Multiparameter Flow Cytometry Identifies Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes: Correlation with Cytomorphology and Cytogenetics.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2376-2376
Author(s):  
Wolfgang Kern ◽  
Daniela Voskova ◽  
Claudia Schoch ◽  
Wolfgang Hiddemann ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Median Number ◽  
Multiparameter Flow Cytometry ◽  
Strong Correlations ◽  
Trisomy 8 ◽  
Prognostic Information ◽  
Accurate Identification ◽  
Hla Dr

Abstract Myelodysplastic syndromes (MDS) are diagnosed and classified based on cytomorphology (CM) and cytogenetics. The accurate identification of dysmyelopoiesis and quantification of bone marrow blasts requires a high degree experience in CM. Multiparameter flow cytometry (MFC) has been shown capable of identifying dysplastic features in all lineages and to reveal prognostic information in MDS. We analyzed 23 bone marrow samples from patients with untreated MDS by MFC, CM, and cytogenetics in parallel. In addition, four normal bone marrow samples and one bone marrow sample with hereditary sideroachrestic anemia (HSA) were analyzed by MFC for comparison. Diagnoses included RA (n=4), RARS (n=1), RAEB-1 (n=7), RAEB-2 (n=6), 5q- syndrome (n=2), and CMML (n=2). CM revealed dysgranulopoiesis and dyserythropoiesis in 16 and 14 cases, respectively, and blasts counts ranging from 3% to 20% (median, 9%). Karyotype aberrations included 5q- (n=3); trisomy 8 (n=2); del(11q) (n=2); t(3;11) (n=1); loss of chromosome X (n=1); and trisomy X, trisomy 8, and 5q- (n=1); while n=13 cases had normal karyotypes. Blast counts as determined by MFC ranged from 1.9% to 14.8% (median, 4.7%; correlation with CM: r=0.466, p=0.029). The median number of aberrant features detected by MFC were 4 for blasts (range, 2 to 9), 3 for granulocytes (1 to 8), 3 for monocytes (0 to 7), and 0 for erythrocytes (0 to 2). The presence of dysgranulopoiesis (CM) correlated with hypogranulation (MFC; 50% vs. 17%, p>0.05) and expression of HLA-DR (38% vs. 17%, p>0.05) and CD7 (13% vs. 0%, p>0.05) in granulocytes as well as with the lack of CD16 expression (63% vs. 0%, p=0.035), the expression of CD2 (25% vs. 0%, p>0.05), and the dim expression of CD66 (63% vs. 40%, p>0.05) on monocytes. Combining these parameters led to strong correlations between dysgranulopoiesis (CM) and at least one dysplastic feature by MFC for granulocytes (75% vs. 17%, p=0.023) and monocytes (81% vs. 33%, p=0.054). Combining the granulocytic and the monocytic MFC scores revealed dysplastic features in 100% cases with dysgranulopoiesis (CM) and in 50% without (p=0.013). No clear-cut correlations were found between dyserythropoiesis (CM) and dysplastic features in erythroid cells as detected by MFC. The most striking MFC features of cases with 5q- syndrome included CD2-positivity (100% vs. 6%, p=0.003) and CD34-positivity (100% vs 28%, p=0.042) in monocytes as well as hypogranulation (100% vs. 32%, p=0.055) and HLA-DR-positivity (100% vs 21%, p=0.023) in granulocytes. Dysplastic features that were detected by MFC were related to each other. Thus, in blasts the expression of CD56 was strongly related to the expression of CD64 (p=0.0003) and the dim expression of CD45 (p=0.0003). In addition, the expression of CD11b was associated with the expression of CD15 (p=0.001). In granulocytes, the lack of CD33 was associated with a lack of CD64 expression (p=0.0004). In monocytes, the dim expression of CD45 was associated with a lack of CD14 expression (p=0.0005). The case with HSA displayed an aberrant pattern of CD71 expression. These data suggest that MFC is capable of reproducibly detecting dysplastic features in different compartments of the bone marrow. Strong correlations between CM and cytogenetic finding and MFC are detectable. Future studies will define the role of MFC in optimizing the diagnosis of MDS and the prognostication in patients with MDS.

Application of Multiparameter Flow Cytometry for the Identification of Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2615-2615
Author(s):  
Wolfgang Kern ◽  
Claudia Schoch ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Erythroid Cell ◽  
Multiparameter Flow Cytometry ◽  
Cell Populations ◽  
Aberrant Expression ◽  
Hla Dr ◽  
Cd16 Expression

The diagnosis and classification of myelodysplastic syndromes (MDS) are based on cytomorphology (CM) and cytogenetics. A high degree of experience in CM is required to allow the accurate identification of dysmyelopoiesis and quantification of bone marrow blasts. The identification of dysplastic features in all lineages by multiparameter flow cytometry (MFC) has been shown feasible. To further analyze the potential role of MFC in the diagnostic work-up of MDS we analyzed 224 bone marrow samples from patients with suspected of proven MDS by MFC, CM, and cytogenetics in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 5%) and from 0% to 33% (median, 4%; correlation: r=0.192, p=0.018). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 7), 1 for monocytes (0 to 5), and 0 for erythrocytes (0 to 2). The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (20.5%), CD15 (14.3%) and CD64 (14.3%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (71.4%), aberrant coexpression of CD56 (29.0%), aberrant pattern of CD13/CD16 expression (26.3%), aberrant pattern of CD11b/CD16 expression (25.9%), reduced expression of CD64 (17.0%), and aberrant expression of HLA-DR (14.7%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (31.3%), aberrant coexpression of CD16 (26.3%), an aberrant pattern of CD11b/HLA-DR expression (6.7%), and aberrant coexpression of CD2 (5.8%). The most frequent dysplastic features observed in the erythroid cell populations included an aberrantly strong expression of CD71 and CD235a (23.7%), a lack of CD71 expression (10.7%), and an aberratly homogeneous expression of CD71 (7.1%). The presence of dysplastic features by CM as well as the presence of cytogenetic aberrations tended to be associated with a higher number of dysplastic features by MFC. These data suggest that the identification of dysplastic features by MFC is feasible although there is a large heterogeneity in aberrantly expressed antigens. Thus, a comprehensive panel of antibodies must be applied to allow the detection of dysplasia. Future studies will define the role of MFC in optimizing the diagnosis of MDS in cooperation with CM and cytogenetics.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

Analysis of the Immunophenotypic Features of Blast Cells, Lymphocytes and Granulocytes by Multiparametric Flow Cytometry in Bone Marrow (BM) Cells of Myelodysplastic Syndromes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4843-4843
Author(s):  
Xin Du ◽  
Jing Huang ◽  
Maohua Zhou ◽  
SuXia Geng ◽  
Jianyu Weng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Flow Cytometric Analysis ◽  
Control Group ◽  
Consistent Finding ◽  
Multiparametric Flow Cytometry ◽  
Hla Dr ◽  
Number Of Patients ◽  
Blast Cells

Abstract Abstract 4843 Background The myelodysplastic syndromes (MDS) are a group of clonal heterogeneous bone marrow disorders characterized by peripheral cytopenias, ineffective hematopoiesis, and unilineage or multilineage dysplasia. Multiparametric flow cytometry is increasingly being used as an adjunct to the establishing of MDS. While many antigens have been described to be aberrantly expressed in MDS the findings are generally heterogeneous and there is no consistent finding that would be present in all cases with MDS. Aim To investigate the immunophenotypic features of MDS and non-MDS patients and the characteristic of subtypes of MDS. Methods BM samples were collected from 22 MDS patients including 3 RA (2 male, 1 female, median age 57), 3 RAS (2 male, 1 female, median age 72), 12 RAEB (6 male, 6 female, median age 67.5), 4 MDS-AML (2 male, 2 female, median age 69.5) and 20 non-MDS (11 male, 9 female, median age 32.5, 7 AA, 5 PNH, 3 IDA, 1 ALL, 2 CML, 2 MM). The multiparametric flow cytometric analysis was performed using an extensive panel of monoclonal antibodies and using the conventional and secondary gating strategies to analysis the immunophenotypic features of BM cells. Results This study showed that the proportion of blast cells increased significantly than non-MDS group (P=0.002). As the disease progressing, the percentage of blast cells became higher and significantly difference compared to the non-MDS group (P=0.226, P=0.464, P=0.001 and P=0.000, respectively). The expressions of CD34+ and CD7+ on blast cells were significantly difference between MDS and non-MDS groups (P=0.005, and P=0.002, respectively). Compared with the subtypes of MDS and non-MDS group, the expressions of CD34+ and CD7+ on blast cells became high gradually (P=0.534, P=0.487, P=0.009, P=0.004 and P=0.294, P=0.166, P=0.002, P=0.001) and the high percentage of blast cells and high expression levels of CD34+ and CD7+ might indicate poor prognosis. The expression of CD7+ on lymphocytes was similar with CD34+ and CD7+ on blast cells, but the expressions of CD19+ and CD56+ on lymphocytes were no significantly difference (P=0.076, P=0.252, respectively). The expressions of antigens on granulocytes showed that the expressions of CD15+CD11b+, CD10+ and HLA-DR were significantly difference between MDS and non-MDS groups(P=0.000, P=0.009 and P=0.007, respectively), meanwhile, as the disease progressing, the expression rates of CD15+CD11b+, CD10+ and HLA-DR in subtypes of MDS increased gradually and the survival time of these patients who had over-expression of these antigens was shorter than control group(P=0.002). However, the expressions of CD33+, CD13+, CD56+ and CD15+CD11b- were significantly difference between subtypes of MDS and non-MDS group (P=0.059, P=0.588, P=0.063 and P=0.207, respectively). Conclusions Our results showed that using multiparametric flow cytometry to analyze the immunophenotypic features of BM cells could provide clinically useful information for the diagnosis, classification and prognosis of MDS patients. Particularly, the percentage of blast cells, the expression of CD34+ and CD7+ on blast cells, the expression of CD7+ on lymphocytes and the expression of CD15+CD11b+, CD10+ and HLA-DR on granulocytes may provide the much more useful information. However, further studies including larger number of patients with a longer follow-up are necessary to confirm these results. Disclosures No relevant conflicts of interest to declare.

Multiparameter Flow Cytometry in Patients with Suspected Myelodysplastic Syndromes Adds Significant Prognostic Information: A Study on 1,013 Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1722-1722
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Continuous Variable ◽  
The Other ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Information ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Equity Ownership ◽  
Cox Analysis

Abstract Abstract 1722 Backgroundn: Multiparameter flow cytometry (MFC) has been demonstrated capable of identifying aberrant antigen expression related to myelodysplastic syndromes (MDS). The exact role and place of MFC in the diagnostic work-up of patients with suspected MDS, however, remains to be defined. Aim: Evaluation of the diagnostic impact of MFC in relation to cytomorphology (CM) and cytogenetic (CG) by determining the association of MFC results to overall survival (OS). Patients and Methods: In 1,013 patients with suspected MDS bone marrow samples had been analyzed in parallel by MFC, CM, and CG. CM confirmed MDS in 511 patients, excluded MDS in 277 patients, and showed dysplastic features but not sufficient to unequivocally diagnose MDS by CM in 225. The MFC diagnostic result was in agreement with MDS (“MDS by MFC”) in 446 patients including 382/511 patients with MDS proven by CM. CG revealed an aberrant karyotype in 245/1,013 patients. The median follow-up time amounted to 14.8 months, a total of 156 deaths was recorded. Results: The first set of analyses was performed on the cohort of 511 patients with MDS confirmed by CM. The median total number of aberrantly expressed antigens amounted to 3 (range, 0–11) and included expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal (SSC) in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. A higher total number of aberrantly expressed antigens as a continuous variable correlated with a shorter OS (Cox analysis, p=0.008). Next, patients were categorized based on the three parameters i) at least 3 aberrantly expressed antigens, ii) significantly reduced SSC in granulocytes, and iii) >5% myeloid progenitor cells in MFC. Patients with at least one of these criteria had a significantly shorter OS than those without (median 48.5 months vs. not reached (n.r.), p<0.001). Overall, the global diagnostic rating of “MDS by MFC” was the strongest MFC parameter: Patients with “MDS by MFC” had a shorter OS as compared to patients without (median 56.8 months vs. n.r., p=0.001). Non-MFC parameters related to OS in univariable Cox analysis included WBC count, thrombocyte count, CG (grouped according to IPSS), % blasts by CM (p<0.001 each), Hb level (p=0.001), and age (p=0.002). In order to determine the clinical relevance of “MDS by MFC” a multivariable analysis for OS was performed on this parameter together with non-MFC parameters (blood counts excluded due to incomplete data sets). It revealed an independent relation between “MDS by MFC” and OS (p=0.045). This was also true for relation of OS to the other parameters (CG, p<0.001; age, p=0.001, % blasts by CM p=0.014). Given this strong prognostic value of “MDS by MFC” in cases with MDS proven by CM a second set of analyses on the relation between MFC findings and OS were performed for the complete cohort of 1,013 patients, i.e. additionally including all cases with a diagnostic result by CM of “no MDS” or “dysplastic features not sufficient to diagnose MDS”. Again, significant relations to OS was found for the total number of aberrantly expressed antigens as a continuous variable (Cox analysis, p<0.001), for at least one of the above mentioned criteria i), ii) or iii) (median 75.6 months vs. n.r., p<0.001), as well as for “MDS by MFC” (median 60.5 months vs. n.r., p<0.001). Again, “MDS by MFC” proved to be the most relevant MFC parameter. Multivariable Cox analysis for OS including “MDS by MFC” and non-MFC parameters revealed a trend only for “MDS by MFC” (p=0.135) and significance for the other parameters (age, p<0.001; CG, p<0.001; blasts by CM, p=0.045). Conclusions: 1) The present data indicates the diagnostic use of MFC for MDS results in independent prognostic information for cases with MDS as proven by CM. 2) Furthermore, the diagnosis of MDS by MFC has a strong prognostic impact even without prove of MDS by CM which strengthens the diagnostic value of MFC even more. 3) This analysis therefore argues in favour of diagnosing MDS not only based on a combination of CM and CG but of adding also MFC for better classification and even prognostication in the future. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Multiparameter flow cytometry provides independent prognostic information in patients with suspected myelodysplastic syndromes: A study on 804 patients

2015 ◽  
Vol 88 (3) ◽  
pp. 154-164 ◽  
Author(s):  
Wolfgang Kern ◽  
Ulrike Bacher ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Information

scholarly journals Developing a Simple Algorithm Based on Multiparameter Flow Cytometry for Fast Screening of Acute Promyelocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-26
Author(s):  
Vitória Ceni Ceni ◽  
Katia B Pagnano ◽  
Gislaine B O Duarte ◽  
Marina DB Pellegrini ◽  
Bruno Kosa Duarte ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Discriminant Analysis ◽  
Acute Promyelocytic Leukemia ◽  
Simple Algorithm ◽  
Diagnostic Algorithm ◽  
Promyelocytic Leukemia ◽  
Advisory Board ◽  
Multiparameter Flow Cytometry ◽  
Fast Screening ◽  
Hla Dr

Introduction: Acute promyelocytic leukemia (APL) is a genetically and molecularly well-defined type of acute leukemia that is curable but has a frequent early mortality due to bleeding. So, there is a need for a fast diagnostic screening in order to start appropriate therapy. Multiparameter flow cytometry (MFC) is usually performed in all types of acute myeloid leukemias (AMLs) but only few features have been described as characteristic of APL. Aim: to develop a diagnostic algorithm based on the intensity of expression of several antigens examined by MFC in AML that could reliably discriminate between APL and the other types of AML. Material and Methods: Consecutive newly diagnosed AMLs treated in our Institution during the last 2 years entered the study. Immunophenotyping was included in the diagnostic workup. An 8-color platform based on the Euroflow recommendations was used. The mean fluorescence intensity (MFI) of each antigen tested was assessed and those best discriminating between APL and all other types of AML were obtained by a discriminant analysis. Phenotypic characteristics of normal myeloblasts taken from examinations of bone marrow (BM) MFC performed for the diagnosis of cytopenias were used as controls. Results: 24 cases of APL and 56 cases of other primary AML entered the study. Median age: 39 (23-56) and 62(26-81) years respectively. Concerning ELN risk groups of non-APL cases, 13 were favorable risk, 26 were intermediate and 09 were adverse risk. In 8 cases risk assessment was not possible due to the absence of cytogenetics. Moreover, among APL patients, 7 cases had a FLT3-ITD mutation. Among non-APL AMLs, 4 had FLT3-ITD mutation, 4 had NPM1 and 10 had FLT3-ITD and NPM1mutation. Concerning antigen expression, CD34 was expressed in only 1/24 APL samples, and in 18/56 samples from non-APL AMLs. The following flow features were differentially expressed in both groups: SSC (p &lt;0.0001), CD45 (p=0.02), CD13 (p=0.001), CD64 (p=0.004), HLA-DR (p&lt;0.0001) and CD33 (p&lt;0.0001) (Table 1). In the discriminant analysis, MFI CD34 and MFI HLA-DR were able to accurately classify APL and non-APL AML in only 62.5%. However, after the addition of the ratio of SSC between blasts and lymphocytes, these 3 parameters were able to differentiate APL from non-APL AML in 91.2% of the cases. Conclusion: MFC was adequate for a fast screening of APL in most cases. Expression of CD34 was not very useful, as many AMLs do not express this antigen, similar to APL, but SSC, together with HLA-DR could discriminate both types of leukemia in most cases. Disclosures Pagnano: Astellas: Other: Advisory Board and lecture; Novartis: Other: Advisory Board; Pintpharma: Other: Lecture; EMS: Other: Lecture. Duarte:Janssen: Other: Lecture; Astellas: Other: Lecture.

scholarly journals Usefulness of Dual Immunohistochemistry Staining in Detection of Hairy Cell Leukemia in Bone Marrow

2019 ◽  
Vol 153 (3) ◽  
pp. 322-327 ◽  
Author(s):  
Gaurav K Gupta ◽  
Xiaoping Sun ◽  
Constance M Yuan ◽  
Maryalice Stetler-Stevenson ◽  
Robert J Kreitman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Hairy Cell Leukemia ◽  
Predictive Value ◽  
Lymphoid Cells ◽  
Multiparameter Flow Cytometry ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Flow Cytometry Data ◽  
Flow Cytometric Data

Abstract Objectives We evaluated efficacy of two dual immunohistochemistry (IHC) staining assays in assessing hairy cell leukemia (HCL) involvement in core biopsies and compared the results with concurrently collected flow cytometric data. Methods Overall, 148 patients with HCL (123 male, 25 female; mean age: 59.8 years; range: 25-81 years) had multiparameter flow cytometry performed using CD19, CD20, CD22, CD11c, CD25, CD103, CD123, surface light chains, CD5, and CD23. In parallel, bone marrow IHC was done using PAX5/CD103 and PAX5/tartrate-resistant alkaline phosphatase (TRAP) dual IHC stains. Results Overall sensitivity of dual IHC stains was 81.4%, positive predictive value was 100%, and negative predictive value was 81.7%. All IHC-positive cases concurred with flow cytometry data, even when HCL burden was extremely low in the flow cytometry specimens (as low as 0.02% of all lymphoid cells). Conclusions Dual IHC stain is a sensitive tool in detecting HCL, even in cases with minimal disease involvement.

Use of 5-Fold Staining for Multiparameter Flow Cytometry-Based Quantification of Minimal Residual Disease in Acute Myeloid Leukemia Results in Improved Sensitivity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2025-2025
Author(s):  
Wolfgang Kern ◽  
Daniela Voskova ◽  
Claudia Schoch ◽  
Wolfgang Hiddemann ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Normal Bone Marrow ◽  
Prognostic Information ◽  
Normal Bone ◽  
Triple Staining ◽  
Marrow Cells

Abstract The quantification of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) using triple staining has been shown to yield prognostic information independent of other parameters in patients with acute myeloid leukemia (AML). Due to the immunophenotypic heterogeneity of AML the application of 5-fold staining may result in a better characterization of the leukemia-associated aberrant immunophenotype (LAIP) and thus in an improved sensitivity of the method as compared to triple staining. We analyzed bone marrow samples from 114 patients with newly diagnosed and untreated AML by MFC using a comprehensive antibody panel with 5-fold combinations. Sensitivity was estimated by quantification of LAIP-positive cells for each LAIP in 18 normal bone marrow samples. In each patient at least one LAIP was identified (total, 203 LAIPs). The LAIPs were present on a median of 15.88% of the bone marrow cells at diagnosis (range, 2.11% to 79.64%). The median number of normal bone marrow cells displaying the LAIPs ranged from 0.001% to 0.065% (median, 0.010%). As a result, the logarithmic difference (LD) in LAIP-positive cells between leukemic and normal bone marrow amounted to a median of 3.33 (range, 1.96 to 4.88). Similarly, if only the most sensitive LAIP was considered for each patient the median frequencies of LAIP-positive cells were 14.07% (range, 2.11% to 77.57%) in leukemic bone marrow and 0.010% (range, 0.001% to 0.065%) in normal bone marrow. Importantly, however, in this setting the resulting LD amounted to a median of 3.45 (range, 1.96 to 4.88). In order to estimate the impact of applying 5-fold staining on the sensitivity the information of each of the applied colors was skipped once while the results of the other four colors, respectively, were used. Skipping one color resulted in an increase of LAIP-positive normal bone marrow cells (median, 0.050%; range, 0.001% to 3.6%) while the percentages of LAIP-positive leukemic cells changed only marginally (median, 22.65%; range, 2.25% to 90.06%). The gain in LD by applying 5-fold staining in comparison to 4-fold staining amounted to a median of 0.58 (maximum gain, 3.14). In 32 patients a total of 120 follow-up samples have been analyzed appyling the combination of antibodies that allowed the best LAIP definition. The LD from diagnosis to follow-up amounted to a median of 2.82 (range, 0.77 to 4.82). Clinical follow-up data is available in 26 of these 32 patients. MRD assessment after completion of consolidation therapy has been performed in 15 patients. The median LD between diagnosis and follow-up assessment is 2.84 (range, 1.07 to 4.33). Separating patients according to this median LD identified a group of patients with no relapses yet (LD >2.84) while patients with an LD <2.84 had an event-free survival of only 50% at one year (p=0.075). These data confirm that flow cytometrically-based assessment of MRD is feasible in AML and results in prognostic information. It is suggested that the application of 5-fold staining significantly improves the sensitivity and thereby the overall accuracy of the method.

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