Keywords

Specimen Collection Techniques [1]

  • Slides preparation:

  • The appropriate sample should be collected sufficiently for any cytology specimen to prepare slides (Table 1.1).

  • Each slide should be labeled with at least two patient identifiers [2].

  • A small amount of sample should be gently expelled onto a slide with the needle tip pointing down towards the slide near the frosted edge to give the most space for subsequent preparation [3].

  • “Two-Slide Pull” method: a new clean slide can be used to spread the sample across the first slide to create a thin, evenly distributed smear with constant gentle pressure, or place the specimen close to the midpoint of the slide and place another slide on the top of the specimen and gently pull both slides on the opposite direction. This will generate two slides containing the specimen. One slide can be used for rapid onsite evaluation (ROSE) by airdrying first and then staining with Diff-Quik stain. In contrast, the other slide should rapidly be fixed using 95% ethyl alcohol or cytology spray fixative.

  • For staining details, refer to the specimen preparation section.

  • All specimens received directly into the cytopathology lab should be tightly secured in their respective containers, labeled, and accompanied by an appropriately completed patient requisition form.

  • Fixation is in 95% alcohol (ethyl alcohol) for the majority of cases. Alcohol shrinks the cells making nuclear details clearer. 100% alcohol may be used in place of 95% ethyl alcohol.

Fine Needle Aspiration (FNA)

  • This procedure is used to evaluate either superficial or deep-seated lesions. FNA of superficial lesions is with or without image guidance, while FNA of deep-seated lesions is performed under image guidance.

  • Pathologists often perform FNA of superficial palpable lesions or fat-pad FNA.

  • Superficial palpable lesions are generally aspirated with a 22–25 G needle attached to a 10–20 cc syringe.

  • Some aspirated contents (2–3 drops) are placed onto a glass slide, and two direct smears are usually prepared using another clear slide. One slide is used for ROSE, while the other is fixed in alcohol for further evaluation.

  • Needles should be rinsed in an appropriate solution or formalin to prepare a cell block that can be used for ancillary testing.

Exfoliative Cytology

  • Gynecologic cytology: Liquid-based gynecological pathology specimens are collected using SurePath or Cytyc ThinPrep preparations for Pap tests.

  • Voided urine: Discard first early morning urine; patient voids into specimen container; collect urine starting at midstream. Specimens must be submitted immediately to the lab to avoid cellular degeneration. Specimens can be submitted fresh or in 70% alcohol fixative for possible ancillary testing (FISH analysis). Cytology preparations of samples are examined for infections or malignancy.

  • CSF: Aspirated CSF is evaluated for infection or malignancy.

  • Sputum: Specimen is evaluated for infection or neoplastic process; complete series: early morning specimens each day for 3 days (if suspected tuberculosis).

  • Brushings: Specimens are cellular, and mostly CytoLyt solution is used for preservation, but direct smears can also be prepared. The specimen is examined to evaluate for infection, nonneoplastic, or neoplastic processes.

  • Washings: Bronchial washing is mostly used to evaluate for infection or malignancy. Peritoneal washing is to assess for a neoplastic process.

  • Body cavity fluids: ThinPrep or cytospin preparations are commonly used. They are examined to determine the cause of fluid accumulation (nonneoplastic versus neoplastic).

    • Direct scrapings: Herpes virus detection: Tzanck test.

    • Touch imprint: Touch imprint cytology is mostly used either for ROSE or during intraoperative frozen sections. The biopsy material is touched or scraped on the glass slide and is immediately fixed in alcohol with subsequent staining (Papanicolaou or rapid H&E) or air-dried and then stained with Diff-Quik stain. Squash preparations are also used (mostly in brain-frozen sections).

Table 1.1 Specimen collection techniques
Full size table

Specimen Preparation

  • Cytology specimen received in the lab is processed based on the specimen site and volume. Direct smears from an FNA procedure usually have an air-dried preparation (Diff-Quik stained) and an alcohol-fixed preparation (Papanicolaou stain).

  • Specimens may be concentrated by centrifugation to obtain a cell pellet.

  • Papanicolaou (Pap) stain.

    • This staining technique is helpful for optimal visualization of cancer cells exfoliated from epithelial surfaces of the body.

    • The polychrome staining reaction is helpful in highlighting cytologic features, chromatin and cytoplasmic details.

    • Procedure.

      • Fixation: Coplin jar with 95% ethyl alcohol (5–15 min).

      • Hydration: running water.

      • Nuclear staining: Harris hematoxylin (1–5 min).

      • Rinse: running water.

      • Scott’s tap water/distilled water.

      • Rinse: running water.

      • Rinse: 95% ethyl alcohol, two changes (10 dips).

      • Cytoplasmic staining: OG-6 for 30 s to a min.

      • Rinse solution: 95% ethyl alcohol, three changes (10 dips).

      • Cytoplasmic staining: EA-50 for 5 min.

      • Rinse: 95% ethyl alcohol, three changes (10 dips).

      • Final dehydration: 100% ethyl alcohol (10 dips).

      • Clearing: xylene, three changes.

      • Coverslip.

  • Diff-Quik stain.

    • Use for ROSE, evaluate the cytoplasmic details, infectious organisms, and hematological elements.

    • Procedure.

      • Fixative solution (methanol) for 30 s to 1 min.

      • Solution I (eosinophilic) 10 dips.

      • Solution II (basophilic) 10 dips.

      • Rinse slides in running water.

      • Coverslip.

  • Rapid H&E stain.

    • The method is used mostly for intraoperative frozen sections and ROSE.

    • Nuclei stain blue; overstaining with hematoxylin results in dark nuclear staining and vice versa.

      • Cytoplasm/other tissue elements, including RBCs, stain pink; overstaining with eosin results in dark cytoplasmic staining.

  • Cell block.

    • A cell block may be prepared from any fluid, washing, brushing, or FNA, with the added benefit that this procedure obtains any tissue which may be present in the specimen. Cell block material can be used for ancillary testing if required.

    • Procedure.

      • Centrifuge the specimen to obtain a pellet which includes the following procedure: Add 10 mL of absolute alcohol and centrifuge it, and gradually decant the supernatant. Add 15–20 mL formalin, again centrifuge it and pour supernatant. Add HistoGel drops to the pellet and then refrigerate it for a few minutes. Put the pellet in the cassette and submit it for processing.

Special Stains

Workup

Stain

Uses

Positive interpretation

Infectious organisms

Ziehl-Neelsen

Acid fast organisms

Red rod-shaped organisms on a blue background

Modified Ziehl-Neelsen

Acid fast organisms

Kinyoun

Acid fast organisms

Red organisms on a blue-green background

Fite

Mycobacteria leprae

Red organisms on a blue background

Auramine-rhodamine

Acid fast organisms

Yellow-red fluorescence

Grocott’s methenamine silver (GMS)

Fungi especially PJP

Black organisms on pale green background

Periodic acid Schiff (PAS)

Fungi; polysaccharides; neutral mucosubstances, basement membrane

Magenta-red organisms on blue-green background

Brown-Hopps

Bacteria

Gram+: Blue

Gram–: Red

Brown-Brenn

Bacteria

Gram+: Purple-blue

Gram–: Pink-red

Warthin-starry

Spirochetes, helicobacter pylori

Dark brown-black organisms on pale yellow background

Other

Alcian blue

Acid mucopolysaccharides; for Cryptococcus spp.

Acid mucins: Blue

Cresyl violet

Neurons (Nissl substance)

Granular purple-blue neuropil

Fontana Masson

Melanin, argentaffin cell granules (of carcinoid tumor)

Melanin: Brown-black; on pale pink background

PAS with diastase digestion

To differentiate mucin from glycogen, fungal infections, Whipple’s disease

Light pink (replaces deep magenta of PAS only)

Masson’s trichrome

Connective tissue/vasculature, collagen, fibrosis

Collagen: Blue; nuclei: Brown/black; muscle: Red; cytoplasm: Pink

Von Kossa

Calcium deposits

Gray-black calcium deposits on light pink background

Reticulin

Reticulin fibers (tumor architecture)

Black fibers on gray-pink background

Luxol fast blue

Myelin in nerves

Myelin: Blue-green

Neuropil: Pink

Verhoeff’s elastic stain

Elastic fibers

Elastic fibers: Black

Collagen: Red

Bielschowsky silver stain

Nerve fibers (neurofibrillary tangles in Alzheimer’s disease)

Black nerve fibers

Oil red O

Lipid, lipid laden macrophages

Red

Prussian blue

Iron (hemosiderin)

Bright blue on pink background

Congo red

Amyloid

Salmon-pink and green birefringence under polarized light

Thioflavin-T

Amyloid

Bright yellow-green fluorescence on black background

Mayer Mucicarmine

Acid mucopolysaccharides; epithelial mucin; for Cryptococcus spp.

Red on yellow background

Bile

Bilirubin

Emerald green

Rhodanine

Copper

Bright red

Myeloperoxidase (MPO)

Myeloid lineage, MPO deficiency

Blue-green or brown, if present

Sudan black B

Neutral lipids

Black granular

Leukocyte alkaline phosphatase

Alkaline phosphatase activity

Brown-black granules

Toluidine blue

Mast cells

Dark rose-violet on a blue background

Movat pentachrome

Collagen, elastic fibers, muscle, and mucin

Elastic fibers-black

Collagen-yellow

Mucin- blue

Muscle-red

Immunohistochemistry

  • An important development in cytopathology is the application of immunohistochemical staining (IHC) techniques on direct smears and ThinPrep slides, along with the traditional use on cell block material.

  • Specimens with limited material or with no cell block can further be evaluated by performing stain on additional ThinPrep slide (such as PTH stain for parathyroid, calcitonin stain for medullary thyroid carcinoma).

  • On cytology preparation (direct smear or ThinPrep), interpretation of IHCs with nuclear staining pattern is more straightforward.

Limitation

  • Polyclonal antibodies are more sensitive and bind with more epitopes. Titration should be done cautiously to avoid overstaining (false positives).

  • Monoclonal antibodies are more homogeneous and specific against a single epitope.

  • Cytoplasmic or membranous staining interpretation on ThinPrep or direct smear slides is often challenging.

  • Fixative-related issues: Alcohol fixative versus formalin fixative.