Introduction: Acute leukemias (AL) are clonal diseases classified into two large groups: acute lymphoid leukemia (ALL), more common in children, and acute myeloid leukemia (AML), more rare in childhood, besides the rare phenotype leukemias such as acute biphenotypic leukemia (ABL) and undifferentiated acute leukemia (UAL). Although the cytomorphology still be relevant in theses leukemias diagnoses, the immunophenotyping by flow cytometry (FC) have become essential in the diagnosis, classification and follow-up of these neoplasms, standing out as a modern and practical methodology, presenting characteristically as a method of multiparametric and quantitative analysis of the leukemic cells. Objective: The objective of this study was realize a retrospective study of immunophenotyping in 371 patients with AL. Methodology: Immunophenotyping was performed biological samples by FC after labeling with a panel of monoclonal antibodies specific for AL directed against lymphoid antigens (B, T and NK cells), myeloid, and markers related to other cell immaturity. At the same time, information was obtained regarding patients such as age, sex, clinical data related to the disease, and previous hematological analysis. Results: From 371 cases, 127 were ALL (71 B lineage and 55 T=ALL), 239 AML, 04 ABL and 2 UAL. In the ALL, it was observed a higher frequency in children, contrasting with the cases of AML, ABL and UAL more prevalent in adults. In ALL, clinical signs and laboratorial data related to disease were more present in the ALL-T and mature-B corroborating with information in the literature. In AML, ABL and UAL the most clinical parameters observed were splenomegaly, hepatomegaly and bleeding. The classification FAB subtypes of AML more predominant were M1, M2 and M4 and lower incidence of the M7 subtype. Conclusion: These data demonstrate the importance of FC technology in the diagnosis, classification and establishment of prognostic factors of these neoplasms. Disclosures No relevant conflicts of interest to declare.
BACKGROUND: Acute Myeloid Leukemia (AML) is the most frequent acute leukemia in older patients (over 60 years of age) with more than 50% of the cases, accounting for 15 to 20% of childhood leukemia and 80% of adult leukemias, with poor prognosis, especially in elderly patients. Myelodysplastic syndromes (MDS), another spectrum of clonal myeloproliferative disorders that also involve mainly older age groups and are characterized by ineffective hematopoiesis, peripheral cytopenia's, chromosomal abnormalities and predisposition for progression to AML (AML/MDS). Elderly patients with AML are part of a biological and clinically distinct group with presence of high-risk cytogenetic abnormalities, hematological abnormalities and immunophenotypic aberrations. OBJECTIVE: Evaluation of clinical, demographic, hematological and immunophenotypic parameters in elderly patients with AML and determination of correlation these parameters with disease status. METHODS: Bone marrow (BM) and peripheral blood (PB) samples from 80 elderly patients diagnosed with AML were evaluated for hematological, immunophenotypic and karyotype parameters. The hematological parameters evaluated were: count of leukocytes, platelets and blastic cells and also determination of hemoglobin levels, cytomorphologic analysis of bonne marrow, immunophenotyping by flow cytometry for immunologic AML classification, determination of aberrant lymphoid cell markers, and detection of antigens related to multidrug resistance (MDR) such us P-glycoprotein (Pgp) and multidrug resistance proteins type 1 (MRP1), p53 protein and also karyotype analysis for confirmation of AML/MDS cases. At the same time, demographic and clinical data were obtained from all patients. RESULTS: Data analysis showed that 56 patients had "de novo" AML, 6 recurrent disease, 15 transformed MDS and 3 refractory AML. In relation to clinical aspects, there was a predominance of splenomegaly (91.2%), followed by hepatomegaly (76.2%). Laboratory findings showed a predominance of hyperleukocytosis (91.3%), thrombocytopenia (85%) and anemia (86.2%), with cytopenias being more pronounced in cases of AML/MDS. Most cases were classified as FAB M1 (36.6%), M2 (17.5%) and M4 (23.7%). Our data analysis was statistically significant (p < 0.05) and showed a correlation with aberrant T-lymphoid antigens (CD3 and CD7), Pgp, MRP1, p53 protein and transferrin receptor (CD71) with the increase in the unfavorable status of the disease, contributing to a worse prognosis in these patients. CONCLUSIONS: Our results demonstrate the importance of clinical and laboratory investigations of these patients in order to obtain further information on these cancers. Disclosures No relevant conflicts of interest to declare.
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.
Background: The Philadelphia chromosome is a cytogenetic change resulting from a reciprocal translocation of genetic material between ABL genes from chromosome 9 and BCR from chromosome 22 or t(9; 22) (q34; 11), forming the chimeric gene BCR- ABL, being associated with chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). The p190 variant is usually associated with acute forms of leukemia, including AML and ALL, whereas the p210 variant is associated with the chronic phases of CML. Due to the high sensitivity and specificity, nucleic acid amplification techniques by real-time PCR have replaced the conventional cytogenetic techniques for the identification of the Philadelphia chromosome and its p190 and p210 variants. Molecular analysis has been indicated in the initial diagnostic phase and also for the therapeutic monitoring defining the percentage of neoplastic cells present in the patients during the different phases of the treatment (Minimum Residual Disease or MRD).The aim of this study was the transcript BCR-ABL identification in patients with suspected of CML and evaluation of the gene frequency in these patients. Methods: The presence of BCR-ABL gene was investigated in blood samples from 42 patients with suspected CML. The RNA extraction was performed by phenol/chloroform method. The cDNA was submitted to PCR, using specific primers for and BCR-ABL genes by Real time PCR. Results: From all studied patients, 16 (38.10%) were negative, and 26 (59.09%) positive for one of rearrangements: p210 b3a2 and b2a2 in 18 cases (40.91%) and p190 a1a2 in 2 cases (4,76%) and double positive p120/190 in 6 cases (14,28%). We observed that the most common rearrangement was the p210 b3a2, and the molecular results were compatible with clinical and hematologic suspicion. Conclusions: The Real-timePCR, because of its specificity and sensitivity, can be considered the most used technique in routine diagnosis and investigation of MRD of CML patients. Disclosures No relevant conflicts of interest to declare.
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