Immunophenotypic Analysis and Rapid Diagnosis of Acute Promyelocytic Leukemia by Flow Cytometry.

Acute promyelocytic leukemia is a unique type of disease for which the all-trans retinoic acid is selected as the first line of induction treatment. Conventional AML regimen containing anthracycline + Ara-C with or without etoposide is dangerous to the patients with APL for its induction of disseminated intravascular coagulation (DIC) and intracranial hemorrhage while otherwise those complications would not happen if all-trans RA were used for induction. Furthermore, morphological diagnosis is usually not always informative at the time of early stage when cytogenetic and fusion gene results are usually not available at the first few weeks. Therefore, rapid and confirmative diagnosis of APL is crucial to the initial treatment of choice.

Objectives The aims of this study were to investigate the new immunophenotypic marker(s) of APL and the quick diagnostic assays.

Patients and Methods 83 cases of patients (45 males and 38 females, age range of 5 – 52 yrs with a median of 24 yrs) with APL were enrolled into this study. Leukemia cells were analyzed by multi-parameter flow cytometry with CD45/SSC gating strategy and 10% or more positive cells were considered positive. Samples from 21 patients with CML, 29 patients with MDS and 11 normal BMs were used as controls.

Results Unsurprisingly, myeloperoxidase (MPO), CD13, CD33 were the most frequent expressing markers for APL cells accounting for 100%, 96% and 99% of the APL cases, respectively, while CD34 and HLA-DR were negative in almost all cases of this disease, with the positive rates of only 3.6% and 2.4%, respectively for these two antigens. The phenotype of high SSC, MPO+CD33+CD13+CD34−DR- strongly suggested the diagnosis. Interestingly, 12 APL cases (14.5%) showed positive for CD56, a neurogenic adhesion molecule, which could be used as leukemia associated phenotype for minimal residual disease (MRD) detection. The stem cell factor (SCF) receptor antigen CD117 was expressed in 78% of APL cases while it was not expressed on CML, MDS and normal bone marrows. Only 17% of APL cases expressed CD11b, while 100% of normal bone marrow neutrophils were positive for this antigen. A multi-color flow cytometry analysis revealed that a phenotype of CD117+CD11b− represented 72% of APL cases while no such phenotype existed in CML(29 cases, P < 0.01), MDS (21 cases, P < 0.01) and normal bone marrows (11 cases, P < 0.01) indicating the diagnostic effect of this particular phenotype. Immunophenotypic analysis of mononuclear cells from the APL patients consecutively detected at 1, 2, 3 and 4 months after RA induction showed that a phenotype switch from CD117+CD11b− to CD117−CD11b+ were clearly observed, indicating the gradual differentiation phenomenon during the induction treatment. Co-existence of CD117 expression and PML-RARα fusion gene presented in 14/16 (87.5%) of long(L)-type cases detected while the co-existence of these two markers presented only in 4/9 cases with short(S)-type fusion gene (P = 0.058), indicating the significant tendency of this two markers.

Conclusions Immunophenotypic analysis with FCM is able to rapidly diagnose the APL patients. High SSC, MPO+CD33+CD13+CD117+CD11b−CD34−DR- can be considered as the useful phenotype suggesting APL, which may provide a quick diagnosis of APL.