exfoliative cytology

EXFOLIATIVE CYTOLOGY

a branch of General Cytology which deals wit the microscopic study of cells that have been desquamated from the epithelial surfaces.

recommended for :1. detection of malignant cells or precancerous lesions in the

body

2. detection of asymptomatic or precancerous cervical lesions in women

3. assessment of female hormonal status in case of sterility and endocrine disorders

4. Determination of genetic (phenotypic) sex

5. Detection of the presence of infectious microorganisms

SPECIMENS FOR EXAMINATION

1. peritoneal, pericardial and pleural fluids

2. CSF

3. nipple discharge

4. bronchial brushings / washings

5. sputum

6. gastric washings

7. urine sediment

8. prostatic secretions / fluid

9. cervicovaginal (paps) smear

SPECIMENS THAT REQUIRE ADHESIVE AGENT

1. Urinary Sediment2. Bronchial Ravage Specimen3. Specimens that utilizes proteolytic enzymes

during processing (eg. Trypsin, conc. Sputum and

enzymatic lavage sample from the GIT)

• CHARACTERISTICS OF A GOOD ADHESIVE

1. must be permeable to both fixative and the stain2. must not retain the stain

• GOOD ADHESIVES FOR CYTOLOGIC METHOD

1. Pooled human serum or plasma2. Celloidin Ether-Alcohol3. Leukonostoc culture

• Collection Procedure:

- Using standard paracentesis technique, obtain a fluid

specimen from the desired body cavity. A minimum of

10 mL of specimen is desirable for optimal cytologic

evaluation. Heparin may be added to the specimen to

reduce clotting.

- Place three (3) units of heparin per mL capacity of

the collection container. Gently agitate to thoroughly

mix the specimen and heparin. - - Submit the specimen along with the completed cytology

request form.

- The specimen should be refrigerated or kept on wet

ice until transported to the lab.

COLLECTION OF SPECIMEN

A pleural smear examines a sample of pleural fluid under the microscope to detect for abnormal organisms.

COLLECTION OF SPECIMEN

During a pericardial fluid culture, a small needle is inserted into the chest between the ribs into the

pericardium (the thin sac that surrounds the heart) and a small amount of fluid is withdrawn. Samples of the fluid are placed in various culture media in the laboratory, and the media is observed for the growth of microorganisms. The test is performed when an infection of the heart is suspected or when a pericardial effusion is present.

COLLECTION OF SPECIMEN

A peritoneal culture is a procedure where peritoneal fluid is withdrawn with a needle from the peritoneal cavity. The peritoneal cavity is the space between the two membranes lining the abdominal cavity. The test is

done to determine the cause of ascites, fluid accumulation in the peritoneal space.

COLLECTION OF SPECIMEN

CSF is usually obtained through a lumbar puncture (spinal tap). During the procedure, a needle is

inserted usually between the 3rd and 4th lumbar vertebrae and the CSF fluid is collected for testing.

COLLECTION OF SPECIMEN

A breast biopsy can help diagnose breast cancer early in the disease process.

COLLECTION OF SPECIMEN

COLLECTION OF SPECIMEN

1. Gently strip the sub-areolar area and nipple with the thumb and forefinger.

2. Place the slide upon the nipple and draw it quickly across the nipple. ***If 1 drop is obtained, get another slide and do the pull-apart technique.

3. Immediately immersed the slide into a bottle of 95% isopropyl ROH or use spray fixative.

4. If the secretion is very little in amount, the smear should be restricted to a small area only to prevent drying.

5. Label the secretions obtained indicating from where they’re collected. (left or right breast)

a. bronchial washing

b. bronchial lavage

c. bronchial brushing

COLLECTION OF SPECIMEN

•Specimen:- Bronchoscopically-directed brushing of the

identified lesion.

•Collection Procedure:

- Using standard bronchoscopy technique, identify the lesion in question and obtain a brushing sample of the lesion.

- Gently apply the sample on to glass slide and immediately immerse slideinto 3% glacial acetic acid alcohol fixative.

- The brush tip should also be cut off into the solution.

COLLECTION OF SPECIMEN

Bronchial Brushings

COLLECTION OF SPECIMEN

Bronchial Washings

•Specimen:

Bronchscopically-obtained washing (preferable at least 10 mL) of the bronchi in the region of thesuspected lesion.

•Collection Procedure:

-Using standard bronchoscopy technique, lavage the distribution of the bronchus to be sampled.

-Collect the wash in a clean container. Label the container with correct patient information and

COLLECTION OF SPECIMEN

NORMAL

-obtain at three consecutive morning sputum specimens by deep cough method.

-Collect the sample in a wide-mouth container containing Saccomano fluid (50% EtOH and 2% carbowax)

INDUCED

- Inhalation of aerosol solution for 20 mins to produce deep cough sample.

- Collect the sample in a wide-mouth container containing Saccomano fluid (50% EtOH and 2% carbowax)

Gastric suction is perform to empty the contents of the stomach before it passes through the rest of the

digestive tract

COLLECTION OF SPECIMEN

COLLECTION OF SPECIMEN

Washings (Esophageal, Gastric, Other)

•Specimen: Endoscopically obtained washing (preferably at least 10 mL) of the region of the suspected lesion.

Collection Procedure:

-Instruct the patient to fast overnight or for a minimum of six hours prior to theprocedure.

- Using standard endoscopy technique, lavage the area of interest using a physiologic solution. Aspirate the solution and place in a clean specimen container.

- If transport of the specimen will be delayed more than four (4) hours, thespecimen should be refrigerated or kept on wet ice until transported to the lab.

COLLECTION OF SPECIMEN

Brushings (Esophageal, Gastroesophageal Junction, Gastric, Duodenal, Bile Duct, Other)

•Specimen:

- Endoscopically-directed brushing sample of the identified lesion.

•Collection Procedures:

-Instruct the patient to fast overnight or for a minimum of six hours prior to the procedure.

- Using standard endoscopy technique, identify the lesion in question and obtain a brushing sample of the lesion.

- Gently apply the sample on to glass slide and immediately immerse slide into 3% glacial acetic acid alcohol fixative.

-The brush tip should also be cut off into the solution.

• Collection Procedure:

-Use an unlubricated speculum (saline or warm water may be used).

-After visualization of the cervix is accomplished, insert the sampling device (e.g. cytobrush®) into the endocervical canal and rotate one complete turn.

-Withdraw the cytobrush and spread the collected material quickly and evenly onto the slide opposite the frosted end.

COLLECTION OF SPECIMEN

The endocervical mucus will prevent air-drying during collection of the subsequent cervical component.

Using the extended-tip spatula ( e.g. Ayerst®), scrape material with the spatula from the whole circumference of the cervix.

Withdraw the spatula and spread the collected material quickly and evenly onto the slide adjacent to the frosted end.

Fix Immediately (drop slide into fixative or spray with fixative, holding the spray bottleapproximately 8 to 12 inches from the slide).

COLLECTION OF SPECIMEN

COLLECTION OF SPECIMEN

COLLECTION OF SPECIMEN

COLLECTION OF SPECIMEN

Click to view the video:

•Insertion of speculum

•Physical examination of the genitalia

•Collection of cervico-vaginal specimen

smears should be from fresh material

see requisition form (patient’s ID: name, age; date and type of specimen requested

label the slide

-- Methods of Smear Preparation:

1. streaking

2. spreading

3. pull apart

4. touch or impression smear

SMEAR PREPARATION

- used for preparing mucoid secretions vaginal secretions, sputum and gastric content)

- use a spatula, dissecting needle or applicator stick and streak in a zigzag fashion

SMEAR PREPARATION

- used for thick mucoid secretions (smears of fresh sputum and bronchial aspirates)

SMEAR PREPARATION

- for serous fluids, concentrated sputum, and enzymatic lavage form the GIT, smears of urinary sediment, vaginal pool and breast secretions

SMEAR PREPARATION

- for preparation of direct impression from the cut surface of tissue like the lymph nodes and other surgical or autopsy secretions.

Impression cytology being collected From a patient with herpes simplex keratitis, using a sterile glass slide with polished edges.

SMEAR PREPARATION

•WHY FIX?

- exfoliated cells decompose rapidly which may destroy cellular and nuclear details, in turn will give inadequate results for diagnosis.

•COMMON FIXATIVES

1. equal parts of 95% EtOh and ether

2. 95% EtOH

3. Carnoy’s fluids

4.equal parts of tertiary butyl alcohol and 1 part 95% EtOH

5. SCHAUDINN’S FLUID – sat. aq. Hg2Cl, absolute HoAc

6. MeOH – for dried films

1. PAPANICOULAOU or Pap’s Smear

Advantage:

- transparent blue staining of cytoplasm is observed

- excellent nuclear staining

- color range is predictable and of great value in identification of cells

Disadvantage:

- procedure is lengthy and complicated

- does not give accurate acidophilic index

Stains for Pap’s:

1. Harris Hematoxylin

2. OG 6 Stain

Orange Green 6, 0.5 solution in 95% ROH 100 ml

Phosphotungstic acid0.015gm

3. EA 50

light green SF, yellowish 0.1% solution in 95% ROH 45ml

Bismarck brown 0.5 in 95% ROH 10 ml

Eosin Y, 0.5% in 95% ROH 45 ml

Phosphotungstic acid 0.2 gm

Lithium Carbonate. Sat. aq. Solution 1 drop

*** EA 50 is comparable to EA 36

*** EA 65 differs from EA 50 or EA 36 only with respect to the concentration of the light green stock solution

Procedure for Pap’s Stain:

1. Fix in ether-ROH and pass thru 80% ROH, 40% ROH and distilled H2O.

2. Stain in Harris Hematoxylin for 4-5 minutes.

3. Wash with H2O.

4. Pass thru 0.25% HCl in 50% ROH.

5. Immerse in 1.5% NH4OH in 70% ROH for 1 minute.

6. Rinse in 70% ROH and pass thru 80% and 95% ROH.

Procedure for Pap’s Stain:

7. Stain with OG 6 for 1.5-a minutes.

8. Pass thru 3 changes of 95% ROH.

9. Stain with EA 65 or EA 50 for 3 minutes.

10. Pass thru 3 changes of 95% ROH.

11. Dehydrate and clear in: a. absolute ROH,

b. equal parts of ether and absolute ROH, c. 2 changes of xylol

12. Mount in Canada Balsam.

Results:Cytoplasm – either bright red or

greenish blue

vesicular nucleus – blue

pyknotic nucleus – dark blue to black

bacteria – dark blue

mycelia – violet

Trichimonas vaginalis – pale greenish blue blob of cytoplasm

Cytoplasm – either bright red or greenish blue

vesicular nucleus – blue

pyknotic nucleus – dark blue to black

Trichimonas vaginalis – pale greenish blue blob of cytoplasm

NUCLEI CHANGES

Altered nuclear-cytoplasmic ratios – due to the enlargement of nuclei and a most important criteria of

malignancy. However this is benign in certain tissues, e.g. endocervical and renal pelvic cells.

Hyperchromasia – although common in malignant cells, one must be careful to exclude

overstaining and not to mistake pyknosis for hyperchromasia.

Increased mitotic activity – frequently seen in reactive pleural and peritoneal mesothelial cells, in

histiocytes and in any tissue undergoing active repair and benign growths

Atypical mitoses –triple or quantiple spindles are highly suspicious and important signs of

malignancy.

NUCLEI CHANGES

Multinucleate cells – with irregular hyperchromatic or bizarre nuclei should be suspicious,

but caution is warranted in certain situations, e.g. after abortion, or expulsion of H-mole, Herpes complex infection

Anisokaryosis – considerable variation in nuclear size and shape is common in

malignant cells.

giant single nucleus (polyploidy) – often seen in malignant cells, polypoidic cells may also occur in benign

conditions, especially in thyroids of older women and less in adrenal cortex and hyperactive islets of Langerhans. In vaginal smears, they may

result from pregnancy as well as malignancy.

CYTOPLASMIC CHANGES

- cells of epidermoid carcinomas frequently show a tendency to cytoplasmic eosinophila.

- adenocarcinoma cells may enclose PMNs, eg. endometrial and colonic cancers)

- cytoplasmic vacuolation is common in adenocarcinoma cells, but may be also be seen in endometrial cells following

cutterage.

APPERANCE OF WHOLE CELLSIn general:

- malignant cells show reduced cohesiveness, possibly related to a defect of the intercellular “zippers” i.e., desmosomes.

-Cancer cells are larger than their normal counterparts and frequently show bizarre and grotesque shape.

Occasionally:- exfoliated cells from epithelial tumors assume a greatly

elongated, fibrocyte-like appearance.

CELL PATTERN

•Examine for the small groups or clusters of cells:-Oblivious patterns

e.g. acini in adenocacinoma arising in glandular tissues-Rosettes in neuroblastomas and ependymoma

-Stratification in squamous epithelial growths-Whorls in mesotheliomas

•If there are no patterns revealed, focus up and down the on cell clumps

-In strips epithelium, irregular stratification of anisokaryotic, hyperchromatic cells are helpful in

diagnosing carcinoma of cervix and bronchus.

OTHER CRITERIA

1. In bronchial secretion smear, abundant lymphocytes are common in presence of malignancy, but may also

be found in certain inflammatory conditions and in leukaemia. 2. The presence of old blood and blood pigments is a minor indirect clue, but has many other etiologies.

The vaginal epithelium is responsive to sex steroids, particularly estrogen, and undergoes predictable changes through the cycle in response to changes in blood concentrations of ovarian hormones. Rising levels of estrogen cause the vaginal epithelium to become

"cornified" - the surface cells become large and flattened, with small or absent nuclei.

In essence, vaginal cytology is a type of endocrine assay. Tracking changes in the morphology of desquamated vaginal epithelial cells

provides a convenient means of assaying changes in estrogen levels.

•Vaginal smears may be taken regularly and often.

•Hormonal changes are best mirrored in the upper third of the vagina.

•They can also be taken from the lateral walls because their more accessible and less likely to be contaminated by cellular debris or discharge.

-Large (30-60u)

-Polyhedral flat cells

-Cytoplasm: may be acidophilic or basophilic

-Presence of small dark pyknotic nuclei (less than 6u)

SUPERFICIAL CELLS

SUPERFICIAL CELLS

SUPERFICIAL CELLS

SUPERFICIAL CELLS

Anucleate cells are abnormal which may be derived from:

1. smear contamination by the cells from the vulva

2. epidermization of the vagina or cervix resulting from prolapse

3. leukoplakia of the cervix

4. ruptured membranes in pregnant women

5. marked hyper-estrinism

- medium large (20-30u)

- Polyhedral or elongated

- Cytoplasm: basophilic with vacuoles

- Vesicular nuclei (6-9u)

INTERMEDIATE CELLS

INTERMEDIATE CELLS

- Boat-shaped intermediate cells with a strong tendency to fold and curl their edges.

- Expression of the combined estrogen-progesterone effect

-found in the latter half of menstrual cycle, during pregnancy, menopause

- may also be found as a result of abnormal androgen stimulation, either endogenous or exogenous

NAVICULAR CELLS

NAVICULAR CELLS

- Round or oval to oval shaped cell

- has a translucent basophilic cytoplasm (due to glycogen accumulation)

- cytoplasm stains deep blue or blue green + cell membrane = a double cell wall appearance

PREGNANCY CELLS

PREGNANCY CELLS

-Round to oval cells

-Smaller than intermediate (15-25 u)

-Thick

-“sunny-side up” like cells

-Have strong basophilc cytoplasm and vesicular nucei (6-9 u )

-Found from 2 weeks of age to puberty, after childbirth, abortion or miscarriages and after menopause.

PARABASAL CELLS

PARABASAL CELLS

-Slightly cylindrical appearance

- occurs in groups and strips of three or more cells

- cytoplasm: deeply basophilic the that of the parabasal cells

ENDOCERVICAL CELLS

ENDOCERVICAL CELLS

-small (13-20u)

-Round, slightly oval cells, with relatively large nucleus that occupying half or more of the cell volume

-Cytoplasm: strongly basophilic

-Found in vaginal smears only before pregnancy and after menopause

BASAL CELLS

BASAL CELLS

-Found during menstruation period ( in groups) and 1-4 days after the cessation of the period (single)

-Endometrial stromal cells: seen in tight clusters of small, oval dark cells; Glandular cells: slightly larger.

-Nucleus: small and moderately dark

-Cytoplasm: basophilic and maybe vacuolated

ENDOMETRIAL CELLS

ENDOMETRIAL CELLS

- “Lactobacillus acidophilus”

-Gram + slender rod bacteria

- Predominant organism of the vaginal normal flora: establishes the

low pH that inhibits the growth of pathogens

-Stains pale blue to lavender

-Energy is obtained by the fermentation of glycogen derived from disintegrating epithelial cells

-Numerous in the luteal phase and during pregnancy

DODERLEIN BACILLUS

DODERLEIN BACILLUS

CYTOLYSIS IN VAGINAL CYTOLOGY

- Infection

- estrogen

-low vaginal pH (below 4.2)

Occurs in the last trimester of pregnancy, more common in diabetic patients

shows large numbers of naked nuclei and very abundant Doderlein bacilli.

QUANTITATION IN VAGINAL CYTOLOGY

ACIDOPHILIC INDEX (A.I.) - percentage of cells that stain pink-orange to red with Pap’s and red in Shorr method

PYKONTIC INDEX (P.I) – “karyo-pyknotic index”; percentage of cells having shrunken, dark, small (less than 6μ) structures nuclei.

MATURATION INDEX (C.H.M.I.) – percentage proportion of cells from the three layers of the vaginal epithelium.

CLASSIFICATION OF PAP TEST RESULTS

Class I Negative for Malignant cells

Class IIAtypical cells present, but Negative for Malignancy

Class III Suspicious for Malignant Cells

Class IV Strongly Suggestive for Malignant Cells

Class V Conclusive for malignant Cells

The BETHESDA SYSTEM

Specimen AdequacySatisfactoryLimitedUnsatisfactory

General Categorization:Negative for Intraepithelial lesion or malignant cellEpithelial cell abnormality

Descriptive Diagnosis:Atypical squamous cells of unknown significanceLow grade squamous intraepithelial lesionHigh grade squamous intraepithelial lesion

Squamous Cell Carcinoma Glandular cell abnormality

Atypical glandular cellsAdenocarcinoma

Others

Pap smear specimens are considered satisfactory for interpretation if there are:

•Adequate numbers of well-visualized squamous cells present •Adequate numbers of well-visualized endocervical cells or squamous metaplastic cells (from the transformation zone). •Less than 50% of the cells obscured by blood or inflammation •Properly labeled specimens

Specimen SatisfactoryADEQUACY

Pap smear specimens are considered unsatisfactory for interpretation if there are:

•Inadequate numbers of well-visualized squamous cells present •Inadequate numbers of well-visualized endocervical cells or squamous metaplastic cells (from the transformation zone). •More than 75% of the cells obscured by blood or inflammation •Improperly labeled specimens

Usually, these smears are recommended for repeat sampling.

Specimen UnsatisfactoryADEQUACY

Specimen Rejected/Not Processed

ADEQUACY

Specimens to which the following conditions apply will be rejected:

1. Specimen is submitted without a requisition.

2. Specimen is not labeled with the patient name.

3. The patient name (or other identifying information) on the specimen and requisition do not correspond.

4. The specimen is labeled appropriately but the requisition is not labeled.

5. The specimen slide(s) is (are) irreparably broken.

6. Specimen is submitted from an unauthorized source.

Negative for IntraepithelialLesion or Malignancy

   * Atypical squamous cells      - of undetermined significance (ASC-US)      - cannot exclude HSIL (ASC-H)   

EPITHELIAL CELL ABNORMALITIESsquamous

Atypical squamous cell (ASC-US) Atypical squamous cell (ASC-US),favor mild dysplasia

   * Low grade squamous intraepithelial lesion (LSIL)    (encompassing: HPV / mild dysplasia / CIN 1)  

Mild Dysplasia Low Grade SquamousIntraepithelialLesion encompassing HPV cytopathic effects

EPITHELIAL CELL ABNORMALITIESsquamous

* High grade squamous intraepithelial lesion (HSIL)   (encompassing: moderate and severe dysplasia, CIS, CIN 2   and CIN 3)      - with features suspicious for invasion (if invasion        is suspected)   

EPITHELIAL CELL ABNORMALITIESsquamous

Severe Dysplasia or CIS Severe Dysplasia or CIS

* Squamous cell carcinoma

EPITHELIAL CELL ABNORMALITIESsquamous

Atypical Glandular Cells

Adenocarcinoma in situ of the cervix (upper left), next to normal glandular

epithelium (lower right).