Novel Leukemia Associated Immunophenotype (LAIP) Absent from Normal and Regenerating Bone Marrow Identified by Multiparameter 6-Color Flow Cytometry.

Flow cytometry analysis of minimal residual disease (MRD) in acute myeloid leukemia (AML) is based on the detection of aberrant phenotypes responsible for the relapse. Until now, all studies were performed by 3 or 4 color immunostaining, allowing the identification of LAIP in 80% of cases. Moreover, no data is available regarding the existence of such phenotypes in regenerating bone marrow. The new generation of cytometers allows the study of 8 parameters that permit a better distinction of malignant from normal phenotypes. In our study we analyzed 20 bone marrow samples from allogeneic donors, 20 ALL regenerating bone marrows after chemotherapy and 53 AML samples at diagnosis. Multiparameter 4 colour and 6 colour flow cytometry was used in order to define antigen combinations which are totally absent or present at very minimal levels in normal and regenerating hematopoiesis. “Blast cells” were gated according to CD45/SSC properties.For the first time we describe by 6 color flow cytometry 47 phenotypes totally absent from “blasts” gate in all normal bone marrow (ex: CD34+DR−117+33−15+, CD34+38+33−56+19−, CD14−DR+4+11B+64+). Another 41 phenotypes were identified as presents at a frequency < 0,05% of total cells (ex: CD34+DR+117−33+15+, CD14−DR+4+11B+64−, CD34+65−56+4−16−). There was no significant difference between normal and regenerating marrows. The 4 color panel of moAbs allowed us to identify only 30 phenotypes presents at a frequency < 0,05% of total cells (ex: CD34+33−13+, CD34+117+11b+, CD34+DR−13+). 53 AML at diagnosis were studied using 6 color immunophenotyping and 58 % of phenotypes described as aberrant or infrequent in normal myeloid hematopoiesis were found in at least one AML at diagnosis in more than 1% of total cells. All AML cases show at least one LAIP but frequently we observed more than one LAIP blast subpopulation in the same sample. Some examples of LAIP observed are CD34+ 38+ 33+ 56+ 19−, CD34+ 38+ 33+ 56− 19+, CD34− DR− 117+ 33+ 15−. In conclusion our results shows that (1) the ability to clearly distinguish leukemic from the healthy cells is considerably increased by 6 color approach (8 parameters analyzed) than 4 color. (2) Furthermore that these aberrant or infrequent phenotypes in normal or regenerating bone marrow samples are identified in AML cases and can be utilized in AML minimal residual disease study. (3) Knowledge of the expression of different markers in normal hematopoietic development provides a frame of reference for identification of abnormal differentiation patterns.