Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients

Ten years ago, we made an incidental flow cytometric observation while immunophenotyping biopsy and marrow samples from children suspected to have leukemia/non‐Hodgkin's lymphoma, but were subsequently diagnosed with neuroblastoma. The samples contained neoplastic CD45 − cells that had an extremely bright CD56 + (beyond the fourth decade on a four‐decade scale) population distinguishable from CD45 + CD56 usual density+ natural killer lymphocytes as well as other CD45 − CD56 usual density+ nonhematopoietic tumors such as small cell carcinoma or melanoma. Following the “rare event” philosophy of selecting one negative and two positive antigens, we initially tried a “cocktail” of CD45 − CD56 very bright+ neuron‐specific enolase (NSE) cytoplasmic+ . We later modified the procedure to a more clinically applicable “lysed whole blood” CD45 − CD56 very bright+ ganglioside GD2 + cocktail to improve turnaround time (eliminating the cell permeabilization step for cytoplasmic NSE analysis), specificity, and sensitivity of the assay. A total of 123 marrow/tissue/fluid samples were analyzed by the various forms of the assay. Clearly interpretable samples had an 83% specificity and a 100% sensitivity. The three‐color GD2 assay has successfully detected cells in marrow samples to a level of 0.002% (1 per 10 5 cells) using patient samples (not artificially “spiked” material). We added CD81 expression of the neuroblastoma cells as a fourth color and now use this rare event clinical test to help stage and monitor all patients with neuroblastoma. Cytometry (Clin. Cytometry) 50:298–304, 2002. © 2002 Wiley‐Liss, Inc.