10.3 Cytologic Preparations, Cell Blocks & the Papanicolaou Stain
Key Takeaways
- The Papanicolaou stain is a polychrome method using Harris hematoxylin for nuclei, OG-6 for keratin, and EA-50 containing eosin Y, light green SF, and Bismarck brown for cytoplasm.
- Papanicolaou preparations must be wet-fixed immediately in 95 percent ethanol or coated with a polyethylene glycol spray fixative; air drying destroys the nuclear detail the method exists to show.
- Romanowsky stains require the opposite handling: the smear must be fully air-dried before the methanol in the stain fixes it.
- Cell blocks convert residual fluid or aspirate material into a paraffin block using plasma-thrombin, agar, or HistoGel, which allows immunohistochemistry and molecular testing on cytology specimens.
- Cornflake artifact is refractile brown air trapped over squamous cytoplasm under the coverslip; it is corrected by soaking the coverslip off in xylene and remounting from fresh xylene.
10.3 Cytologic Preparations, Cell Blocks & the Papanicolaou Stain
ASCP HT Exam Focus: The ASCP BOC content outline lists cytologic specimens under both Fixation and Processing, and cytological stains (e.g., Papanicolaou) under Staining. The Summary of Stains names Papanicolaou directly. The histotechnician is expected to prepare, fix, stain, and coverslip these specimens even when a cytotechnologist screens them.
Cytology specimens arrive without a block, without a cassette, and often without a fixative. Everything that determines diagnostic quality happens in the first 30 seconds.
Specimen Types
Gynecologic
- Conventional smear: cervical sample spread directly on a glass slide and fixed immediately.
- Liquid-based cytology: the collection device is rinsed into a preservative vial and a monolayer is deposited by filter transfer or sedimentation. ThinPrep uses a methanol-based preservative; SurePath uses an ethanol-based preservative with a density-gradient step.
Non-gynecologic
- Fine-needle aspiration (FNA) of thyroid, breast, lymph node, salivary gland, pancreas.
- Fluids: pleural, peritoneal, pericardial effusions, cerebrospinal fluid, urine, bronchoalveolar lavage.
- Brushings and washings from bronchus, biliary tree, or esophagus.
- Touch imprints (roll preparations) taken from a fresh biopsy or resection margin, including intraoperative use.
Fluid handling: fresh fluids are delivered promptly and refrigerated at 2 to 8 degrees C if processing is delayed. If refrigeration is unavailable, add an equal volume of 50 percent ethanol. Cerebrospinal fluid is fragile and low in cellularity and is concentrated by cytocentrifugation (cytospin) or membrane filter.
Fixation: the Decision That Cannot Be Undone
Cytology has two mutually exclusive fixation routes, and choosing the wrong one ruins the preparation.
| Route | Handling | Stain | Cells look like |
|---|---|---|---|
| Wet fixation | Slide into 95 percent ethanol immediately, while still wet, or sprayed with a coating fixative | Papanicolaou | Smaller, transparent cytoplasm, crisp chromatin |
| Air drying | Slide waved dry completely before staining | Romanowsky (Wright, Giemsa, Diff-Quik) | Larger, flattened, opaque cytoplasm, prominent granules and stromal material |
- Spray coating fixatives are alcohol solutions containing polyethylene glycol (Carbowax), which leaves a protective wax film. Hold the nozzle 6 to 10 inches from the slide; spraying too close blasts cells off the glass. The wax film must be removed by soaking in 95 percent ethanol before staining.
- Air-dried Papanicolaou slides show swollen, pale nuclei with smudged chromatin and are non-diagnostic. This is the single most common preventable cytology defect.
- Wet-fixed Romanowsky slides show shrunken cells with weak granule color, so rapid on-site evaluation smears are deliberately air dried.
The Papanicolaou Stain
The Pap stain is polychrome and regressive, and its purpose is cytoplasmic transparency so that overlapping cells in a thick smear can still be evaluated.
| Solution | Contents | Stains |
|---|---|---|
| Harris hematoxylin | Alum hematoxylin, usually used regressively with acid differentiation and bluing | Nuclei blue to blue-black |
| OG-6 | Orange G with phosphotungstic acid in alcohol | Keratin and keratinized squamous cytoplasm orange |
| EA-50 (or EA-36) | Eosin Y, light green SF yellowish, Bismarck brown Y, phosphotungstic acid, lithium carbonate | Superficial squamous cytoplasm pink; intermediate, parabasal, and glandular cytoplasm blue-green; nucleoli red |
Expected results: superficial squamous cells pink to orange; intermediate and parabasal squamous cells blue-green; glandular and metaplastic cells blue-green; nuclei blue-black with visible chromatin texture; erythrocytes red-orange.
Why alcohol matters throughout: OG-6 and EA-50 are alcoholic solutions. Water carried into them dilutes the alcohol, swells the cells, and mutes the orange and green. Every rinse between the alcoholic dye baths is 95 percent alcohol, not water.
Cell Blocks
A cell block turns a cytology specimen into a paraffin block, which is what makes immunohistochemistry and molecular testing possible on an aspirate or an effusion.
- Plasma-thrombin method: patient or pooled plasma plus thrombin clots the cell pellet into a handleable button.
- Agar or HistoGel method: the pellet is suspended in warm gel that solidifies on cooling.
- Collodion bag and automated (Cellient) methods are also in routine use.
- The resulting button is fixed in 10 percent neutral buffered formalin or alcohol-formalin, wrapped in lens paper, and processed exactly like a tissue biopsy.
Because the cell block is formalin-fixed and paraffin-embedded, it behaves like tissue for antigen retrieval and for nucleic acid extraction. Laboratories that need predictive marker testing on an FNA plan the cell block at the time of collection, not afterward.
Cytology-Specific Artifacts
- Cornflake artifact: refractile golden-brown "crazed glass" overlying squamous cytoplasm. Air was trapped beneath the coverslip because the slide dried between the last xylene bath and mounting. Correction: soak the coverslip off in xylene, return the slide through fresh xylene, and re-coverslip without allowing it to dry.
- Air-drying artifact: pale, swollen nuclei with lost chromatin detail on a Pap-stained slide.
- Spray fixative blasting: cells stripped from the center of the smear where the nozzle was held too close.
- Corn-starch glove powder: round, refractile bodies with a central Maltese cross under polarized light.
- Cross-contamination (floaters): cytology stain dishes are a classic source; filter or change solutions on a documented schedule and stain gynecologic and non-gynecologic specimens in separate runs when required by the laboratory's procedure.
A conventional cervical smear arrives in the laboratory with a note that the collector spread the sample and set the slide down on the counter for two minutes before placing it in 95 percent ethanol. On the Papanicolaou-stained slide the nuclei are swollen and pale with smudged chromatin. What happened, and what is the correct collection practice?
Which Papanicolaou staining solution is responsible for the orange coloration of keratinized squamous cytoplasm?
A pulmonary fine-needle aspiration yields material that the pathologist wants tested by immunohistochemistry for TTF-1 and by molecular sequencing. Which preparation should the laboratory make at the time of collection, and why?