Immunophenotyping of acute leukemia by flow cytometry: Use of CD45 and right angle light scatter to gate on leukemic blasts in three color analysis

Department of Pathology, Duke University Medical Center, Durham, North Carolina.
American Journal of Clinical Pathology (Impact Factor: 2.51). 12/1993; 100(5):534-40.
Source: PubMed


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. When CD45-peridin chlorophyll alpha protein was combined with other pairs of fluorescein isothiocyanate- and phycoerythrin-conjugated reagents, it was possible to set an analysis window on the leukemic blasts and display dual-parameter (ie, green vs. red fluorescence) data regarding expression of two additional markers on the leukemic population. This gating strategy was superior to traditional forward-angle versus RALS displays in that it did a better job of isolating the leukemic cells analytically.

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    • "The data analysis was performed using FCS Express software (De Novo Software, Los Angeles, CA, USA, Initial cell subpopulations were established using the levels of CD45 expression and side-scatter (SSC) properties [21,22]. After defining immunophenotypes of leukemic cells, antibodies for CD45, CD34, CD117, CD33, HLA-DR, CD64 or CD14 (Becton Dickinson, NJ) were used to select cells of interest to determine fluorescence levels of bound aptamers for individually gated subpopulations. "
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    ABSTRACT: The majority of patients with acute myelogenous leukemia (AML) still die of their disease. In order to improve survival rates in AML patients, new strategies are necessary to discover biomarkers for the detection and targeted therapy of AML. One of the advantages of the aptamer-based technology is the unique cell-based selection process, which allows us to efficiently select for cell-specific aptamers without knowing which target molecules are present on the cell surface. The NB4 AML cell line was used as the target cell population for selecting single stranded DNA aptamers. After determining the affinity of selected aptamers to leukocytes, the aptamers were used to phenotype human bone marrow leukocytes and AML cells in clinical specimens. Then a biotin-labelled aptamer was used to enrich and identify its target surface protein. Three new aptamers were characterized from the selected aptamer pools (JH6, JH19, and K19). All of them can selectively recognize myeloid cells with Kd in the low nanomole range (2.77 to 12.37 nM). The target of the biotin-labelled K19 aptamer probe was identified as Siglec-5, a surface membrane protein in low abundance whose expression can serve as a biomarker of granulocytic maturation and be used to phenotype AML. More importantly, Siglec-5 expression can be used to detect low concentrations of AML cells in human bone marrow specimens, and functions as a potential target for leukemic therapy. We have demonstrated a pipeline approach for developing single stranded DNA aptamer probes, phenotyping AML cells in clinical specimens, and then identifying the aptamer-recognized target protein. The developed aptamer probes and identified Siglec-5 protein may potentially be used for leukemic cell detection and therapy in our future clinical practice.
    Journal of Hematology & Oncology 01/2014; 7(1):5. DOI:10.1186/1756-8722-7-5 · 4.81 Impact Factor
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    • "Leukaemic cells were identified based on low expression of CD45 and low side scatter (SSC) properties (CD45low/ SSClow) (Borowitz et al, 1993; Lacombe et al, 1997). As differentiated monocytes express both hMICL and CD123, CD14 de-selection was performed except for cases with CD14+ AML. "
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    ABSTRACT: Real-time quantitative polymerase chain reaction (qPCR) has been extensively validated for the detection of minimal residual disease (MRD) in acute myeloid leukaemia (AML). Meanwhile, multicolour flow cytometry (MFC) has received less attention because the so-called leukaemia-associated immunophenotypes (LAIPs) are generally of lower sensitivity and specificity, and prone to change during therapy. To improve MRD assessment by MFC, we here evaluate the combination of human Myeloid Inhibitory C-type Lectin (hMICL, also termed C-type lectin domain family 12, member A, CLEC12A) and CD 123 (also termed interleukin-3 receptor alpha, IL3RA) in combination with CD34 and CD117 (KIT), as an MRD assay in pre-clinical and clinical testing in 69 AML patients. Spiking experiments revealed that the assay could detect MRD down to 10(-4) in normal bone marrow with sensitivities equalling those of validated qPCR assays. Moreover, it provided at least one MFC MRD marker in 62/69 patients (90%). High levels of hMICL/CD123 LAIPs at the post-induction time-point were a strong prognostic marker for relapse in patients in haematological complete remission (P < 0·001). Finally, in post induction samples, hMICL/CD123 LAIPs were strongly correlated (r = 0·676, P = 0·0008) to applied qPCR targets. We conclude the hMICL/CD123-based MFC assay is a promising MRD tool in AML.
    British Journal of Haematology 10/2013; 164(2). DOI:10.1111/bjh.12614 · 4.71 Impact Factor
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    • "Thus, a pre-selection of cells based on their low side scatter (SSC, i.e. their content of organelles capable of scattering the laser light) and their CD45 expression is often employed in multicolour assays. The latter surface marker has served as a model for protein isoforms generation by alternative splicing (Oberdoerffer et al, 2008) and is usually, but by no means always, expressed in decreased amounts on leukaemic cells (Borowitz et al, 1993; Lacombe et al, 1997; Miyachi et al, 1999). As will be seen from Fig 1, the CD45low/SSClow can be an important way of focusing on the AML blasts and has been proven to significantly increase the sensitivity of the MFC approach (Kern et al, 2004). "
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    ABSTRACT: The concept of minimal residual disease in acute myeloid leukaemia has been steadily developed pre-clinically, with quantitative polymerase chain reaction (qPCR) leading the way with highly validated assays for patient-based risk stratification at post-treatment time points, which are being integrated in clinical trials both at evaluation of first complete remission (CR1) and after attaining CR1. Moreover, multicolour flow cytometry (MFC) has been increasingly employed in identifying leukaemia-associated immunophenotypes (LAIPs) with significant progress being made in standardization. In translating these widely varying methodologies to parameters useful for individualized patient decision-making, one of the obstacles has been that the assays entail varying sensitivities dependent on a number of variables. For qPCR, sensitivity depends on target type (i.e. fusion transcript, mutated gene or even overexpressed gene) and - in the case of overexpressed genes - on expression in healthy haematopoiesis. For MFC, sensitivity is likewise largely a function on whether the same phenotype is seen in normal immature cells and, in addition, antigen drift/shift with LAIPs changing at relapse is a well-known problem. In considering which sensitivity to opt for, a further variable is the situation of patient, most importantly the level of cytoreduction intended. Here we will attempt to give an overview of these pertinent questions intended for the practicing haematologist, focusing on where the field is heading at the clinical level.
    British Journal of Haematology 06/2012; 158(5):569-80. DOI:10.1111/j.1365-2141.2012.09203.x · 4.71 Impact Factor
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