| Solid tissue specimens (e.g., lymph nodes, spleen, tonsil/adenoids, or extranodal sites) should be submitted as thin slices, less than 2 mm thick, in a generous amount of sterile tissue culture media at 4ºC (i.e., on ice). This helps to reduce the rate of autolysis and degradation of cellular proteins and DNA. For in-house cases, where specimens are delivered immediately to the laboratory, it is only required to keep the tissue moistened with a culture media- (or saline-) soaked gauze. There are no set rules for the required amount of sample because this is dependent on several factors, including the cellularity in the sample, the fragility of the cells, and the susceptibility to apoptosis. More is always better, especially in the case of extranodal specimens where there may be a significant proportion of nonlymphoid tissue or fibrocollag-enous stroma. It is important, however, to submit a generous amount of fresh solid tissue to the FCM–hematopathology laboratory, so that an adequate amount of sample may be allocated to various other procedures in addition to the preparation of cell suspensions for FCM analysis. These procedures, potentially necessary for the complete characterization of a particular lymphoid tumor, include the following: • Snap-freezing for immunohistochemistry or molecular genetics. • Fixation in a 1 : 1 mixture of RPMI and ethanol for molecular genetics (optional). • Wright-Giemsa-stained air-dried touch imprints for cytologic evaluation. The cut surface of the tissue slice should be blotted to remove excess fluid prior to making imprints. The imprints would be otherwise unreadable as slow drying produces severe shrinkage artifacts on the cellular elements. • Fixation for histology, preferably with B-5 or a fixative with a heavy metal component (e.g., barium chloride) for morphologic correlation. For referral cases from an outside pathology laboratory, a small fraction of the sample submitted to the FCM laboratory can be used for histology (if possible). Efforts should be made to obtain H&E sections from the referring institutions, so as to achieve immunophenotypic-morphologic correlation in the FCM hematopathology reports.To ensure that the sample is representative, the slices sent to the FCM laboratory should be adjacent to those submitted for routine histology (Figure 2.1). A practice to be avoided is that of submitting the tip of the lymph node for FCM while allocating the central portion for routine histology. It is also not advisable to hold the fresh tissue for FCM analysis until after the his-tologic sections are ready, because the time delay often adversely affects the viability of the sample. Cell suspensions are obtained from the solid tissue by mechanical dissociation whereby the tissue is minced with two scalpels in a Petri dish containing a small volume of culture media, and then passed through a fine-wire-mesh screen. Alternative techniques, such as repeated aspiration of the tissue using an 18-gauge needle or scraping the cut surface of the tissue section with a scalpel blade or glass slide at a 45-degree angle, can also be employed. |
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