Diagnostic Cytology- FINALS L14

DIAGNOSTIC CYTOLOGY

• Microscopic examination of cells from different body sites for diagnostic purposes

• Includes exfoliative cytology & fine needle aspiration (FNA)

METHODS OF SMEAR PREPARATIONS

• Streaking

• Spreading

• Pull- apart

• Touch/ Impression/ Imprint/ Abraded

STREAKING

For preparation of mucoid secretions such as vaginal secretion aspirated from posterior fornix, sputum & gastric secretion

SPREADING

For preparation of thick mucoid secretion, for smears of fresh sputum & CSF

PULL- APART

Secretion such as serous fluids, concentrated sputum & enzymatic lavage sample from GIT, smears of urinary sediment, vaginal pool & breast

TOUCH/ IMPRESSION/ IMPRINT/ ABRADED

Smear-cut surface of tissue such as lymph node & other surgical or biopsy specimens; cells are directly taken form surfaces of excised/incised specimens by touching them with clean glass slide

EXFOLIATIVE CYTOLOGY

• Deals with microscopic study of cells that have been desquamated from epithelial surfaces

• Exfoliated cells either came from:

  • Spontaneously or physically removed from epithelial or mucous membrane

• Has been proven to be of great value in early detection of cancer

PURPOSES OF EXFOLIATIVE CYTOLOGY

• Detection of malignant cells in body fluids, mainly used for cervix

• Detection of precancerous cervical lesion women

• Assessment of female hormonal status in case of infertility & endocrine disorders

• For determination of genetic sex

• Diagnosis of infection

5 TECHNIQUES IN PROCESSING FLUIDS FOR CYTOLOGIC EVALUATION

1. Smear Technique

2. Cell Block Technique

3. Membrane Filter Method

4. Concentration Technique

5. Aspiration Cytology

SMEAR TECHNIQUE

Smear is the product of a diagnostic technique in which cells & other components are spread out thinly in a clean glass slide

AIR DRYING

Smears are allowed to be completely air dried & stained using Diff-Quick, Giemsa, or May Grunwald-Giemsa

CELL BLOCK TECHNIQUE

• Cell block is a paraffin-embedded specimen derived from different fluids & aspirated materials

• Considered as “microbiopsy”

• Mainly used with smears as an adjunct for establishing more definitive cytopathologic diagnosis

MEMBRANE FILTER METHOD

• Technique for collecting cells using a filter with a specialized pore size

• Filter is made up of polycarbonate & cellulose esters

• Also utilized in any procedure that requires body fluids in very small amounts (in sex chromatin determination on amniotic fluids)

CONCENTRATION TECHNIQUE

• Uses cytospin & sedimentation preparations

• Cells are isolated via a series of centrifugation steps to concentrate cells into small suspension

ASPIRATION CYTOLOGY

• Used to obtain specimens that don’t shed cells spontaneously

• Can be carried out in diagnosing any palpable lesions (FNAB / Cytology) & deep-seated / non palpable lesions (image guided biopsy)

COLLECTION & PREPARATION OF SPECIMEN

• Cervicovaginal smear

• Nipple discharge

• Gastric or bronchial secretions

• Pleural & peritoneal fluids

• Sputum

• Urine sediment

• CSF

SMEAR PREPARATION

• Smears should be made from fresh materials & smeared evenly on a clean slide

• Fixation

• For effusion fluids, it’s spin

• Supernatant, sediments & extra sediments

FIXATION

• Through fixative solution & spray

• If smear can’t be made immediately, collected materials should be placed in 50% alcohol or Saccomano fluid

COMMON FIXATIVES USED

• Equal parts of 95% ethyl alcohol & ether

• 95% Ethyl Alcohol

GYNAECOLOGIC SPECIMENS

• TRANSFORMATIONAL ZONE- most cancer cell arise

• An optimal sample should include squamous, columnar, & metaplastic cells

COLLECTION DEVICES

• Endocervical brush

• Plastic spatula

• Wooden spatula

• Plastic “broom-type” sampler

PAPANICOLAU METHOD

Staining method of choice for exfoliative cytology

ADVANTAGES OF PAPANICOLAU METHOD

• Transparent blue staining of cytoplasm is obtained due to action of high alcoholic content of cytoplasmic counterstain, allowing overlapping cells to be seen & identified

• Excellent nuclear detail is produced

• Color range is predictable & of great value in identification & classification of cells, producing good differential coloring of basophilic & acidophilic cells

CELLS FOUND IN CERVICO-VAGINAL SMEARS

• Mature superficial Cells

• Intermediate Cells

• Parabasal Cells

• Navicular Cells

• Pregnancy Cells

• Endometrial Cells

• Endocervical Glandular Cells

• Doderlein bacillus

MATURE SUPERFICIAL CELLS

• Polygonal squamous cells (45-50 µm)

• Have pale pink staining cytoplasm & dark pyknotic nuclei (<6 µm)

INTERMEDIATE CELLS

Medium-sized polyhedral or elongated cells (20-30 µm) with basophilic vacuolated cytoplasm

PARABASAL CELLS

• Round oval cells (15-30 µm) with dense basophilic cytoplasm

• Smaller than intermediate cells & have lager vesicular nucleus from 2 weeks of age of puberty, after childbirth, abortion & menopause

NAVICULAR CELLS

• Boat-shaped intermediate cells with strong tendency to fold or curl on edges

• Their presence suggests a combined estrogen progesterone effect

• Found in latter half of menstrual cycle, during pregnancy & menopause

PREGNANCY CELLS

• Round, oval or boat-shaped cells with translucent basophilic cytoplasm observed greatest at the center of cell, due to glycogen accumulation, pushing nucleus to the side or towards cell membrane

• This appearance is characteristics due to deeper blue stain of cytoplasm at the periphery

ENDOMETRIAL CELLS

• Small cells, slightly cylindrical with less basophilic cytoplasm, occurring in tightly packed groups of 3 or more

• Found during & 1-10 days after menopause

ENDOCERVICAL GLANDULAR CELLS

• Cytoplasm is usually stained pale blue/gray & is finely vacuolated, often with indistinct cell borders & nuclei with finely granular chromatin

• They may present honeycomb appearance when viewed on end

Doderlein bacillus

• Gram-positive, slender rod-shaped

• Most common organism of normal vaginal flora

PAP’S STAIN Doderlein bacillus

• Blue to lavender; numerous naked nuclei with many Doderlain bacilli

• Last trimester of pregnancy infection

• Estrogen deficiency

• Diabetes mellitus

QUANTITATION IN VAGINAL CYTOLOGY

1. Acidophilic Index

2. Pyknotic Index

3. Maturation Index

ACIDOPHILIC INDEX

Percentage of cell staining pink orange to red with Pap’s method

PYKNOTIC INDEX

• Ak.a karyo-pyknotic index

• Percentage of cell having shrunken, dark, small (<8 µm), structureless nuclei

MATURATION INDEX

• Consist of the proportion of cells from three layers of vaginal epithelium; superficial, intermediate & deep

• Provided pyknotic nuclei of <6 µm in diameter are taken as criteria for mature superficial cells

• Based on careful differential counts, is probably the most accurate index available

NON-GYNAECOLOGIC SPECIMENS

• Respiratory Tract Specimens

• Bronchoscopy Specimens

• Cell Suspensions*

• Gastrointestinal Specimens

• Peritoneal, Pleural, and Pericardial Fluids

• Breast Secretions

• Urinary Tract Specimens

• Body Cavity Specimens

RESPIRATORY TRACT SPECIMENS

Obtained to exclude possibility of malignancy or infectious agents, especially from patients with immunodeficiency syndrome

SPECIMENS OF RESPIRATORY TRACT SPECIMENS

• Sputum

• Bronchioalveolar lavage / bronchial washing (BAL/BW)

• Bronchial brushing (BB)

COLLECTION AND PREPARATION

• Obtain consecutive morning sputum specimens

• In a wide-mouthed jar containing Saccomano fluid

SPUTUM INDUCTION

• Aerosol 20 minutes

• For more extensive study, it requires slides, one is air-dried for Giemsa staining

• At least 2 slides should be stained with Pap’s method

• Sputum is examined first for blood (flecked or solid particles)

• With the end of wooden applicator stick, samples are evenly spread on slides & immediately placed in fixative for minimum of 1 hour

BRONCHOSCOPY SPECIMENS

• Bronchial Brushing

• Bronchial Washing

• Bronchial Aspirates

BRONCHIAL BRUSHING

Prepared by pull-apart technique & fixed

BRONCHIAL WASHING

Specimens are freshly collected in bronchoscopy collection container & hand delivered to laboratory

BRONCHIAL ASPIRATES

Collected by aspiration or by washing

CELL SUSPENSIONS

• From direct tap of pleural/peritoneal effusions, CSF, & synovial fluid

• Optimum amount is 20 mL

• Cells can remain viable for up to 2 days if specimen is kept at 4oC

PREPARATION OF CYTOSPIN SLIDES

• Specimen is centrifuged ASAP at 2000 rpm for 2 minutes & supernatant fluid is removed

• Smear is made of the sediment on glass slide which has been previously coated with thin layer of egg albumin, spreading smear with another slide as evenly as possible

• When it begins to dry around edges, but still moist in center, smear is fixed in 95% alcohol

• If smears can’t be prepared immediately, sediment should be covered with absolute alcohol & placed in refrigerator

GASTROINTESTINAL SPECIMENS

• Gastric smear preparations are quite difficult to make

• Specimen should be examined ASAP since a delay of 30 minutes will digest cell & make it unsatisfactory for evaluation

• Patient should have fasted for 6 hours before gastric washing is performed

SPECIMENS FOR GASTROINTESTINAL SPECIMENS

• Gastric lavage

• Gastric brush

• FNA

PERITONEAL, PLEURAL, & PERICARDIAL FLUIDS

• Presence of malignant cells in serous fluid usually indicate metastasis

• Clots can formed after collection, it can be prevented by heparin

BREAST SECRETIONS

• Nipple discharge has extremely low diagnostic yield for diagnosis of breast cancer

• When secretion is bloody a benign intraductal papilloma should be considered

• Except during lactation & immediate post lactation period, any discharge from nipple is abnormal

BREAST SECRETIONS COLLECTION TECHNIQUE

• Gently strip sub-areolar area & nipple using thumb & forefinger

• Place slide upon nipple & draw it quickly across nipple

• Immediately drop slide in a bottle of 95% isopropyl alcohol or use spray fixative

• Secretions obtained from both breast should be properly labelled as left or right

• For localized breast lesions producing discharge upon pressure, secretions may be smeared directly on slide after expressing material from breast

URINARY TRACT SPECIMENS

Major goal: urothelial malignancy

SPECIMENS FOR URINARY TRACT SPECIMENS

• Voided urine

• Catheterized specimen

• Washing from bladder or renal pelvis

URINARY TRACT SPECIMENS IMPORTANT NOTES

• To obtain a more reliable cytological evaluation, specimen may be collected & examined twice

• At least 2 mL is needed.

• In general urine cytology alone offers a very low diagnostic yield for detection of urothelial carcinomas

• Collection technique should be always mentioned on the requisition form

BODY CAVITY SPECIMENS- EFFUSIONS

• Positive effusion for malignancy is the first presentation of cancer of unknown origin

• Has a diagnostic value in patient with known history of cancer

BODY CAVITY SPECIMENS- EFFUSIONS SPECIMENS

• Cavity fluids include PF

• Ascitic/AF

• Peritoneal washing

• Pericardial & CSF

EFFUSION COLLECTION TECHNIQUE

• Place it in a clean, non-sterile dry container, & submitted fresh to the lab

• For delay place the specimen to isopropyl alcohol

• Avoid preservatives, except to heparin

• For CSF, minimum of 2 mL is necessary for cytologic evaluation

• Wright-Giemsa Staining (W-G); useful when diagnosis of lymphoma, also useful for CSF

• Air dried material is stained to enhance cytoplasmic details