scholarly journals Quantitative flow cytometry for the differential diagnosis of leukemic B-cell chronic lymphoproliferative disorders

2000 ◽  
Vol 64 (4) ◽  
pp. 275-281 ◽  
Author(s):  
Giovanni D'Arena ◽  
Pellegrino Musto ◽  
Nicola Cascavilla ◽  
Matteo Dell'Olio ◽  
Nicola Di Renzo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cell ◽  
Lymphoproliferative Disorders ◽  
Quantitative Flow

scholarly journals Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study

2021 ◽  
Author(s):  
Sebastian Böttcher ◽  
Robby Engelmann ◽  
Georgiana Grigore ◽  
Paula Carolina Fernandez ◽  
Joana Caetano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Diagnostic Algorithm ◽  
Lymphocytic Leukemia ◽  
B Cell Lymphomas ◽  
Multicentric Study ◽  
Predictive Values ◽  
Large B Cell

Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing nine disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis we subsequently utilized Canonical Correlation Analysis of 176 training cases to project the multi-dimensional space of all 26 immunophenotypic parameters into 36 two-dimensional plots for each possible pair-wise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%) and mantle cell lymphoma (95.4%). Burkitt and CD10+ diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD10- diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms.

Substantially High Frequency of Chromosome 7q Interstitial Deletion in B-Cell Lymphoproliferative Disorders.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2006-2006
Author(s):  
Gary Lu ◽  
John Zhang ◽  
Katherine Latorre ◽  
Michele Hibbard ◽  
Sing-Tsung Chen
Keyword(s):  
Flow Cytometry ◽  
Patient Care ◽  
B Cell ◽  
Hematological Malignancies ◽  
B Cell Lymphoma ◽  
Disease Diagnosis ◽  
Test System ◽  
Lymphoproliferative Disorders ◽  
Interstitial Deletion ◽  
Chromosome 7Q

Abstract It has been well known that cytogenetics study plays an important role in cancer patient care, in particular in the diagnosis, treatment and prognosis of hematological malignancies. The MCIM test system, a combination of studies of cell morphology (M), cytogenetics (C), immunophenotype (I) and molecular genetics (M), has become a useful test system in hematologic disease diagnosis. It provides physicians more comprehensive and accurate diagnostic laboratory information. Deletions in the long arm of chromosome 7 are among the most common abnormalities observed in hematologic disorders. These changes were usually viewed as markers for myeloid malignancies, in particular myelodysplastic syndrome and acute myeloid leukemia. Recently, interstitial 7q deletions, a subset of the 7q deletions, have been found to be associated with B-cell lymphoproliferative disorders (LPDs). The correlation of narrow 7q deletions with specific subsets of B-cell LPDs has been extensively studied. However, the frequency of interstitial 7q deletions associated with B-cell LPDs has not been well investigated. We recently studied 7q deletions collected from our clinical cases done by the MCIM system at US Labs over the past two years. Deletions of chromosome 7q were observed in 85 of 19,483 cases. Interstitial 7q deletions were detected in 46 of the 85 cases with 7q deletions. In combination with the findings of flow cytometry, interstitial 7q deletions were found to be associated with B-cell LPDs in 10 out of the 46 cases, accounting for 21.7% of all the observed interstitial 7q deletions and 11.8% of all the 7q deletions. The B-cell LPDs associated with 7q interstitial deletion are diverse, including hairy cell leukemia, atypical chronic lymphocytic leukemia, splenic marginal zone lymphoma, and large B-cell lymphoma. Interestingly, the deleted region in the 10 cases with B-cell LPDs in this study was solely confined to 7q22-32. This is so far, to our knowledge, the largest series reported in the literature. Our investigation showed that the frequency of 7q interstitial deletions associated with B-cell LPDs is substantially high. We conclude that (1) association of B-cell LPDs must be taken into account when an interstitial 7q deletion is observed, in particular when the deleted region is confined to 7q22-32; (2) together with the flow cytometry analysis in the MCIM system within a clinical laboratory diagnostic facility, cytogenetics study can provide clinicians more accurate diagnostic information, thus strengthening patient care by improving the management of hematological malignancies. (The authors are grateful to Deanna Collins for her cooperation to collect the data in this study.)

Multiparameter Flow Cytometry (MFC) for Identification of the Waldenström's Clone in IgM MGUS and Waldenstrom's Macroglobulinemia (WM): New Criteria for Differential Diagnosis and Risk Stratification

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 936-936
Author(s):  
Bruno Paiva ◽  
Maria-Carmen Montes ◽  
Ramón García-Sanz ◽  
Jennifer Alonso ◽  
Natalia de las Heras ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Conflicts Of Interest ◽  
Phenotypic Characterization ◽  
International Prognostic Scoring System ◽  
Multiparameter Flow Cytometry ◽  
Risk Of Progression

Abstract Abstract 936 Demonstration of bone marrow (BM) infiltration by lymphoplasmacytic lymphoma is essential to the diagnosis of WM, and a trephine biopsy is considered mandatory for this assessment. Multiparameter flow cytometry (MFC) has demonstrated its clinical relevance in MGUS and myeloma; however, immunophenotypic studies on IgM monoclonal gammopathies are scanty, and focus only in patients with WM. Herein, MFC immunophenotyping was performed on BM samples from 244 patients, including 67 IgM MGUS, 77 smoldering, and 100 symptomatic WM newly diagnosed patients according to the Second International Workshop. A four color panel that systematically allowed the identification of B cells and plasma cells (PC), and their phenotypic characterization for a total of 24 antigens was used. We first analyzed the percentage of B cells and PC in BM and the percentage of light chain restricted cells in both compartments. Our results show a progressive increment of B cells from IgM MGUS to smoldering and symptomatic WM (medians of 2%, 9% and 12%; P<.001), as well of light chain restricted B cells (75%, 96% and 99%; P<.001). In contrast, no differences were found for the percentage of PC (median of 0.3%), but light chain restricted PC progressively increased from IgM MGUS to smoldering and symptomatic WM (70%, 85% and 97%; P<.001). Accordingly, only 1% of IgM MGUS patients showed >10% B cells, in contrast to 34% and 55% of smoldering and symptomatic WM (P<.001). Likewise, only 1% of IgM MGUS patients showed 100% light chain restricted B cells, in contrast to 19% and 40% of smoldering and symptomatic WM (P<.001); similar results being also found using a cutoff of 100% light chain restricted PC. Subsequently, we explored whether the percentages of BM and light chain restricted B cells and PC could predict time to progression (TTP) from smoldering into symptomatic WM, as well as overall survival (OS) in symptomatic WM. In smoldering WM, B cells (>10% vs ≤10%: median TTP of 47m vs 145m; P=.016) and light chain restricted B cells (100% vs <100%: 26m vs 145m; P<.001) but not PC, predicted risk of progression. On the multivariate analysis that included serum M-spike (±3g/dL), BM infiltration (±50% lymphoplasmacytic cells), BM B-cells and light chain restricted B cells (by MFC), only the later retained independent prognostic value (HR: 19.8, P=.001). Upon analyzing factors influencing survival in symptomatic WM patients, cases with >10% B cells showed a trend for inferior OS (P=.080), and significant differences emerged when comparing patients with 100% vs <100% light chain restricted B cells (median OS 44m vs 78m; P=.001). The later marker was independent (HR: 2.6; P=.004) of the International Prognostic Scoring System (HR: 2.2; P=.006). Focusing on the antigenic profiles of B cells and PC, we noted that within the B-cell compartment there was a progressive increment of CD22dim (69%, 92% and 88%; P<.001), CD25+ (61%, 88% and 90%; P<.001) and sIgM+ (88%, 95% and 97%; P=.002) B cells from IgM MGUS to smoldering and symptomatic WM. This underlies that the accumulating light chain restricted clonal B cells show a characteristic Waldenstrom's phenotype (CD22dim/CD25+/IgM+). Of note, a bimodal (from - to +) expression for the B cell memory marker CD27 was found in >50% of WM patients, which raises the possibility that the WM clone may arise, at least in some cases, before antigenic stimulation; subsequent maturation of the clone into PC would explain the typical presence of somatic hypermutations. On the other hand, B-cells from IgM MGUS and WM patients were negative in ≥90% of cases for CD5, CD10, CD11c and CD103, which can be useful to differentiate between WM and other B-NHL. Finally, the antigenic profile of PC in IgM MGUS and WM was similar to that of normal PC, and different from myeloma PC by consistently showing a CD27+ and CD56- phenotype, in addition to sIgM+ expression in ≥87% of all cases. Similarly to B-cells, we also noted that within the PC compartment there was a progressive increment of CD19+, CD45+ and sIgM+ CD20+ PC from IgM MGUS to smoldering and symptomatic WM. This underlies that this transition is asssociated with an accumulation of light chain restricted clonal PC displaying an immature/plasmablastic phenotype. In summary, our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM. Disclosures: No relevant conflicts of interest to declare.

Multicentric Analyses of the CD148, CD180, and CD200 Combination for the Diagnosis of Mature B-Cell Neoplasm Using Flow Cytometry

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2662-2662 ◽  
Author(s):  
Laurent Miguet ◽  
Luc Fornecker ◽  
Marie Wyrwas ◽  
Sarah Cianferani ◽  
Raoul Herbrecht ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cell ◽  
Expression Profile ◽  
Standard Error ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Strong Expression ◽  
Group A ◽  
Weak Expression

Abstract Introduction Diagnosis of mature B-cells proliferations, especially those involving the spleen, do not always falls into any of the WHO types of B-cell neoplasms using standart diagnosis tools. This situation in notably encountered in the case of the differential diagnosis of marginal zone lymphoma (MZL), atypical chronic lymphocytic leukemia (aCLL), mantle cell lymphoma (MCL), and lymphoplasmacytic lymphoma (LPL), mostly due to the lack of immunological positive markers. In order to find new markers to discriminate between these different malignancies, we have previously developed a proteomic strategy based on the analyses of plasma membrane microparticles and proposed two new specific markers: CD148 and CD1801,2 for MCL and MZL respectively. The simultaneous use of these two markers, together with the CD200 that is positive in most cases of CLL and negative in MCL could be of great interest to better assess the differential diagnosis. Methods Flow cytometry analyses have been realized in Nancy and Strasbourg hospitals by combining these three markers: CD148 (Clone 143-41 FITC); CD180 (Clone G28.8 PE) and CD200 (Clone OX104 APC). Expression profile of these proteins was established on a well characterized set of patients (N=287): CLL with a Matutes score > 3 (N=81); MCL harboring t(11;14) translocation or CCND1 overexpression (N=44); LPL (N=58) classified following cytological morphology, IgM peak and the positivity of CD38 and/or Myd88 mutation, MZL (N=84), displaying a CD5- CD23- immunophenotype associated to a splenomegaly and 20 controls. For each group the mean of fluorescence intensity and Standard Error have been determined. Results MCL exhibited a strong expression of CD148 combined with a weak expression of CD180 and CD200. A weak expression of CD148 and CD180 coupled to a strong expression of CD200 was typical of the CLL group and a weak expression of CD148 and CD200 coupled to a strong expression of CD180 was observed in the MZL group. A moderate expression of these three markers was observed in the LPL group. A threshold corresponding to MFI +/- 4 standard error was then calculated for each group, and patients were categorized following the expression profile of these 3 markers (see figures). In this cohort, the above described profiles correctly identified MCL cases with a specificity of 92% and a sensitivity of 64%, aCLL cases with a specificity of 100% and a sensitivity of 47%, LPL cases with a specificity of 90% and a sensitivity of 54% and MZL cases with a specificity of 99% and a sensitivity of 60%. Conclusion These results strongly suggest that the incorporation of these three markers CD148 CD180 and CD200 in addition of the routinely used flow cytometry panel can be helpful in a number of cases for which the diagnosis is difficult. References: 1) Miguet et al leukemia 2013 2) Miguet et al journal of proteome research 2009 Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Quantitative flow cytometric evaluation of CD200, CD123, CD43 and CD52 as a tool for the differential diagnosis of mature B-cell neoplasms

2017 ◽  
Vol 39 (3) ◽  
pp. 252-258 ◽  
Author(s):  
Elissandra Machado Arlindo ◽  
Natália Aydos Marcondes ◽  
Flavo Beno Fernandes ◽  
Gustavo Adolpho Moreira Faulhaber
Keyword(s):  
Differential Diagnosis ◽  
B Cell ◽  
Flow Cytometric ◽  
Flow Cytometric Evaluation ◽  
Quantitative Flow

Proposal For a Novel Scoring System For The Diagnosis Of CLL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4150-4150 ◽  
Author(s):  
Thomas Köhnke ◽  
Veronika K Wittmann ◽  
Daniela Sauter ◽  
Veit Bücklein ◽  
Zlatana Pasalic ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
B Cells ◽  
B Cell ◽  
Sensitivity And Specificity ◽  
Validation Cohort ◽  
Diagnostic Value ◽  
Internal Validation ◽  
Expression Intensity ◽  
Higher Sensitivity

Abstract Background Immunophenotyping is essential for the diagnosis of chronic lymphocytic leukemia (CLL). The scoring proposed in the modified Matutes score has been the basis of diagnosis for the past 15 years and is defined by strong expression of CD5 and CD23, low or absent expression of CD79b, sIgM and FMC7. However, some markers within the current score such as sIgM display a high variability in staining patterns and thus the interpretation of expression intensity is not easily reproducible. Furthermore, the newly identified marker CD200 is not included in the current score in spite of its highly informative value in the differential diagnosis of B-cell disorders. In the study presented here we aimed to improve the current score through the addition of highly informative markers such as CD200 and the omission of sIgM as a less informative, error-prone marker. Methods Between February 2011 and May 2013, peripheral blood or bone marrow aspirates of patients with suspected B-cell lymphoproliferative disorders were subjected to evaluation by flow cytometry. Immunophenotyping was performed using a Navios flow cytometer (Beckman Coulter) and samples were stained by monoclonal antibodies targeting the antigens CD45, CD19, CD5, CD10, CD23, CD79b, CD200, FMC7, sIgM, kappa and lambda. Corresponding isotype controls were used. The modified Matutes score was calculated as described previously (Moreau et al., Am J Clin Pathol 1997) with positivity defined as ≥20% positive cells. Mean Fluorescence Intensity (MFI) ratio (MFI sample/MFI isotype) was calculated as a measure of expression intensity. For our new score, optimized cut-offs for positivity vs. negativity (CD5, CD23, CD200, FMC7) and low or absent expression (CD79b) as well as sensitivity and specificity were calculated by receiver operating characteristics (ROC). The final clinical diagnosis was defined as the diagnosis established by the treating physician taking into account clinical symptoms as well as all results from diagnostic procedures, including cytomorphology, flow cytometry, cytogenetics, molecular genetics and immunohistochemistry, if available. In order to perform an internal validation of our proposed score, we divided the patient cohort into an exploratory and a validation cohort by a 2:1 ratio based on the date of receipt of the samples. Result Flow cytometry data of 371 patients with B-cell disorders were available for analysis. 247 patients were assigned to the exploratory cohort and 124 patients were assigned to the validation cohort. 84.2% and 82.1% of patients, respectively, were diagnosed with CLL. In the exploratory cohort, sIgM-expression intensity on CD19+ B-cells (as measured by MFI ratio) was significantly lower in CLL versus non-CLL cases (p=0.001). However, low or absent sIgM-expression displayed poor specificity in distinguishing CLL from non-CLL cases (51,3%; sensitivity 83,7%). Absent or low CD79b-expression on CD19+ B-cells showed a higher sensitivity and specificity (94.2% and 71.8%, respectively). Positivity for CD200 as well as lack of FMC7-expression showed high diagnostic value (sensitivity and specificity all above 80%). Interestingly, positivity for CD5 on CD19+ B-cells did not have a strong diagnostic value (sensitivity and specificity 69.7% and 76.9%, respectively), but double positivity for CD5 and CD23 on CD19+ B-cells showed higher sensitivity and specificity (79.8% and 87.2%, respectively). Therefore, CD200+, CD23+/CD5+, FMC7- and low or absent CD79b on CD19+ B-cells were included in a new diagnostic score. The resulting score showed comparable sensitivity (97.1% for our score versus 98.6% for the Matutes score, McNemar’s test p=0.38), but markedly increased specificity (87.2% versus 53.8%, p<0.001). These results were confirmed in the internal validation cohort (sensitivity 97.0% versus 100%, p=N/A; specificity 86.4% versus 59.1%, p=0.03). Conclusion The data support the use of the improved score for the differential diagnosis of CLL. This novel scoring system exhibits significantly higher specificity while maintaining very high sensitivity and might therefore contribute to less false positive results. Finally, the surface markers contained in the novel score show more consistent staining patterns, which might further improve reproducibility. External validation of the proposed score will be pursued. Disclosures: No relevant conflicts of interest to declare.

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