| Peripheral blood can be collected in either ethylenediamine tetraacetic acid (EDTA) or heparin. Collection in EDTA is preferred, however, because a hemogram and a blood smear can be obtained from the same sample. The volume of blood required depends on the white blood cell (WBC) count; 10 mL of blood is adequate in most instances. The blood specimen is preferably maintained at room temperature. Referred blood specimens from outside institu-tions should be accompanied by a hemogram and a fresh blood smear (i.e., free of storage artifacts), either unstained or stained with Wright-Giemsa, for cytologic evaluation. For quality control, however, the FCM laboratory should also make a smear from the FCM blood sample. Similarly, EDTA is the anticoagulant of choice for bone marrow specimens sent to the FCM–hematopathology laboratory. Bone marrow smears, cytochemistries where appropriate, and FCM studies can all be performed from the same tube. Ideally, the hematopathology staff should be personally involved with the collection of the bone marrow specimen to ensure that the sample obtained is adequate enough to cover all the desired tests (e.g., FCM, cytogenetics, morphology). Two of the authors routinely performed the bone marrow procedure themselves. Using the previously described recommended approach (Nguyen and Diamond, DiagnosticHematology: A Pattern Approach), an ample quantity of marrow aspirate and admixed blood is collected in EDTA tubes. The marrow spicules are allowed to rise to the top, from where they can easily be harvested virtually free of blood contamination and allocated in appropriate amounts for FCM, morphologic smears, and any other necessary studies. This optimal approach eliminates one of the most frequent problems encountered in the laboratory, the marked dis-crepancy between the cellular bone marrow smears made at the bedside and those made from the aspicular, severely hemodilute marrow sample submitted for FCM analysis. Referred bone marrow aspirate samples received from outside institutions should be accompanied by a fresh bone marrow smear containing an adequate number of spicules. Preferably, a fresh blood smear and hemogram should also be included, so that a complete diagnostic evaluation of the bone marrow can be carried out properly. Approximately 3 to 5 mL of representative marrow aspirate is usually sufficient for a comprehensive FCM analysis, unless the marrow is severely hypo-cellular. Because bone marrow aspirates have a much higher cell density than peripheral blood specimens, degenerative changes tend to occur more quickly. Refrigeration and the addition of nutrient media containing serum proteins to the aspirate will help to maintain cell viability in bone marrow samples that cannot be processed soon after collection. Once received in the laboratory, the bone marrow sample is poured out onto a Petri dish to check if spicules are present. A small portion of the spicules is taken to prepare an “in-house” marrow smear for quality control. Two scalpels are applied to mince the remaining spicules to release cellular elements (especially neoplastic lymphoid cells and plasma cells) that tend to adhere to the spicules.In the case of an aspicular aspirate (“dry tap”), the FCM marrow sample should consist of at least two 2-cm-long core biopsies submitted in sterile tissue culture media, preferably RPMI supplemented with fetal calf serum and a mixture of antibiotics. Cell suspensions can be obtained from the biopsies by the same mechanical dissociation procedure applied to solid tissue. In body cavity effusions, it is important to collect samples with good cell viability. This can be achieved by draining off the existing effusion, and later obtaining the reaccumulated “fresh” effusion. For deep-seated lesions (e.g., mediastinal or retroperitoneal), the preferred specimen for FCM analysis is a fine-needle aspiration rather than a core biopsy. Multiple passes (at least 3 to 4), performed by an experienced person, usually provide a higher cell yield than a tissue core biopsy, and minimize sampling error. Body fluids such as CSF, vitreous humor or peri-cardial fluid carry a scant number of cells and do not require much initial processing. A cell count can be obtained and cytospins prepared directly from the submitted sample. |
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