Immunophenotyping of acute leukemia and lymphoproliferative disorders: a consensus proposal of the European LeukemiaNet Work Package 10

Béné, Marie C.; Nebe, Thomas; Bettelheim, Peter; Buldini, Barbara; Bumbea, Horia; Kern, Wolfgang; Lacombe, Francis; Lemez, P; Marinov, Iuri; Matutes, Estella; Maynadié, Marc; Oelschlägel, Uta; Órfão, Alberto; Schabath, Richard; Solenthaler, Max; Tschurtschenthaler, G.; Vlădăreanu, Ana-Maria; Zini, Gina; Fauré, G; Porwit, Anna

doi:10.1038/leu.2010.312

Cited by 175 publications

(102 citation statements)

References 76 publications

(72 reference statements)

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“…Analysis was performed with CXP software (Beckman Coulter). Lymphocytes were first gated on a CD45/Side Scatter (SS) histogram (28). Normal T-cells (CD5þ, CD19-) and clonal CLL B-cells (CD5þ, CD19þ) were then gated on the CD5/CD19 biparametric histogram (Fig.…”

Section: Flow Cytometrymentioning

confidence: 99%

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

Rizzo

Bouvier

Youlyouz-Marfak

et al. 2012

Cytometry Part B Clinical

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Flow cytometry is the reference technique for assessing ZAP70 expression, a marker of poor prognosis in CLL. One of the most common methods is to assess ZAP70 levels in CLL cells by calculating the ratio between ZAP70 mean fluorescence intensities (MFIs) in residual T-cells and CLL B-cells (ZAP70 T/B ratio). In this study, we developed a new method for ZAP70 labeling. Cells were labeled with a combination of anti ZAP70 phycoerythrin-conjugated SBZAP monoclonal antibody (mAb) and mAbs against CD45, CD19, and CD5. The latter three were used to specifically gate on different lymphocyte subsets. Staining was performed in absence (test) or in presence of excess unconjugated SBZAP mAb (isoclonic control). A so-called ZAP70 isoclonic ratio between SBZAP MFIs in the test and isoclonic control was calculated. A series of 32 patients with CLL and 10 normal controls were studied. Prediction of IGHV mutation status by ZAP70 isoclonic and T/B ratios was similar. By using the ZAP70 isoclonic ratio, we showed that ZAP70 expression was increased in T-cells from CLL patients. Nearly all cases with increased ZAP70 expression in CLL cells were associated with high ZAP70 expression in cognate T-cells. Therefore, the ZAP70 isoclonic ratio was more likely to closely reflect the biology of ZAP70 dysregulation rather than the T/B ratio. These results also explained why ZAP70 T/B ratios were artefactually close to normal in cells from CLL patients with high levels of ZAP70. V C 2012 International Clinical Cytometry Society

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Section: Flow Cytometrymentioning

confidence: 99%

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

Rizzo

Bouvier

Youlyouz-Marfak

et al. 2012

Cytometry Part B Clinical

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“…39 The European LeukemiaNet group has published recommendations regarding standardized pre-analytical steps and antibody combinations for the flow cytometric evaluation of acute leukemia. 40 However, even the use of standardized procedures may not eliminate small variations in fluorescence intensities, instrument performance and immunophenotypic profiles across different laboratories. 41 …”

Section: Limitations Of Multi-color Flow Cytometry-based Mrd Detectionmentioning

confidence: 99%

Multi-color flow cytometric immunophenotyping for detection of minimal residual disease in AML: past, present and future

Jaso

Wang

Jorgensen

et al. 2014

Bone Marrow Transplant

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Current chemotherapeutic regimens achieve CR in a large percentage of patients with AML. However, relapse after CR remains a significant problem. The presence of leukemic cells at levels too low to be detected by conventional microscopy, termed minimal residual disease (MRD), has been associated with an increased risk of relapse and shortened survival. Detection of MRD requires the use of highly sensitive ancillary techniques. Multi-color flow cytometric immunophenotyping is a sensitive method for quick and accurate detection of MRD. Use of this method in patient management may result in lower rates of relapse and improved survival, and is an effective means of assessing novel therapeutic agents. This method can be used in the vast majority of patients with AML, regardless of the immunophenotypic, cytogenetic and molecular genetic abnormalities present. Unfortunately, conflicting data regarding optimum methods of measurement and reporting, as well as the expertize required to interpret results have limited broad application of this technique. We provide a broad overview of this technique, including its advantages and limitations, and discuss the methods employed at our institution. We also review several possible areas of future investigation. INTRODUCTION AML is characterized by clonal expansion of myeloid precursors in the BM, peripheral blood and/or extramedullary tissues. 1 Current treatment regimens achieve CR in the majority of adult patients; however, approximately 65% of patients develop relapsed disease. 2,3 Thus, there is a need to effectively identify which patients are at most risk for impending relapse. Low levels of leukemic cells, termed minimal residual disease (MRD), have been shown to correlate with an increased risk of relapse and shortened survival believed to be a major cause of relapse. 4 These cells are present at levels below the sensitivity of conventional microscopic examination and as such, their detection requires the use of sensitive ancillary techniques. Detection of MRD by multi-color flow cytometric immunophenotyping (MFC) has been shown to be useful in the detection and characterization of MRD. However, several important concepts must be understood in order to ensure optimal application of this technique in both the clinical and research settings so that the information provided can be translated into better patient outcomes.

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“…12 All these lymphoproliferative disorders rely heavily on immunophenotyping to characterize their lineage, apply proper scoring and define light chain restriction lineage. 13 These diseases are being diagnosed with increasing frequency, quite often fortuitously in elderly people hospitalized for a fall, a stroke or other unrelated reasons. Follow up after initiation of therapy is also increasingly benefitting from the efficient application of flow cytometry.…”

mentioning

confidence: 99%

“…All subtypes of MDS and AML are reported as possibly therapy-related and, therefore, classical morphological and immunophenotypic diagnostic criteria apply. 13,16 Many patients will present in the stage of overt AML that differs from de novo AML primarily by the high incidence of trilineage involvement, difficulty in classification, frequent cytogenetic abnormalities and poor response to antileukemic therapy. The bone marrow is reported as hypercellular in up to 52% of patients, normocellularity or hypocellularity may occur at diagnosis, while marrow fibrosis is reported in approximately 15% of patients.…”

mentioning

confidence: 99%

Morphology and immunophenotyping issues in the integrated diagnosis of hematologic disorders of elderly patients

Zini

Béné²

2014

Haematologica

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Immunophenotyping of acute leukemia and lymphoproliferative disorders: a consensus proposal of the European LeukemiaNet Work Package 10

Cited by 175 publications

References 76 publications

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B‐cells due to ZAP70 overexpression in patient T‐cells

Multi-color flow cytometric immunophenotyping for detection of minimal residual disease in AML: past, present and future

Morphology and immunophenotyping issues in the integrated diagnosis of hematologic disorders of elderly patients

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