Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia (original) (raw)
Related papers
American journal of clinical pathology, 1993
This article describes a procedure for performing routine three-color flow cytometric analysis for acute leukemia on lysed whole bone marrow preparations. This technique uses the combination of CD45 intensity and right-angle light scatter (RALS) to distinguish leukemic cells from normal lymphocytes, monocytes, neutrophils, eosinophils, and nucleated red blood cells. On this display, leukemic cells occupy a unique blast region characterized by intermediate CD45 density and low RALS, which, in normal marrows, contains less than 5% of the total cells. This approach was applied to 39 cases of acute leukemia and 8 cases of myelodysplasia or myeloproliferative disorders. The estimate of blasts by flow cytometric analysis was correlated highly with morphologic leukemic cell counts over a wide range. Moreover, the pattern seen on the CD45-RALS display was different for different French-American-British subtypes of leukemia, suggesting that this pattern might be useful for categorization. Wh...
Cluster of Differentiation 45: An Adjunct to Flowcytometric Diagnosis of Leukemias
Background: This Cross sectional comparative study was performed to compare the mean fluorescence intensity of cluster of differentiation 45 between healthy individuals and patients with acute lymphoblastic leukemia. Patients and methods: Thirty three healthy individuals (mean age 11 years) and 41 patients with B cell acute lymphoblastic leukemia (mean age 7 years) were enrolled in the study in department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, from Jan 2009 to April 2011, after ethics committee approval and obtaining informed consent. For immunophenotyping analysis, blood was stained with monoclonal antibodies using lyse wash procedure. For each subject, panel for staining consisted of cluster of differentiation 3, cluster of differentiation 5, cluster of differentiation 7, cluster of differentiation 10, cluster of differentiation 19, cluster of differentiation 20, , cluster of differentiation 13, cluster of differentiation 34, cluster of differentiation 1314, cluster of differentiation 1333, cluster of differentiation 45, HLA-DR, and intracytoplasmic myeloperoxidase as well as terminal deoxynucleotidyl transferase . Cells were then analyzed on Becton Dickinson FACSCalibur flow cytometer using Cell Quest Pro software. For each subject, mean fluorescence intensity was calculated in software along with geometric mean and standard deviation. Results: Mean of geometric means of cluster of differentiation 45 expression on blasts of patient population was considerably low (145) as compared to lymphocytes of healthy population (764), being statistically significant (p<0.0001). Conclusion: cluster of differentiation 45 is useful in differentiating mature lymphocytes from blasts in ALL cases.
Side scatter versus CD45 flow cytometric plot can distinguish acute leukaemia subtypes
Indian Journal of Medical Research, 2016
Background & objectives: Flow cytometry is an important tool to diagnose acute leukaemia. Attempts are being made to find the minimal number of antibodies for correctly diagnosing acute leukaemia subtypes. The present study was designed to evaluate the analysis of side scatter (SSC) versus CD45 flow dot plot to distinguish acute myeloid leukaemia (AML) from acute lymphoblastic leukaemia (ALL), with minimal immunological markers. Methods: One hundred consecutive cases of acute leukaemia were evaluated for blast cluster on SSC versus CD45 plots. The parameters studied included visual shape, CD45 and side scatter expression, continuity with residual granulocytes/lymphocytes/monocytes and ratio of maximum width to maximum height (w/h). The final diagnosis of ALL and AML and their subtypes was made by morphology, cytochemistry and immunophenotyping. Two sample Wilcoxon rank-sum (Mann Whitney) test and Kruskal-Wallis equality-of-populations rank tests were applied to elucidate the significance of the above ratios of blast cluster for diagnosis of ALL, AML and their subtypes. Receiver operating characteristic (ROC) curves were generated and the optimal cutoffs of the w/h ratio to distinguish between ALL and AML determined. Results: Of the 100 cases, 57 of ALL and 43 cases of AML were diagnosed. The median w/h ratio of blast population was 3.8 for ALL and 1 for AML (P<0.001). ROC had area under curve of 0.9772.The optimal cutoff of the w/h ratio for distinction of ALL from AML was found to be 1.6. Interpretation & conclusions: Our findings suggest that if w/h ratio on SSC versus CD45 plot is less than 1.6, AML may be considered, and if it is more than 1.6, ALL may be diagnosed. Using morphometric analysis of the blast cluster on SSC versus CD45, it was possible to distinguish between ALL and AML, and their subtypes.
Role of blast cell immunophenotyping for the diagnosis and prognosis of acute myeloid leukemia
Biology of the Cell, 1992
Bone marrow blast cell antigen expression from 86 patients with cle n o w acute myeloid leukemias (AML) was studied and corrclated with FAB classification and clinical outcome. Among a panel of 14 monoclonal antibodies routinely used for the diagnosis of acute leukemias we studied the expression of six antibodies (CD13, CD15, VIM2, CU33, CD14, CD34) of the granulomonocytic lineage and found that some of them were useful for diagnosis and/or prognosis. For FAB subclassification of AML, the CD13 or VIM2 antigen expression was of no benefit. Monocytic leukemias (M4+ M5PD+ M5WD) more frequently expressed CD34 antigen (28/31) than granulocytic (MI +M2+M3) subtypes (33/53) (P<O.OI). Finally, the most striking differences were found with CD14 antigen expression: CD14 antigcn was more frequently expressed in M4+ M5 leukemias (21/31) than in M1 + M 2 + M3 subtypes (12/33) (P<O.OI). The mean percentage of CD14 positive blast cells was accordingly higher in monocytic leukemias than in granulocytic leukemias and the difference was highly significant (P<O.OOOI). The CD15 antigen was more frequently expressed in differentiated leukemias (M2 + M3 + M4+ M5WD) (35/44) than in poorly differentiated forms (M 1 + M5PD) (17/37) (P< 0.001). The statistical difference was higher when the mean percentage of CD15 positive blast cells were compared (P<0-0003). Moreover these latter percentages were different in M1 and M2 subtypes (P < 0.003). The blast cell expression of CDI 3, CD14, C D I5 or CD33 was not predictive of the length of CR or survival. Moreover, our results support previously published findings suggesting a longer overall survival duration for patients whose leukemic cells do not express the CD34 antigen (P<O.OI). We also confirm that patients with the more differentiated subtypes of AML (CD13-, CD34+) tend to survive longer than patients with the less differentiated subtypes ofAML (CD13--, CD34+) (P<O-OOl).
The hematology journal : the official journal of the European Haematology Association / EHA, 2004
Multiparameter flow cytometry (MFC) is capable of quantifying minimal residual disease (MRD) in acute myeloid leukemia (AML). Its broad application, however, is limited by a lack of sensitivity in about 20% of patients. CD45 gating may improve sensitivity. A broad panel of four-fold combinations of monoclonal antibodies including CD45 in each was used to define leukemia-associated aberrant immunophenotypes (LAIP), to define their sensitivities in normal bone marrow samples, and to compare results to data obtained without CD45 gating using triple staining. In 45 patients, a LAIP was defined, 11 normal bone marrow samples were analyzed as controls. The median percentage of LAIP-positive AML cells with and without CD45 gating was 21.95% (range, 3.31-82.52%) and 20.52% (range, 3.22-81.94%). The median percentage of LAIP-positive normal bone marrow cells ranged from 0.01 to 0.42% (median, 0.02%) and 0.02 to 0.58% (median, 0.15%) with and without CD45 gating. The difference of LAIP-positi...
New methodologic approaches for immunophenotyping acute leukemias
Haematologica, 2001
Flow cytometry is nowadays the preferred method for immunophenotypic identification, enumeration and characterization of blast cells at diagnosis. Despite widespread application of standardized protocols, inter-laboratory reproducibility has still not been achieved. The complexity of diagnosis and evaluation of minimal residual disease, in immunophenotyping acute leukemia, demands the use of a test that provides all the necessary information. The information given here is derived from the experience of the authors and from literature files. The most relevant studies with adequate conclusions were considered. We report on the current status of multiparametric immunophenotyping using simultaneous three and four-color staining and the applications of this technique. Multiparametric immunophenotyping is a powerful method for achieving a clear discrimination between normal and pathologic cells. The specific identification of leukemic cells by immunologic gating forms the basis for immuno...
Background: Immunophenotyping by flowcytometry is well conceived & fundamental tool to diagnose & subtype hematological malignancy, especially acute leukemia. By detecting various antigens presenting in various parts of cell, it is possible to know cell lineage & immaturity of the cell or group of cells. Apart from diagnostic importance, this specialized tool is also useful in prediction of prognosis & detection of minimal residual disease. Now, immunophenotyping can diagnose and type also those acute leukemias where morphology and cytochemistry fail. Aim: Study of Immunophenotypic patterns and their correlation with morphology and cytochemistry in North Indian Population. Materials and methods: Short clinical details and complete blood count of 150 patients were noted in the department of hematology of tertiary health centre. Sample of each patient was processed as per protocol and run on FACS CALIBUR OF BD BIOSCIENCES, USA. Dot plot data of each patient was analyzed and result was released. Results: AML, BALL and TALL comprised 38%, 49%, and 13% of all cases. Almost all blasts were expressing dimCD45 with no significant differences between the subtypes. CD34 have different expressions in AML subtypes, usually negative in APML. Aberrant expression of CD7 and CD19 were expressed in 5% and 3.4%of all cases of AML respectively. In 40% cases, morphology and Cytochemical studies clinched the diagnosis. 60% cases essentially needed Flowcytometric evaluation for diagnosis and subtyping of acute leukemias. Conclusion: Flowcytometric analysis of the patterns and intensity of antigen expression in blasts improved the diagnosis of AML and ALL in our centre. All cases do not require Immunophenotyping for diagnosis. Simultaneous use of conventional morphology, cytochemistry and flowcytometry reduce diagnostic cost of acute leukemia. Immunophenotyping results of our acute leukemia patients were comparable to international published studies.