CYTOLOGY

 INTRODUCTION

Definition 

Cytology is the study of individual cells and/or tissue fragments.

Uses of Cytology

•• Diagnosis

–– The diagnosis of benign, dysplastic and malignant lesions.

–– Intraoperative diagnosis

–– The diagnosis of inflammatory lesions and specific infections (e.g. tuberculosis)

•• Assessment of hormonal status in women

•• Cytogenetics, DNA cytometry and DNA ploidy analysis.

Steps Involved in Processing of Cytological Smears

Three steps are involved namely: 

(1) specimen collection, 

(2) fixation and 

(3) staining of smears.

Specimen Collection

The cytological sampling techniques to obtain the cells for study may be divided into five main groups.

••Exfoliative cytology: It is the study of cells which shed off spontaneously/exfoliated(shed) from the lining of an organ into a body cavity. The principle of exfoliative cytology is that, the cells normally exfoliate from any surface lining and this exfoliation increases in pathological conditions.

 Source: The different sources of exfoliated cells are:

–– Surface of mucosal or epithelial lining

◆◆ Female genital tract: Cervix, vagina

◆◆ Respiratory tract: Sputum

◆◆ Urinary tract: Urine

–– Body fluids

◆◆ Effusions: Pleural, peritoneal and pericardial

◆◆ Other fluids: Synovial fluid and CSF.

Uses: Exfoliative cytology is useful for the diagnosis of various diseases, identification of dysplasia and malignancy. The malignant cells become less cohesive and are shed easily.

•• Abrasive cytology: In abrasive cytology viable cells are artificially obtained by abrasive techniques from the surface of an organ. The instrument used to obtain the cells includes scraper or spatula (cervical, oral cavity-buccal smear for sex chromatin) and cytological brush (bronchial, gastrointestinal tract). The cells may also be obtained by washing or lavage (peritoneal, bronchoalveolar), where small amount of saline or similar solution is instilled into the target organ and aspirated. The cells obtained by abrasive technique do not show degenerative changes or necrosis, in contrast to that of exfoliative cytology.

•• Fine needle aspiration cytology: By this technique, cells are obtained from various organs or masses by needle aspiration (fine needle aspiration cytology—FNAC). FNAC is the most widely and commonly used procedure for the assessment of localized and readily palpable lesions of different organs or sites. It is less invasive, rapid, extremely reliable and useful procedure which can be performed on an outpatient basis. In FNAC, small amount of tissue/cells are aspirated with a small-bore needle (22G/23G) using syringe holders such as Cameco syringe pistol (Fig. 56.1) or Franzen handle.

Fig. 56.1: Cameco syringe pistol (syringe holder) with disposable 5 cc syringe and 22G needle

 Aspirated material is spread on the slide and smears are stained with Pap/Giemsa/H&E stain.

–– Common sites for FNAC

◆◆ Lymph nodes

◆◆ Breast

◆◆ Salivary glands

◆◆ Thyroid

◆◆ Soft tissue masses

◆◆ Intra-abdominal masses under ultrasound/CT guidance (kidney, liver, etc.).

Modern imaging techniques have extended its use to lesions in deep-seated structures like pelvic lymph nodes, pancreas and lungs.

•• Intraoperative cytology: Procedure by which the cells are obtained during operation, which includes:

–– Touch (imprint) cytology: Smears are prepared by forcefully pressing the clean glass slide to the cut surface of the organ.

–– Scrape cytology: Smears are prepared from material obtained by scraping the cut surface of the biopsy specimen with clean scalpel.

–– Crush preparation (crush cytology): Small fragments of fresh unfixed surgical tissue are crushed between two slides. This technique is mainly used in neuropathology.

Uses: It can be used for obtaining cells from any organ or tissue. It supplements or replaces the frozen section.

Advantage: Easier, cheaper and faster to prepare smears and useful for rapid diagnosis.

Disadvantage: Interpretation of smears requires training and experience.

•• Liquid-based cytology (LBC): It is a newer technology in which cells are immersed in a conserving liquid before being fixed on the slide. This avoids desiccation and reduces the quantity of obscuring material. One of the methods is Thin prep Pap test.

Advantages:

–– Additional slides can be prepared from the liquid

–– Molecular testing of infectious agents

–– DNA cytometry

–– DNA ploidy analysis.

Disadvantage: Higher cost.

Fixation of Cytological Smears

Different methods of fixation of cytological smears are:

•• Wet fixation: Cytological smears are fixed immediately (when smears are wet) in any of the following fixatives in a coplin jar.

–– 95% alcohol

–– A mixture of equal parts of 95% alcohol and ether.

•• Spray fixation: In this method, the cytological smears are immediately fixed with a spray fixative. This type of fixative usually contains alcohol (e.g. hair spray).

•• Air dried smears: Smears are air dried and fixed in 95% alcohol for 5 minutes (for May-Grunwald Giemsa stain).

Staining of Smears

Different stains that can be used for cytological specimen are: 

Papanicolaou (Pap) stain, May-Grunwald Giemsa (MGG), hematoxylin and eosin (H&E) and Leishman stain. 

Most commonly used stain is the Pap stain. Papanicolaou (Pap) smear and Pap stain: Dr George N Papanicolaou (an American of Greek descent) is one of the pioneers who observed certain changes in cells of vaginal smears. He incidentally observed cancer cells in vaginal smears of women and the cytological technique was applied for the first time to discover the occult cancer of cervix. Papanicolaou’s name was applied to cytologic procedure done for detection of the cervical cancer. Thus, the term Pap (cytologic) smear is usually applied to smears obtained from female genital tract. The Pap stain is also invented by Papanicolaou and bears his name.

Procedure for Pap Stain

•• Take the smears which are fixed in any of the above mentioned fixatives and hydrate them by processing through descending grades of alcohol (80, 70 and 50%) to distilled water.

•• Stain in Harris’ hematoxylin for 4 minutes.

•• Wash in running tap water for 1–2 minutes.

•• Dehydrate the smears up to 95% ethyl alcohol.

•• Stain in OG-6 (Orange-G) for 1–2 minutes.

•• Rinse the slide 10 times in 95% alcohol.

•• Stain in EA-36 (Eosin A) for 2–3 minutes.

•• Rinse the slide in 3 separate jars containing 95% alcohol.

•• Dehydrate, clear and mount the slide.

Uses

•• Staining of cervical smears (Pap smears) for diagnosis of carcinoma of the cervix (often at insitu stage).

•• Smears from other organs with suspected malignancy (e.g. endometrial carcinoma, bronchogenic carcinoma, tumors of bladder and prostate and gastric carcinomas).

•• For the identification of tumor cells in body fluids (abdominal, pleural, joint and cerebrospinal fluids).

•• Assessment of hormonal status and inflammatory conditions.

CYTOLOGY OF FEMALE GENITAL TRACT

In the female genital tract, the cells can be obtained by different techniques depending on the purpose (e.g. hormonal assay).

The different types of smears are:

•• Lateral vaginal wall smear

•• Cervical smear

•• Vaginal pool smear

•• Endocervical and endometrial smears: These are not done routinely.

Lateral Vaginal Wall Smear

The cells are obtained by scraping the upper third of lateral vaginal wall and smears are prepared. It is suitable for cytohormonal assessment and is preferable to the posterior fornix pool smears. 

The cytological features and appearance of cells in normal vaginal smear are shown in Table 56.1 and Figures 56.2A to C, respectively.

Uses of Vaginal Smear

•• For detecting the following inflammatory conditions which may reveal either the causative agent or morphological features associated with them.

–– Trichomonas vaginalis

–– Candida albicans

These causative agents can be detected in the smears

–– Human papillomavirus (HPV)

–– Herpes simplex virus

•• Diagnosis of cervical intraepithelial neoplasms and malignancies.

•• For hormonal assessment

–– Maturation index (MI): It represents a differential count of the different squamous cell populations. It is expressed as a percentage of parabasal, intermediate and superficial cells (e.g. 10/20/70).

–– Karyopyknotic index (KI): It is useful in assessing ovulatory/anovulatory cycles. It is the percentage of superficial squamous epithelial cells having pyknotic nuclei and expressed as percentage of total mature squamous cells.

–– Cornification index: The percentage of acidophilic squamous cells is termed as cornification index (CI) or eosinophilic index (EI).

Cervical Smear

The cells are obtained from cervical os by using an Ayre’s spatula (wooden or plastic) which is rotated through 360°, so as to obtain the cell sample from both ectocervix and squamo-columnar junction. The scraped material on the Ayre’s spatula (Fig. 56.3) is smeared on glass slides and smears are prepared. Smears are wet fixed, stained by Pap stain and are usually recommended for detection of dysplasia and carcinoma of cervix.

Bethesda (2001) system: 

Currently the cytological smears are reported according to the Bethesda (2001) system. According to this system, the cytological features of the precursor lesions of carcinoma cervix are divided into:

•• LSIL (low grade squamous intraepithelial lesion).

•• HSIL (high grade squamous intraepithelial lesion).

Squamous cell carcinoma cervix (Fig. 56.4): Characteristic cytological features of invasive squamous cell carcinoma are:

•• Cells present singly, in sheets and syncytial aggregates.

•• Marked variation in cell size and shape. Elongated and bizarre shaped malignant cells, tadpole/caudate cells and spindle squamous cells are often seen.

•• Cytoplasm is eosinophilic in tadpole shaped cells.

•• High nuclear cytoplasmic ratio.

•• Nuclei have irregular membranes with coarse chromatin.

•• Background shows tumor diathesis (necrosis and old degenerated blood).Vaginal pool smear: Cells are obtained by aspirating material from the posterior fornix of vagina and smears are prepared on the glass slide.

CYTOLOGY OF OTHER SYSTEMS

Cytology of Respiratory Tract

The source of cells from respiratory tract may be:

•• Sputum

•• Bronchoscopy

–– Bronchial aspirates and washing

–– Bronchial brushing (BB)

–– Bronchoalveolar lavage (BAL)

•• Fine needle aspiration including trans bronchial fine-needle aspiration.

•• Pleural fluid.

Sputum examination

•• Cytological examination of sputum is usually done for diagnosis of primary and metastatic malignancies of lung. At least three samples of sputum (preferably early morning samples) should be examined.

•• The solid particles/blood specked areas from sputum are smeared over slides and stained with Papanicolaou stain.

Cytology of Gastrointestinal Tract

Cells from lesions of the oral cavity can be obtained by scraping the surface with a metal or wooden spatula. Cell samples from esophagus, stomach, small and large intestines can be obtained either by brushing or lavage during fibre optic endoscopy.

Cytology of Urinary Tract

Cells from urinary tract lesions may be obtained from either urinary sediment prepared from voided urine/catheterized urine or bladder washings obtained at cystoscopy. The deposit is stained and examined. Specimen may also be obtained by aspirates and brushings. Cytological examination of body fluids and CSF are discussed in our other post.

BUCCAL SMEAR FOR BARR BODY

Buccal smears for Barr body (Fig. 56.3) are prepared with a thin wooden spatula, by scraping the buccal mucosa. Smears are stained by Papanicolaou stain. Barr body is seen as a solitary condensation on nuclear membrane in the intermediate squamous cells from buccal smears. In normal females, Barr bodies are present in 4–20% of nuclei of squamous cells, where as in males, their count is less than 2% of nuclei. In XXX female, 2 Barr bodies are found while in Turner syndrome (XO), the Barr body is absent. In males with Klinefelter syndrome (XXY) the Barr body is present. Neutrophils in the peripheral smear may also be examined for nuclear sexing.

Leukocytes: Nuclear Sexing (Figs 56.5A and B) 

Abnormalities of sex chromosomes can be diagnosed by nuclear sexing. In a normal female (XX), the neutrophils in a peripheral smear show a drumstick which is counter part of Barr body in buccal smear. Absence of drumstick is observed in Turner syndrome(XO), while one drumstick is found in males with Klinefelter syndrome (XXY).

FNAC APPEARANCE OF SOME COMMON LESIONS 

Granulomatous (Tuberculous)Lymphadenitis (Fig. 56.6)

•• Smears are cellular and composed of epithelioid cells in cohesive clusters forming granulomas.

•• Epithelioid cells have elongated (oblong/slipper shaped) nuclei and abundant pale cytoplasm.

•• Multinucleated Langhans type of giant cells may be seen.

•• The background shows lymphocytes and eosinophilic granular caseous necrotic material.

•• Ziehl-Neelsen stain for acid-fast bacilli may show positivity for the tubercle bacilli.

Pleomorphic Adenoma (Fig. 56.7)

•• Epithelial cells in clusters, sheets and singly scattered.

•• Regular ovoid nuclei with bland nuclear chromatin and well-defined cytoplasm.

•• Fibrillary chondromyxoid ground substance.

•• Spindle-shaped cells seen in stromal matrix.

Fibroadenoma Breast (Fig. 56.8)

•• Moderately cellular smears.

•• Large, branching monolayered sheets of uniform benign ductal epithelial cells having antler horn configuration.

•• Numerous single bare bipolar nuclei of benign type.

•• Fragments of loose fibromyxoid stroma.

Infiltrating Duct Carcinoma Breast(Fig. 56.9)

•• Highly cellular smear consisting of clusters of loosely cohesive malignant cells of variable size and shape.

•• High nuclear cytoplasmic ratio; nuclei are enlarged, hyperchromatic with irregular nuclear membranes.

•• Absence of single bare nuclei of benign type.

•• The background may show necrosis.

Points to note:

•• Cytology is the study of individual cells or tissue fragments.

•• Cytology is useful for diagnosis, assessment of hormonal status in women and cytogenetic analysis.

•• Study of cells which shed off spontaneously is known as exfoliative cytology.

•• FNAC is the most widely and commonly used procedure for the assessment of localized and readily palpable lesions of different organs or sites.

•• Liquid-based cytology is a newer technology in which cells are immersed in a conserving liquid before being fixed on the slide and has certain advantages over conventional cytology.

•• Papanicolaou’s name was applied to cytologic procedure done for detection of cervical cancer and the term Pap smear is usually applied to smears obtained from female genital tract and the stain also bears his name.

•• Cervical smears are obtained by using an Ayre’s spatula and are reported by using Bethesda (2001)system.