CYTOLOGYCYTOLOGY

EXFOLIATIVEEXFOLIATIVE

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

recommended for : Detection of malignant cells or precancerous lesions in the body

Detection of asymptomatic or precancerous cervical lesions in women

Assessment of female hormonal status in case of sterility and endocrine disorders

Determination of genetic (phenotypic) sex

Detection of the presence of infectious microorganisms

CYTOLOGY SPECIMENS

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

9. Cervicovaginal (paps) smear

• Collection Procedure:

- Using standard paracentesis technique. 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. - Always Submit the specimen along with the completed

cytology request form.- The specimen should be refrigerated until transported to

the lab.

BODY FLUIDSBODY FLUIDS

A pleural smear

Pericardial fluid

CSF is usually obtained through a lumbar puncture (spinal tap).

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

• 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.

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

NIPPLE DISCHARGENIPPLE DISCHARGE

a. bronchial washing

b. bronchial lavage

c. bronchial brushing

- Bronchoscopically-directed brushing of 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 slide into 3% glacial acetic acid alcohol fixative. - The brush tip should also be cut off into the solution.

Bronchial Brushings

Bronchial Washings

•Specimen:

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

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)

Washings (Esophageal, Gastric, Other)

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

Procedure

Patient should fast overnight or for a minimum of six hours prior to the procedure.

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

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

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

Using the extended-tip spatula scrape material from the whole circumference of the cervix.

Withdraw the spatula and spread the collected material quickly and evenly onto the slide .

Fix Immediately .

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

STREAKING

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

SPREADING

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

PULL APART

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

                        

TOUCH IMPRESSIONTOUCH IMPRESSION

Impression cytology being Impression cytology being collected From a patient , using collected From a patient , using

a sterile glass slide with a sterile glass slide with polished edges.polished edges.

FIXATION

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

Altered nuclear-cytoplasmic ratios

Hyperchromasia

Increased mitotic activity

Atyical mitoses

CHANGES IN MALIGNANCYCHANGES IN MALIGNANCY

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

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.

CYTOPLASMIC CHANGES

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

- adenocarcinoma cells may enclose, endometrial and colonic cancers)

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

cutterage.

In 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 mesotheliomasIf there are no patterns 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.

VAGINAL CYTOLOGY

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.

-SUP{ERFICIAL CELLS

-Large (30-60u)

-Polyhedral flat cells

-Cytoplasm: may be acidophilic or basophilic

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

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

INTERMEDIATE CELLS

-

medium large (20-30u)

-

Polyhedral or elongated

-

Cytoplasm: basophilic with vacuoles

-

Vesicular nuclei (6-9u)

- 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

-PARABASAL 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.

-ENDOCERVICAL CELLS

-Slightly cylindrical appearance

-occurs in groups and strips of three or more cells

-cytoplasm: deeply basophilic the that of the parabasal cells

-ENDOMETRIAL 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

- “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

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.

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 Satisfactory

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.

Specimens subjected to rejection

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 NEGATIVE FOR INTRAEPITHELIAL INTRAEPITHELIAL LESIONLESION

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

Atypical squamous cell (ASC-US)

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

Low Grade SquamousIntraepithelialLesion

•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)   

Severe Dysplasia or CIS

* Squamous cell carcinoma

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

epithelium (lower right).

Endometrial Endometrial carcinomacarcinoma

REFFERENCESREFFERENCES

Pathology.jhu.eduPathology.jhu.edu www.pathologyoutlines.comwww.pathologyoutlines.com icytologywordpress.comicytologywordpress.com er.wikipedia.orger.wikipedia.org