TABLE 2.
Advantages and Disadvantages of Various Cytology Preparation Methods for Application of Immunocytochemistry
| Preparation (Sample Feasibility) | Advantages | Disadvantages |
|---|---|---|
| CB (all types of samples except those with low cellularity) | Morphology and epitope stability comparable to histology | Inconsistent cellularity |
| Multiple sections for ICC panels | Time-consuming | |
| Easier validation using IHC protocols | Different methods of preparation lacking standardization | |
| Histology FFPE controls acceptable (if collected in formalin) | Non-formalin-fixed CBs require revalidation | |
| Direct smears (FNA, brushings, sedimented fresh effusions) | Bulk of the material used in cytopathology practice | Require larger quantities of antibody to cover entire slide16 |
| Excellent cytomorphology | Limited ICC panels | |
| Extra smears easily made during aspiration | ICC revalidation required | |
| ICC can be performed on the same diagnostic smear | ||
| Applicable even for smears with few tumor cellsa | ||
| Coverslipped alcohol-fixed Papanicolaou-stained smears show stable epitopes for ~2 years36 | ||
| Cytospin specimens (low-volume fluid samples and FNA collected in nonfixative solutions, ideally 0.9% NaCl solution) | Rapid | Not suitable for specimens with blood or mucous |
| Several slides from the same specimen for ICC panels16 | Inferior cytomorphology to smearsb | |
| Alcohol-fixed, air-dried, unstained cytospins can be stored at 4°C for a month, at −20°C for several months, and at −70°C for years without loss of antigenicity16 | Cell counting to make aliquots of 0.5–1 × 105 cells/mL for optimum cellularity16 | |
| ICC revalidation required | ||
| LBC (all types of cytology specimens) | Uniform cell collection and standardized processing | Altered cytomorphologyc |
| Clean background due to clearing of blood and mucous | ICC revalidation required | |
| Multiple slides from same specimen for ICC panels | ||
| Smaller quantity of reagent required | ||
| Material easily stored for up to 6–8 weeks in preservative solution at room temperature37 | ||
| Alcohol-fixed, air-dried, unstained LBC slides can also be stored at −70°C for years without loss of antigenicity16 |
Abbreviations: CB, cell block; FNA, fine needle aspirate; ICC, immunocytochemistry; IHC, immunohistochemistry; LBC, liquid-based cytology.
Use of tumor cell mapping either by photocopy of the slide or marking location on a superimposed glass slide, or by saving microscope co-ordinates helps in relocating rare tumor cells on the ICC slide.
Alteration of cell composition by speed and time of centrifugation can occur. We recommend careful centrifugation of 2 min at 400 rpm, followed by fixation in ethanol-acetone for 10 minutes.
Cells more dyscohesive with shrinkage artefacts leading to difficulty in identification of isolated small atypical cells on low power examination.