Introduction to Cytology (MLSC 410)

Introduction to Cytology (MLSC 410)

• Cytology is the study and interpretation of cells from the human body that exfoliate or are removed from tissues.
• Cytology specimens can be categorized as gynaecological or non-gynaecological based on the collection site, referring to female reproductive organs/genitalia or other body parts, respectively.
• Exfoliative cytology examines spontaneously shed cells from organ linings, using nonabrasive removal methods.

Diagnostic Cytology

• Exfoliative cytology relies on the spontaneous shedding of cells from organ linings into body cavities, where they are removed non-abrasively.
• Cell age within samples can vary, ranging from recently shed to weeks old.
• A common example is the vaginal smear, derived from cells in the posterior fornix of the vagina.
• Cells in the vaginal fornix come from the squamous epithelium of the vagina/uterine cervix, the endocervical canal's lining and other sources like the endometrium/peritoneum.
• Mammalian cells consist of three essential parts: the cell membrane, cytoplasm, and nucleus.

Subdivisions of Cytology

• Gynaecological cytology (Gynea-cytology) focuses on cells from the vagina, cervix, and endometrium.
• Non-gynaecological cytology studies cells suspended in body fluids.
• Fine Needle Aspiration Cytology (FNAC) involves analyzing cells extracted from organs and bodily structures.

Gynaecological Cytology Applications

• Gynaecological cytology can detect malignancy like cervical cancer.
• Gynaecological cytology can assess hormonal activity.
• Gynaecological cytology can diagnose vaginal infections and inflammatory conditions.

Importance of Cytology

• Cytology can assess chemotherapy, radiotherapy, and surgeries.
• Cytology can study hormone-influenced cell maturation.
• Cytology helps in detecting cellular changes due to infections.
• Cytology can detect malignancy, especially cervical cancer.
• Cytology can diagnose genetic conditions via sex chromatin markers, such as Barr bodies and drumstick appendages.
• Cytology can diagnose nutritional deficiencies via buccal smears for Vitamin A deficiency.

Sample Collection: Broad Types

• Exfoliative Cytology involves cervical smears and liquid-based cytology.
• Respiratory samples include sputum, bronchial wash, bronchial brush, broncho-alveolar lavage and Fine Needle Aspiration Cytology (FNAC).
• Additionally, gastrointestinal tract samples like gastric brush, lavage, and trans-endoscopic FNAC are collected.
• Other samples include urine (voided, catheterized, ureteric), effusion fluid (pleural, peritoneal, pericardial), cerebrospinal fluid (CSF), vitreous fluid, and joint fluid (in anticoagulant)..

Fine Needle Aspiration Cytology (FNAC)

• FNAC is a diagnostic technique to examine lumps using a thin (23–25 gauge) needle to extract cells.
• Biopsy cells are stained and examined microscopically, sometimes guided by ultrasound in breast cases

FNAC vs FNAB

• Fine-needle aspiration biopsy (FNAB) and fine-needle aspiration cytology (FNAC) both involve aspiration biopsies, with FNAC emphasizing cytopathology over histopathology.
• Aspiration is safer than open biopsy, with rare complications, though it risks inconclusive or false negative results due to limited cells.

Cervical Cytology

• Cervical cytology's accuracy directly impacts cervical cancer screening effectiveness, thus proper sample collection is crucial.
• Patients planning for procedure should schedule it two weeks after their last menstrual period and abstain from intercourse for two days prior.
• Further, patients should avoid vaginal creams, jellies or tampons.

Sample Collection: Device Properties

• The ideal collection equipment should retrieve materials from both ectocervical and endocervical zones.
• Additional device qualities include being non-traumatic, non-sticky, cheap and disposable.

Collection Devices Available:

• Wooden spatula: Inexpensive and easy to use, however, cells may adhere to wood fibers.
• Plastic spatula: More expensive than wooden, and have non-stick properties.
• Endocervical brush: Easy to collect material and is more traumatic due to the stiff bristle.
• Cervix brush: used mainly in liquid-based cytology (LBC) sample collection, can source material from the ecto and endocervical material, however, is costlier than any other device.

Sample Collection: Procedure

• Position the patient in dorsolithotomy position.
• Clean vagina with wet swab and introduce a speculum to visualize cervix.
• Inspect both the ectocervix and transformation zone while avoiding jellies.
• Ectocervix is smooth/pink, and endocervix is dark pink.
• Collect samples from the cervical os.

Sample Collection: Conventional Smear

• For the conventional smear, inserts the spatula within the vagina to fit the contour of the cervix.
• Rotate the spatula 360 degrees and withdraw gently.
• Spread the sample then fix the slide in 95% ethyl alcohol for at least 30 minutes.
• Use a cervix brush for LBC preparation.

Sample Collection: Broom Technique

• Introduce the broom’s central part into the endocervical canal, ensuring shorter bristles touch the ectocervix.
• Rotate four to five times clockwise
• Withdraw and place in fixative; label liquid-based cytology vials or glass slides.

Other Smear Types

• For Vagina smears, gather cytology samples from the upper third of the lateral vaginal wall using a spatula.
• Endometrial aspiration smears: A sterile cannula and syringe is used to aspirate the endometrial sample.

Respiratory Sample: Sputum

• Collect the morning sputum in a wide-mouthed container without fixative.

Respiratory Sample: Bronchial Brush

• Visualize the lesion through the bronchoscope.
• Immediately make the smear on the slide.
• Prepare smears that are either alcohol fixed/air-dried.

Respiratory Sample: Bronchial Wash

• Bronchial washes visualized through the bronchoscope
• Wash involves insertion of 5 ml normal saline and aspiration of the solution.
• Send the fluid to the laboratory immediately.

Respiratory Sample: Bronchoalveolar Lavage(BAL)

• Under local anesthesia, introduce a fiberoptic bronchoscope in a selected subsegmental bronchus.
• Next, flush 20 ml normal saline through bronchoscope and recollect the lavage solution gently.
• Repeat the process four to five times to yield 40-50 ml solution.
• Send the BAL fluids to laboratory immediately.

Respiratory Sample: Endoscopic Ultrasound Guided(EUS) FNAC

• The lesion is visualized by endoscopic ultrasound
• The FNAC needle is introduced through the channel of the fiberoptic endoscope.
• The needle is moved to and fro in the lesion maintaining negative suction.
• Withdraw the collected aspirated material, and spread on the glass slide.

Urine Sample Collection

• Urine samples can be voided, catheterized or ureteric.
• Voided urine is preferable for routine cytology; collect the second voided urine in a clean container without fixative and send immediately for processing.

Processing of Laboratory Samples Steps:

• Receiving
• Preparing smear
• Staining
• Mounting/final submission of the slide.

Sample Requisition

• When processing samples, include: patient ID, collection date/site/method, and clinician details.
• Additional details: Include tests needed, patient history, complaints, physical and Radiological findings, and any relevant history such as surgery, chemotherapy, chemical exposures, etc..

Sample Identification

• Confirm the sample’s unique identification number matches the requisition form during receiving and processing.
• Apply a unique lab bar code number to each sample, distinct from the unique ID, and affix it to the container, smears, and forms..

Common Processing Techniques

• Direct smear
• Centrifugation
• Cytocentrifuge
• Liquid-based preparation
• Millipore technique
• Cell block

Sputum Processing

• Pour sputum in a black Petri dish and look for tissue fragments/grey-white substance/bloody material for smear prep.
• Pick the fragments using forceps.

Body Fluid and Lavage Processing

• Centrifuge 50–100 ml of sample, such as effluent or turbid urine.
• Pour the fluid into clean centrifuged tube, and rotate the tube at 1500 RPM for 10 mins.
• Discard the supernatant liquid, and make multiple smears from the sediments.

Centrifuge Principle

• Circular movement creates centrifugal force, driving particles from the center balanced by buoyant and frictional forces.

Cytological Staining Techniques

• The commonly used stains in cytology are: Papanicolaou's stain and May Grunwald Giemsa stain.

Papanicolaou's Stain

• Dr. George Papanicolaou introduced it in 1942 and is used in cervical and non-gynaecologic smears.
• The components are: Haematoxylin, Orange G, and Eosin Azure (EA)

Papanicolaou's Stain: Components

• Haematoxylin stains nuclei.
• Orange G (OG-6): it is a cytoplasmic counterstain which also stains keratin.
• Eosin Azure (EA) is a polychrome stain with Eosin Y, light green SF yellowish, and Bismarck brown Y.

Papanicolaou's Stain: Properties

• Cytoplasmic differentiation: helps differentiate cells in transparent stains.
• Nuclear details are seen.
• Demonstrates intracytoplasmic keratin.

Principle of Papanicolaou Stain

• Rehydrate smear with graded alcohol.
• Stain nuclei with Harris haematoxylin.
• Remove excess haematoxylin with acid alcohol.
• Treat smear with weak alkaline solution.
• Cytoplasmic staining is done when an alcohol-soluble dye like Orange G (OG) is applied.
• The cell cytoplasm is stained blue-green with EA.
• The preparation is dehydrated with absolute alcohol, cleared with xylene and mounted using DPX.

Papanicolaou's Staining Steps

• 70% ethanol, 1 min
• 50% ethanol, 1 min
• Distilled water, 5 dips
• Harris haematoxylin, 3-5 minutes
• Distilled water, 5 dips
• 0.25% aqueous solution of hydrochloric acid, few dips
• Water, 1 min
• Lithium carbonate, 1-2 minutes
• Water, few dips
• 70% ethanol, 2 min
• 90% ethanol, 2 min
• Orange G, few dips
• 95% ethanol, 2 min
• EA modified, 2 min
• Absolute ethyl alcohol, 2 min
• Absolute ethyl alcohol, 2 min
• Absolute ethyl alcohol and, 2 min
• Xylene, 5 min
• Xylene and mounting in DPX until clear

Papanicolaou's Staining Results

• Nuclei: Dark blue
• Superficial (cornified) cells: Pink
• Intermediate (non-cornified) cells: Green
• Candida (monilia): Red
• Trichomonads: Grey-green
• Cytoplasm of parabasal cells: Deep green
• Red blood cells: Orange

Papanicolaou Staining: Precautions

• Cover the container so the dye will stay concentrated.
• The containers should be washed regularly.
• To increase staining quality, add fresh dye to the Haematoxylin solution.
• Replace OG-6 when the cytoplasmic color appears less crispy due to instability.
• Exchange tainted xylene immediately and routinely monitor the alcohol.
• Ensure that the stain solution is free from deposits and is filtered daily.

May Grunwald-Giemsa Staining

• May Grunwald-Giemsa staining method is used for inspection and differential counting blood smear.
• MGG contains alkaline methylene blue, azures and eosin that produce azures in the result.
• Giemsa staining technique examines blood for malaria parasites and morphologically differentiate the nuclear and cytoplasm of Erythrocytes, leucocytes, Platelets and parasites
• The components for Stock solution are: MayGrunwald dye 0.3gm and 100 ml Methanol.
• The components for Working solution are: 20 par Stock solution and 30 part Phosphate Buffer (pH 6.8).

May Grunwald Stain: Procedures

• Prepare a thin smear before air drying and fixing it with methanol for 5-10 minutes.
• Then stain the smear in May Grunwald working solution for 10 minutes.
• Rinse in pH 6.8 buffer, and stain the slides with diluted Giemsa stain for 30 minutes.
• Wash the smears then let them dry with distilled water; mount the slide with DPX to examine under the microscope.

May Grunwald Stain: Results

• Erythrocytes: Light pink to light purple.
• Platelets: Granules – Reddish purple.
• Lymphocytes/monocytes: Nuclei – Dark purple, Cytoplasm – Sky blue.
• Neutrophils: Nuclei – Dark blue, Granules – Reddish purple, Cytoplasm – Pale pink.
• Eosinophils: Nuclei – Blue, Granules – Red/orange red, Cytoplasm – Blue.
• Basophils: Nuclei – Dark blue, Granules – Purple.

Examination of Hormonal Status

• Karyopyknotic index
• Maturation index
• Eosinophilic index

Karyopyknotic Index (KI)

• The karyopyknotic index (KI) expresses the ratio of superficial squamous cells with small(pyknotic) nuclei to all mature squamous cells.
• Usually, 200 to 400 cells are evaluated in three/four different fields on the smear.
• The peak of KI usually coincides with the time of ovulation and was estimated at 50% to 85% of total cells.
• KPI is calculated by counting at least 200 squamous cells and expressing the percentage with condensed/pyknotic nuclei and pink or red cytoplasm.

Maturation Index (MI)

• The maturation index (MI) is an extension of KPI and needs a differential count of 200 squamous cells.
• Cell maturation, classified as superficial, intermediate, or parabasal, is determined by staining/morphology reactions and expressed as a percentage.
• High or low estrogenic activity is indicated by the preponderance of superficial/parabasal cells..
• The count should be performed on single cells.
• It shows the percentile association of parabasal, intermediate, and superficial cells that represent the maturation of the squamous epithelium.
• For example, an MI of 0:35:65 indicates there are 0% parabasal, 35% intermediate, and 65% superficial.

Other Indices

• The folded-cell index shows correlation of mature superficial/intermediate squamous cells with folded cytoplasm to all mature squamous cells.
• The crowded-cell index is a relationship of cells in clusters of four or more cells to all mature squamous cells.

Eosinophilic and Basophilic Index

• In a normal menstruating woman, the El's peak coincides with peak KI's and may reach 50% to 75% at the time of ovulation.
• The basophilic index is the percentile relationship of immature squamous cells with basophilic cytoplasm to all immature squamous cells, despite nucleus status.