Cytology

Cytology  Study of the microscopic appearance of cells for diagnostic purposes  Can be used as a screening tool for healthy individuals at risk of a particular disease  Exfoliative vs FNA cytology o Exfoliative – cells desquamated from epithelial surfaces  Spontaneous shedding  Physically removed from epithelial and mucous membranes o FNA – fine needle aspiration of palpable and non-palpable masses  Applications of Exfoliative Cytology o Detection of malignant cells in body fluids (for staging cancers) o Detection of precancerous cervical lesions (cervicovaginal or Pap smear) o Assessment of female hormonal status in case of sterility and endocrine disorders   o For determination of genetic sex o Detection of infectious agents  Gynecological vs Non-gynecological cytology o Gyn cytology  specimens from the female genital tract o Non-gyn cytology  Specimens from all other body sites  Cytology: Fixation  Specimen should be fixed immediately for optimal cell preservation o 95% ethanol o Equal parts of 95% ethanol and ether  If smears from effusions cannot be made immediately, place in: o 50% alcohol o Saccomano preservative  If fluid specimen is enough for cytocentrifugation: o Centrifuge at 2000 rpm for 2 mins, decant supernatant and smear the sediment o Prepare at least 2 cytocentrifuged smears and a cell block (similar to tissue processing) Gynecological Cytology  Transformation zone/T-zone o Endocervical-ectocervical junction o Where majority of cervical CA and precancerous lesions of the cervix arise  Cytologic collection and preparation o For conventional pap smears o Endocervical brush

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o Vaginal scrape o Lateral vaginal scrape o Four quadrant vaginal scrape o Vulvar scrape Specimen collection o A bivalve speculum of appropriate size is gently inserted into the patient’s vagina o A cervical brush/broom/spatula is inserted in the endocervical canal o For conventional smears  The brush sample should be rolled over the slide followed by immediate fixation o For liquid-based preparations  Cells are rinsed into a liquid collection media containing fixatives  Ensures the capture of an entire sample from the collection devices o Liquid-based preparations  alternative to conventional cervicovaginal smears  Improved preparation that minimizes cell overlap for better identification of abnormal cells  ThinPrep technique:  SurePath technique: Gynecological Cytology: Staining o Papanicolau method o Nuclear stain  Hematoxylin o Cytoplasmic stains – 2 counterstains  Orange G  Eosin Azure  Components: o Staining technique 1. Fix in 95% EtOH 2. Primary stain with hematoxylin 3. Differentiate with acid alcohol then wash with water 4. Blue in ammonia water then wash with water 5. Counterstain with OG-6 6. Wash with 2 changes of 95% EtOH 7. Counterstain with EA-50 8. Dehydrate (ascending grades of alcohol) 9. Clear with xylene 10. Mount with resinous media Bethesda System Categories for Specimen Adequacy o Satisfactory for evaluation  A satisfactory squamous component must be present.

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Note the presence/absence of endocervical/transformation zone component.  Obscuring elements (inflammation, blood, drying artifact, other) may be mentioned if 50% to 75% of epithelial cells are obscured. o Unsatisfactory for evaluation  Specimen rejected/not processed because [specify reason]. Reasons may include:  Lack of patient identification  Unacceptable specimen (e.g., slide broken beyond repair)  Specimen processed and examined, but unsatisfactory for evaluation of an epithelial abnormality because [specify reason]. Reasons may include:  Insufficient squamous component  Obscuring elements covering more than 75% of epithelial cells Cytohormonal Maturation Index (CHMI) o Assesses ovarian hormonal function  Estrogen and Progesterone  Shows the predominant hormone of the woman at the time of collection of the smears o Correlated with the age of the patient and her last menstrual period o Numerical expression representing the relative proportion of the 3 vaginal cell types:  Parabasals, Intermediates, Superficials  CHMI = P / I / S o Parabasal cells  Round to oval cells with small dense basophilic cytoplasm  Absence of both estrogen and progesterone o Intermediate cells  Polyhedral or elongated cells with basophilic cytoplasm  Influenced by increased progesterone o Superficial cells  Polygonal squamous cells with pale, pink-staining cytoplasm and dark pyknotic nuclei  Influenced by increased estrogen CHMI Results o Premenarche: o Childbearing age  Ovulation:  Menstruation:  Pregnancy:  Postpartum:

 Perimenopausal: After childbearing age  Postmenopausal:  Teleatrophy: Gynecological Cytology: Normal Cell Components o Neutrophils o RBCs o Lactobacillus acidophilus (Doderlein bacilli)  Gram positive bacillus that is a part of the vaginal normal flora o Leptothrix spp  Long, thin, filamentous, hair-like bacilli that are normal commensals and become prolific if vaginal pH increases o Endocervical cells  Columnar epithelial cells that are part of the normal lining of the endocervical canal o Endometrial cells  Small epithelial cells from the shedding of the endometrial lining or due to a proliferating endometrial pathology Gynecological Cytology: Abnormal Cell Components o Candida albicans  A budding yeast that form a branching pseudohyphae that spears clusters of epithelial cells o Trichomonas vaginalis  Sexually-transmitted pear-shaped organism o Gardnerella vaginalis  Tiny pleomorphic coccobacilli that clings to the surface of the cytoplasm of epithelial cells (clue cells) o Koilocytes  Squamous epithelial cells that show HPV cytopathic effects o HSV-II  Herpes simplex virus o High grade squamous intraepithelial lesions (HSIL)  Encompassing severe dysplasia and carcinoma in-situ o Invasive squamous cell carcinoma  Most common form of cervical malignancy Bethesda System for Reporting Cervical Cytology o Negative for intraepithelial lesion or malignancy (NILM)  Organisms  Trichomonas vaginalis  Fungal organisms morphologically consistent with Candida species  Shift in flora suggestive of bacterial vaginosis  Bacteria morphologically consistent with Actinomyces o

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 Cellular changes consistent with herpes simplex virus Other non-neoplastic findings  Reactive cellular changes associated with: inflammation (includes typical repair); radiation; intrauterine contraceptive device (IUD)  Glandular cells status post hysterectomy  Atrophy Epithelial cell abnormalities  Squamous cell  Atypical squamous cells (ASC) o Of undetermined significance (ASC-US) o Cannot exclude HSIL (ASC-H)  Low-grade squamous intraepithelial lesion (LSIL)  High-grade squamous intraepithelial lesion (HSIL)  Squamous cell carcinoma (SQC)  Glandular cell  Atypical glandular cells (AGC) (specify if endocervical, endometrial, or not otherwise specified)  AGC, favor neoplastic (specify if endocervical or not otherwise specified)  Endocervical adenocarcinoma in situ (AIS)  Adenocarcinoma (specify if endocervical, endometrial, extrauterine, or not otherwise specified) Other: Includes sarcoma, malignant lymphoma, others

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Non-gynecological Cytology  Respiratory Tract Specimens o Principle: obtained to exclude the possibility of malignancy or infectious agents o Specimen:  Sputum  Fixative: Saccomanno fluid  bronchoalveolar lavage (BAL)  bronchial washing (BW)  bronchial brushing (BB)  Gastrointestinal Specimens o Principle: Done to exclude the possibility of malignant tumors o Specimen: o delay of more than 1/2 hour before fixation  digestion of cells  specimen unsatisfactory for evaluation  Smears of Breast Secretion o Low diagnostic yield for diagnosis of breast carcinoma o Specimen: Nipple discharge

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Causes:  Benign breast lesion such as duct ectasia and papilloma  Endocrine problems  Detection of malignant cells Urinary Tract Specimen o Principle: Diagnosis of malignancy, usually of urothelial origin o Specimen  Voided urine (second morning)  Catheterized specimen  Washings from bladder or renal pelvis Body Cavity Effusions o Accumulation of fluids within the body cavities usually indicate a pathologic process o Principle: Presence of malignant cells  metastasis o Specimen: Cytopreparation o Cell suspensions  For body cavity effusions, CSF, urine, watery lavages  Cells are viable up to 4 days at ref temp  Standard technique: cytocentrifugation then slides are stained with Pap stain

Fine Needle Aspiration Cytology  FNA of superficial masses and deeply seated lesions  Specimen collection for non-palpable masses o Aspirated under fluoroscopy, computed tomography, ultrasound or other radiologic techniques  Specimen collection for palpable masses o breast, thyroid soft tissue and lymph nodes 1. Palpate the target lesion, sterilize the overlying skin 2. Fix the lesion with one hand between fingers 3. Introduce the needle (22-23 gauge) and then aspirate  Slide preparation o First few drops from the tip of the needle has the most diagnostic material o Prepare about 4 slides using wedge technique or pull-apart technique o Rinse needle in a preservative (Saccomano) and send to the lab  for cytocentrifugation and/or cell block  Slide fixation o 95% alcohol or spray fixative  Stain o Same with Papanicolau method