Because of the uneven distribution of metastases to the bone marrow, at least two marrow aspirates and two biopsies (trephines) from the iliac crests are recommended (Brodeur et al. 1993). Alternatively, four aspirates from four different sites of the iliac crest or in infants from the proximal tibial bone are sufficient to rule out gross marrow involvement. For an adequate biopsy at least 1 cm of marrow (not cartilage, not bone) is necessary (Brodeur et al. 1993) which may not be feasible in young infants. The bone marrow aspiration consists of three sampling steps per site:
— First aspiration (0.1-0.4 ml) for bone marrow smears
— Second aspiration (2-5 ml, anticoagulated with heparin) for immunocytology
— Third aspiration (2-3 ml, anticoagulated with EDTA or directly into extraction medium) for PCR investigations (Ambros and Ambros 2001).
Figure 7.2 demonstrates characteristic syncytia and immunocytologically positive clumps of cells as requested for the bone marrow diagnosis of neuroblas-toma. Although consensus on the specific antibodies for marrow immunocytology has not yet been reached, commercially available anti-GD2 antibodies are widely accepted (Ambros and Ambros 2001) since very few neuroblastomas are GD2 negative. Complementary markers include the neural cell adhesion molecule (NCAM, CD56), NSE, chromogranin A, and
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