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FLOW CYTOMETRY IN IMMUNOLOGY AND HEMATOLOGY
Dipartimento di Scienze Pediatriche e dell’Adolescenza, Università degli Studi,Torino, Italy
The immunophenotyping of acute leukemias is today the preferred method for performing the identification, enumeration and characterization of blast cells at diagnosis. In spite of its widespread present utilization, there does not exist a standardized protocol in clinical laboratories to achieve inter-laboratory reproducibility using the most informative procedures. The complexity of most common questions in acute leukemia immunophenotyping, diagnosis and evaluation of minimal residual disease, demands the utilization of the majority of possible information. The multiparametric immunophenotyping approach using three colors simultaneously is reported here. This approach is based on an adequate choice of reagents, and on sample preparation using whole blood. Recent data show that three color analysis is an adequate method to achieve a clear discrimination between normal and pathological cells, in order to perform the diagnosis, classification and prognosis of acute leukemias. One color is used for the immunological gate, in fact this is the best method for the leukemic cell identification, and the following analysis is performed in blast-gated biparametric histograms. The procedure must be dissimilar in lymphoblastic and myeloblastic leukemias, because the diagnostic questions are different for heterogeneity and clonality of malignant cells. The useful method to define positive antigen criteria is the quantification of antigens, using pre-calibrated sets of fluorescent beads. This method permits a cross evaluation of immunophenotyping results between different investigators and laboratories. Recent reports indicate that the phenotypic aberrations reflect genetic abnormalities of leukemic cells and therefore their definition and identification seem relevant for prognostic implications and for minimal residual disease identification.