Importance of Detection of Intracellular Myeloperoxidase, CD13, CD79a, CD22, CD3 and Terminal Deoxynucleotidyl Transferase By Flow Cytometry Diagnosis of Acute Leukemias
Authors: Paiva Aldair Sousa , Paiva Hugo Diogenes De Oliveira , Cavalcanti Geraldo Barroso , Bahia Frank , Freitas Rodrigo Villar , Silveira Lenilton Silva , Silva Lorena KF , Vasconcelos Roberto C , Silva Dany GKC , Soares Victor lima , Gil Erica Aires , Januario Geisa , Fernandes Maria , Jardim Alessandra Suelen 
Published: October 2019

Abstract
Background: The detection of Intracellular (IC) antigens by flow cytometry (FC) such as myeloperoxidase (MPO), cCD13, cCD79a, cCD22, cCD3 and Terminal deoxynucleotidyl Transferase (TdT) has become the useful tool in the differential diagnosis between acute myeloid leukemias (AML) and acute lymphoid leukemias (ALL). Through detection of myeloid antigens (MPO and cCD13), B cells precursors (cCD79a and cCD22) and precocity T-cells (cCD3) it has been possible to confirm the diagnosis of these acute leukemias. The detection of intracellular cell markers by FC usually requires previous permeabilization of fresh cell suspensions. TdT, also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells. TdT adds N-nucleotides to the V, D, and J exons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity. In humans, terminal transferase is encoded by the DNTT gene. This antigen is expressed mostly in the nucleus cells from primary lymphoid organs, like the thymus and bone marrow. The TdT detection has also been shown to be useful in confirming the acute forms of B and T-lineage lymphoproliferative diseases by FC. The aim of this study was to demonstrate the importance of this cell markers' detection by FC in the differential diagnostic of acute leukemias. Methods: Bone marrow and/or peripheral blood leukemic cells from 50 cases of acute leukemia: 16 ALL and 36 AML. The cells were fixed and permeabilized in briefly exposed to Becton & Dickinson Lyse Solution at concentration of 10%, and subsequently labeled with monoclonal antibodies anti-MPO, TdT, CD3, CD13, CD22 and CD79a. Results: The MPO expression was observed in 35/36(97,22%) and cCD13 in all cases of AML and in none ALL patients. Three cases of MPO-positive ALL (FAB-L2) could be reclassified as M0-AML. These cases were CD34+;HLADR+;CD33-;CD13-;CD7+ and cCD13+. The intensity of TdT expression was observed in 15/16 (93.8%) of ALL and 5/36 (13.9%) of AML. The cCD22 and cCD79a were positive in 15/16 (93.8%) and all of pre-B ALL respectively and cCD3 was expressed in one case of Pre-T ALL that initial phenotype was CD34+/HLADR+/TdT+/CD7+ and sCD3-). Conclusions: These results indicate that monoclonal antibodies anti-MPO, cCD13, cCD79a, cCD22, cCD3 and TdT were excellent cell markers for the diagnosis and classification of acute leukemias and can be reliably detected by FC. This rapid and specific technique should be a valuable addition to routine immunophenotyping of acute leukemia.

Disclosures
No relevant conflicts of interest to declare.