Review Article
Clinical Applications of Molecular Haematology : Flow Cytometry in Leukaemias and Myelodysplastic Syndromes Bakul I Dalal
Abstract Flow cytometry is semi-automated study of antigen profile of cells using the Scatchard principle of antigenantibody binding and fluorochrome-based detection systems. Flow cytometric evaluation of cellular proteomics has become an integral part of the laboratory diagnosis and classification of haematopoietic neoplasms. Recent technical advances in lasers, monoclonal antibodies, fluorochromes, and computer-based color compensation algorithms have expanded the usefulness of flow cytometry. Detection of minimal residual disease by flow cytometry in leukaemias and lymphomas is incorporated in many treatment protocols. Finding of aberrant maturation pattern of granulocytes offers a sensitive screening tool for early diagnosis of myelodysplastic syndromes. Detailed proteomic analysis of leukemias is helping more precise prognostic and biological stratification. ©
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INTRODUCTION
nitial flow cytometers were built for biological warfare during the world war II, to test the efficiency of filter masks in removing bacteria or fungi. The light source was the headlight of Ford model T automobile. When the research was declassified and published in 1947,1 a young engineer, Wallace Coulter promptly acquired a patent for his particle counting machine in 1953. The next major boost to the flow cytometer technology came in 1975,2 when Dr. George Köhler, a post-doctoral fellow working in Dr. Cesar Milstein’s laboratory in Cambridge, UK, accidentally discovered the hybridoma technic for manufacturing monoclonal antibodies. Now that the major technical hurdle was overcome, advances in laser, optics, hydraulics and computer technology have made flow cytometry cheaper and less labor/ skill intensive technic. A search of National Library of Medicine database for word “flow cytometry” showed the number of publications rose from 0 in 1979 to over 7000 in 2006 (Fig. 1). Flow cytometer records physical properties and fluorescence of cells (Fig. 2). If we use antibodies tagged with different coloured fluorochrome dyes, the flow cytometer can tell us if the appropriate antigens are present on the cells. Flow cytometry has revolutionized how we diagnose, treat, prognosticate and monitor acute leukaemias. The Division of Hematopathology, Vancouver General Hospital, Vancouver, BC, Canada. © JAPI • VOL. 55 • AUGUST 2007
Fig. 1 : Number of publications containing word “flow cytometry” since development of hybridoma technology.
However, its role in chronic myeloproliferative disorders and myelodysplastic states is still evolving. Flow Cytometry in quantitation of blasts of acute leukaemia and myelodysplastic syndromes In myelodysplasias as well as in early acute leukaemias, it is sometimes difficult to assess percentage of blasts, due either to blood contamination of the marrow specimen, or hypogranular morphology of the granulocyte precursors. Flow cytometric assessment of blasts is precise and correlated better with overall survival than morphologic blast count.3 Flow cytometry in classification and subtyping of acute leukaemia Roughly a quarter to a third of the leukemias can not be confidently diagnosed as myeloid or lymphoid by
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Fig. 2 : Schema of a typical flow cytometer.
morphology alone. Flow cytometry provides objective and unequivocal diagnosis in these cases. In addition to myeloid and lymphoid designation, flow cytometry also identifies the cases with expression of multiple lineages, enabling the assignment of biphenotypic leukaemia. Several international groups have defined criteria for biphenotypic leukaemia.4,5 Flow cytometry is very useful in subtyping of acute leukaemias according to the WHO 6 classification. Minimally differentiated acute myeloid leukaemias can only be identified by flow cytometry. Acute megakaryocytic leukaemias express platelet associated glycoproteins CD41 and 61, and von Willebrand/ factor VIII antigens, although one should exclude platelet adhesion onto the blast cells. Pure acute erythroleukaemias may express alpha glycophorin (CD235) and transferring receptor (CD71). In addition, characteristic immunophenotypic pattern has been identified in patients with acute myeloid leukaemias with recurrent chromosomal abnormalities. Acute promyelocytic leukaemias with t(15;17) are typically CD9+/HLADR-/CD34-/CD117-. 7 Acute myeloid leukaemias with t(8;21) are CD13+/CD34+/CD19+/ CD56,8 those with inv(16) are CD34bright/CD13bright/ CD33dim,9 while those with 11q23 translocations are CD4+/CD56+/CD34-.10 These highly sensitive and specific imunophenotypic patterns uncover cryptic genetic abnormalities that otherwise would have gone undetected. Flow cytometry in prognostication of acute leukaemia Flow cytometric expression of multidrug resistance proteins MDR1, LRP and MRP have been correlated with induction failure, shorter relapse free periods and shorter overall survival. Coexpression of antigens of other lineage in adult acute leukaemias e.g. CD56 in myeloid leukaemias correlates with aggressive disease and is incorporated into many treatment algorithms. 572
Flow Cytometry in treatment of haematologic neoplasms Several antibodies are being used in therapy of haematologic malignancies, including anti-CD33 for myeloid leukaemias, anti-CD52 and anti-CD20 for lymphoid neoplasms etc. Flow cytometric demonstration in the neoplastic cells of bright expression of appropriate antigens is necessary prerequisite to the initiation of immunotherapy. Flow cytometry in monitoring of acute leukaemia The molecular technics for monitoring of pediatric acute lymphoblastic leukaemia are well established in the therapy protocols. However, adult myeloid leukaemias do not have uniform or identifiable molecular lesions that lend themselves to molecular monitoring. Flow cytometry can identify “blast-specific” patterns in up to 90% of patients, and is routinely used to monitor minimal residual disease of 1 in 104 leukaemic cells.11 Flow cytometry in myelodysplastic and myeloproliferative disorders The incidence of myelodysplastic syndrome is increasing worldwide, especially the early cases. The morphologic abnormalities in these cases are often minimal and subtle, and staining variations between the laboratories make assessment of referred-in cases for hypogranularity etc. difficult. The cytogenetic abnormalities may be absent in half to three-quarter of cases. Flow cytometry helps in these cases by objective demonstration of hypogranularity12 and also abnormal patterns of antigen expression in neutrophils and other granulocytes. In more advanced cases of myelodysplastic syndromes, flow cytometric counting of blast cells is more precise and correlates with overall survival.3 Flow cytometric protein profiling of leukemias Recent demonstration of usefulness of gene expression profiling in acute leukaemias13 has spurred interest in the profiling the translation products of these genes. Such protein profiling14 is technically easier than geneprofiling, and provides functional and translational information to complement gene-profiling.14
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Khoury H, Dalal BI, Nevill TJ, et al. Quantitative Immunophenotype (QUIP) Pattern of CD34high, CD13high and CD33low Identifies Patients with Acute Myelogenous Leukemia (AML) with inv(16). Abstract no 457. Presented at annual meeting of the American Society of Hematology 2001.
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Announcement
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