THE NORMAL CELLULAR ELEMENTS :

SUPERFICIAL CELLS
 INTERMEDIATE CELLS

NAVICULAR CELLS
PARABASAL CELLS
ENDOCERVICAL CELLS
ENDOCERVICAL CELLS
LACTOBACILLI / COCCOBACILLI

GARDNERELLA VAGINALIS
(CLUE CELLS)
M 26
Candida spp.
Candida spp.
Leptothrix
ACTINOMYCES SPP.

M 27
TRICHOMONAS VAGINALIS
M 28
Herpes simplex
NORMAL
 LSIL

LSIL
 HSIL

CIN2 HSIL (Moderate dyskaryosis)

 HSIL

CIN3 HSIL (Severe dyskaryosis)

SCCA
ASCUS
 Atypical squamous cells of undetermined
significance (ASCUS)
• is defined, according to the Bethesda System,
as squamous abnormalities that are more
marked than those attributable to reactive
changes, but that quantitatively or qualitatively
fall short of a definitive diagnosis of a squamous
intraepithelial lesion
• cells as having nuclei about two to three times
normal size with normochromatic or slightly
hyperchromatic nuclei, even chromatin
distribution, and smooth or only slightly irregular
nuclear membranes.