Specimen are obtained by inserting a needle into the pleural space (thoracentesis), pericardial space (pericardiocentesis) or peritoneal cavity (paracentesis). Less commonly, fluid is obtained by suction during thoracic or abdominal surgery.
Fluid is collected in clean containers and sent unfixed to the laboratory. To prevent clotting, which widely disperses cells, thus hindering their evaluation, fluid must be collected in heparinised bottles, or a few drops of heparin must be added into the bottle before the fluid is collected. The specimens can be refrigerated for several days at 4°C without effects on cellular morphology.
Several slide preparation methods are available. The container is shaken to disperse cells, and a 50 mL aliquot of the fluid (or the entire specimen) is centrifuged. The supernatant is discarded and the sediment is used to prepare direct smears, cytocentrifuge preparations, thin layer slides or filter preparations. The slides are alcohol-fixed and Papanicolaou-stained.
If a haematologic malignancy is suspected, air-dried cytocentrifuge preparations are useful. One is stained with a Romanowsky-type stain, the rest can be reserved for immunocytochemical studies. The remainder of the sediment is wrapped in filter paper and used for cell block preparations. It is helpful to coagulate the sediment by adding to it a few drops plasma and a thrombin solution, so that the sediment is congealed into a compact mass. If the fluid was not heparinised and clots are present, they should be removed and processed as cell block material.
To improve sensitivity, most laboratories use two or more preparation methods. Smears alone detect 65-70% of malignant fluids, whereas concentrating methods such as cytocentrifugation, filter and cell block preparations detect about 85% of malignancies. Cell block sections are especially useful for special stains and provide the best morphologic comparison to biopsy material. In some laboratories an unfixed wet smear stained with toluidine blue is prepared first any fluid containing a large number of malignant cells.
Leftover fluid should be stored in the refrigerator in case additional slides are required. Fresh fluid is sometimes useful for other studies, such as flow cytometry, electron microscopy and cytogenetic analysis.
Ficoll gradient technique for removal of red blood cells:
- allows cleaner background
- decreases non-specific antibody binding
- otherwise postfixation in Carnoy