FEATURE
IMMUNOLOGY
a reasonably long period of time and multicolour flow cytometry is of particular importance in this context since it provides a better insight into the antigenic aberration though a detailed profiling of the leukemic cells at presentation. This in turn allows clearer distinction between the leukemic cells and the normal cells in the post-treatment bone marrow even if the former are present in a very small number.
FUTURE OF MULTICOLOUR FLOW CYTOMETRY Bioinformatic merging of immunophenotyping data It is quite apparent that most advanced clinical flow cytometry laboratories have settled for 8-10 colour analysis after a lot of trial and error, and the race for using a larger number of reagents per tube has slowed down following the realization of technical complexities associated with the use of more than 10 colours per tube and after consideration of cost factors. The recent introduction of violet laser
(405 nm) and pacific blue and pacific orange dyes as new fluorochromes has expanded the scope and applications of multicolour flow cytometry. However, there is an increasing awareness of and agreement on the limitations of simultaneous use of more than 10 reagents tagged with different fluorochromes. As a result, the focus is shifting to more intelligent interpretation of the kaleidoscope of phenotypic profile of hemopoietic cells obtained from multicolour flow cytometry using up to 10 reagents per tube. This is being achieved through innovative approaches and use of new computer software. One such software, Infinicyt (developed by the EuroFlow Consortium), merges and magnifies the information on the complex immunophenotype of each normal or dysplastic/neoplastic hemopoietic cell obtained from multicolour flow cytometric
“Conventionally, flow cytometry laboratories use 2-4 antibodies tagged with different fluorochromes per tube”
analysis of each tube to detect even the slightest deviation in the immunophenotype of cells from the normal. The software uses “nearest neighbour population analysis” to compare the backbone markers among different cells to identify the cells and uses the additional markers in the reagent panel to detect the deviation from the normal. Till some significant technological advancements occur in the design and capabilities of clinical flow cytometers and in reagents used in immunophenotyping of hemopoietic cells, this seems to be the right approach for tapping the full potential of the data on flow cytometric immunophenotyping of hematolymphoid neoplasms. This approach could be used for retrospective analysis of older data from two to four colour study of neoplastic hemopoietic cells and obtain insights into hitherto unexplored frontiers of aberrations in cellular phenotype in hematolymphoid malignancies.
Flow cytometric detection of fusion proteins in leukemia An important step in multiparametric diagnosis and monitoring of hematolymphoid malignancies is detection of ‘signature’ and novel chromosomal abnormalities using conventional and/ or molecular cytogenetics. However, the methods are time consuming and require complex technical infrastructure and skilled manpower. In contrast, information on the cellular constituents can be obtained in a few hours by flow cytometry. Therefore, there has been a lot of interest in exploring the possibility of using flow cytometry as a tool for rapid
detection of cytogenetic and molecular abnormalities in neoplastic cells using gene fusion proteins as markers for such abnormalities. This led to the discovery of flow cytometry based techniques for rapid detection of some well known fusion proteins resulting from translocation of chromosomes in neoplastic hemopoietic cells. Using this approach, immunobead assays for detection of a number of fusion proteins have been developed recently. These include BCR-ABL, PML-RARA, TEL-AML1, E2A-PBX1, MLL-AF4, AML1- ETO and CBFB-MYH11. Initial data show a high degree of correlation between results obtained by flow cytometry based methods and those from molecular methods. The advantages of performing both
immunophenotyping and molecular analysis of leukemic cells on the same platform are overwhelming and provide future direction to clinical research in this field. ■
ML
MORE INFO: For more information, please contact the author on
adasgupta@srlworld.com
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The Histopathology and Microbiology track at the Medlab Congress is taking place on January 24th
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MedLab Issue 3 2011 33
January. For more information, please
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