Flow Cytometry Immunophenotypic Analysis Research Articles

Advancing Diagnostic Accuracy and Quality of Patient Care Through the Implementation of a Flow Cytometry Quality Assurance Program.

Flow cytometry immunophenotypic analysis plays an important role in the diagnosis, classification, and disease monitoring of hematologic neoplasms. The interpretation of flow cytometry testing can be challenging. To explore ways to improve diagnostic accuracy and in turn enhance the quality of patient care. A flow cytometry quality assurance (QA) program was developed. Cases from various complex flow cytometry panels were randomly selected and cross-reviewed. The outcomes of the QA review were categorized into 3 groups: complete agreement, minor discrepancy, and major discrepancy. Each discrepancy underwent a process of documentation, discussion, and resolution. Here we summarize our 3 years of experience with this program. In total, 6166 cases were evaluated; 6028 cases (97.7%) showed complete concordance, 120 cases (2.0%) showed minor discrepancies, and 18 cases (0.3%) showed major discrepancies. Among the top 5 panels evaluated, the panel evaluating mature T-cell abnormalities showed the highest rate of discrepancy, whereas the panel for evaluation of myelodysplastic syndromes showed the lowest discrepancy rate. When analyzing the trends of concordance and discrepancy over time, we observed a statistically significant decrease in discrepancy rate over time, from 4% at the beginning of the 6-month period to 1.5% in the final 6-month period. The overall concordance rate was 97.7%. The remaining 2.3% of cases showed discrepancies that required a correction, underscoring the value and necessity of having a QA program. The overall discrepancy rates exhibited a gradual decline over time, indicative of the positive impact of the QA program on enhancing diagnostic competency and accuracy over time.

Integrative immunophenotypic and genetic characterization of acute myeloid leukemia with CBFB rearrangement.

We sought to characterize the immunophenotype of acute myeloid leukemia (AML) with CBFB rearrangement and correlate the results with cytogenetic and molecular data. Sixty-one cases of AML with CBFB rearrangement were evaluated. The sample population consisted of 33 men and 28 women, with a median age of 49 years. Flow cytometry immunophenotypic analysis showed that myeloblasts were positive for CD34 and CD117 in all cases, and myeloperoxidase was positive in 52 of 55 (95%) cases. The most common abnormalities included decreased CD38 in 90%, increased CD13 in 85%, increased CD123 in 84%, and decreased HLA-DR in 84% of cases. Monocytes were increased, with a mature immunophenotype, and accounted for 23.7% of total cells. Among 60 cases with available karyotype, inv(16)(p13.1q22) was most common in 50 (83%) cases, followed by t(16;16) (p13.1;q22) in 6 (10%). Type A CBFB::MYH11 transcript was most common, detected in 84% of cases. Mutational analysis showed mutations of NRAS in 37%, FLT3 in 25%, and KIT in 24% of cases. Comparing cases with type A vs non-type A transcripts, blasts in type A cases more frequently exhibited CD64 positivity and increased CD13 levels while showing a lower frequency of CD7 and CD56 expression. Trisomy 22 and mutations in KIT, NF1, and TET2 were identified only in cases with type A transcript. Myeloblasts of AML with CBFB rearrangement are positive for CD34, CD117, and myeloperoxidase. These neoplasms most frequently carry inv(16)(p13.1q22) and type A fusion transcript. NRAS mutation was the most common mutation. Some immunophenotypic and genetic correlations occurred with different types of transcripts.

Comparative flow cytometry-based immunophenotyping analysis of peripheral blood leukocytes before and after fixation with paraformaldehyde

Flow cytometry based immunophenotyping provides prime insight into cellular population composition and characteristics, and is widely used in basic and clinical research. Challenges in processing peripheral blood samples in a timely manner necessitate protocol adaptations and utilization of fixatives. Fixation, however, may introduce artifacts to the flow cytometry readout. We performed a comparative flow cytometry immunophenotyping analysis of 13 immune cell populations in the whole blood using a staining protocol with and without fixation step. Freshly procured human peripheral blood samples were stained with a panel of 33 fluorochrome-conjugated antibodies. Samples were processed using a protocol with or without a paraformaldehyde-based fixation step, and matching sample pairs were analyzed by flow cytometry. Our results show that paraformaldehyde-based fixation, in comparison to matched unfixed samples, did not significantly affect population distribution and frequency for: B cells, Plasmablasts, Dendritic cells, NK cells, Granulocytes, Neutrophils, Eosinophils, or Hematopoietic Stem/Progenitor Cells. However, fixation led to significant marker shifts in the subpopulation distribution in CD4, T regulatory, CD8, Monocytes, and Basophils. These results indicate the importance of pre-experimental assessment of fixation-introduced artifacts in the flow cytometry output when considering the feasibility of fresh processing. This is especially important for samples analyzed using comprehensive exploratory immunoprofiling panels.

Flow cytometry immunophenotypic features of pure erythroid leukemia and the distinction from reactive erythroid precursors.

The immunophenotype of pure erythroid leukemia (PEL) as determined by flow cytometry immunophenotypic analysis is not well characterized. The immunophenotypic difference between PEL and reactive conditions is under-explored. We assessed and compared the immunophenotype of 24 PEL cases and 28 reactive cases containing early erythroid precursors by flow cytometry. The neoplastic erythroid cells in all PEL cases were positive for CD36 and CD71. CD45 was also positive in all cases, but the expression level was often dimmer than granulocytes. CD117 expression ranged from partial to uniform, and CD235a was often only positive in the CD117-dim to negative cells, corresponding to more differentiated subset. PEL cases frequently (87%) showed decreased or negative CD38 expression, contrasting to reactive early erythroid precursors that showed bright CD38 (p < 0.0001). CD7 (25%) and CD13 (29%) aberrant expressions were only observed in PEL but not in the reactive erythroid cells. Normal early erythroid precursors in all reactive bone marrows showed partial expression of CD4; In contrast, aberrant CD4 expression was detected in 71% PEL cases, either uniformly positive (50%) or completely negative (21%). While normal/reactive bone marrows almost always contained a small subset of CD34-positive early erythroid precursors, the neoplastic pronormoblasts in all PEL cases were CD34 negative. Although not increased in number, CD34-positive myeloblasts were frequently detected in PEL and demonstrated an aberrant immunophenotype in 90% PEL cases. PEL shows a distinctive immunophenotype which can be distinguished from reactive erythroid precursors by flow cytometry immunophenotyping.

Issue Highlights — May 2021

Issue Highlights — May 2021

Determination of immunophenotypic aberrancies provides better assessment of peripheral blood involvement by mycosis fungoides/Sézary syndrome than quantification of CD26- or CD7- CD4+ T-cells.

Blood involvement by mycosis fungoides (MF)/Sézary syndrome (SS) influences prognosis and therapeutic decisions. MF/SS blood stage is currently determined by absolute CD4 + CD26- or CD4 + CD7-cell counts, which quantification method may overestimate MF/SS by including CD26- or CD7- normal CD4+ T-cells, or underestimate disease burden when MF/SS cells show incomplete loss of CD26 and/or CD7. Recently, through the standardization effort led by the International Clinical Cytometry Society (ICCS), recommendation was made to quantify MF/SS by enumerating immunophenotypically aberrant CD4+ T-cells, rather than CD26- or CD7- in isolation. We compared these two quantitation methods in 309 MF/SS patients who had blood samples analyzed by flow cytometry immunophenotyping (FCI) over a 1-year period. Using the European Organization of Research and Treatment of Cancer (EORTC)/International Society for Cutaneous Lymphomas (ISCL) criteria, 221 (71.5%) patients had a blood stage corresponding to B0, 57 (18.4%) to B1, and 31 (10%) to B2. By FCI analysis, a total of 62 patients (20.0%) were found positive for MF/SS. Among EORTC B0 patients, 11/221 (5%) were positive by FCI (false negatives), and among EORTC Stage B1 patients, 35/57 (61%) were negative by FCI (false positives). Regarding patients positive for MF/SS cells by FCI, there was an overall excellent correlation (r = .999, p < .001) between the EORTC/ISCL method and FCI method; however, four (6.5%) patients would have an altered B stage between B0 and B1. The MF/SS cell quantification method using immunophenotypic aberrancies, as recommended by the ICCS, allows to distinguish MF/SS cells from background benign T-cells and enables for more accurate staging, especially among patients currently being considered to have B0 and B1 stage diseases.

Abstract 3702: Establishment and characterization of a novel MYC/BCL2 double-hit high-grade B-cell lymphoma cell line HLA-3

Abstract The majority of MYC/BCL2 double hit lymphoma (DHL) morphologically resembles diffuse large B-cell lymphoma (DLBCL) or unclassified B-cell lymphoma, with intermediate features of DLBCL and Burkitt’s lymphoma. DHL also typically results in shorter survival time than other lymphomas. To further understand DHL’s biology and identify potential effective agents against DHL, we established a unique MYC/BCL2 high-grade B-cell lymphoma cell line with morphological DLBCL features. The HLA-3 DHL cell line was derived from a MYC/BCL2 high-grade B-cell lymphoma patient-derived xenograft (PDX) model. We performed flow cytometry immunophenotypic analysis and established the HLA-3 cell line after 7 months of continuous culture. We utilized molecular and cellular techniques, including flow cytometry and polymerase chain reaction (PCR) analysis, to characterize the HLA-3 cell line. We performed reverse phase protein array (RPPA) analysis to determine its protein signaling profile. To identify agents with anti-DHL activity, we screened the effects of a panel of agents alone and in combination with ibrutinib on the cell viability of this newly established cell line. The HLA-3 cell line was optimally maintained at a density between 1 and 2 × 106 cells/mL and successfully proliferated in a single-cell suspension without cellular clump formation. The HLA-3 cells were EBV-negative and possessed a germinal center B cell immunophenotype. Moreover, the immunophenotypes of the primary patient tumor cells and HLA-3 cells were virtually identical. The HLA-3 cells were positive for CD19, CD45, CD35, CD10 and HLA-ABC, negative for CD3, CD5, CD40, CD34 and TdT, and were dim for CD22, CD56, CD8, CD4 and CD23. Representative flow cytometry histograms showed 99% of the HLA-3 cells were positive for CD19 and CD45, with no detectable surface kappa and lambda light chains. The HLA-3 cells expressed a high level of CD38, an indicator of MYC rearrangement. Similar to the original lymphoma cells, the HLA-3 cells showed dim/low CD20 expression. Based on RPPA data, we constructed a diagram showing activation of the ATM/Chk2 DNA repair and the IGF1R/PI3K/mTOR signaling pathways in the HLA-3 cells. In support of this upregulated activity, we found that 5/7 tested commercial PI3K/mTOR inhibitors significantly reduced the viability of the HLA-3 cells in a dose-dependent manner, with AZD8055 the most sensitive of the tested inhibitors against the cell line. Further, DNA repair pathway inhibitors BMN673 and RG7112 were effective against the HLA-3 cell line. The newly characterized high-grade B-cell lymphoma cell line will provide useful in vitro and in vivo models for biological studies related to human DHL, which is refractory to current therapies and urgently needs novel therapeutic approaches. This work suggests the PI3K/mTOR and DNA repair pathways may be therapeutic targets for MYC/BCL2 double-hit high-grade B-cell lymphoma. Citation Format: Hui Guo, Hui Zhang, Krystle Nomie, Liang Zhang, Kelley P. Murfin, Michael Wang. Establishment and characterization of a novel MYC/BCL2 double-hit high-grade B-cell lymphoma cell line HLA-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3702.

PB1660 FLOW CYTOMETRY CHALLENGE FOR MEASURABLE RESIDUAL DISEASE MONITORING IN B-ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL) PATIENTS RECEIVING TARGETED IMMUNOTHERAPY

Background: Targeted immunotherapy (TI) with Blinatumomab (Bln) or Inotuzumab ozogamicin (IO) are a salvage option for the management of B-ALL patients with measurable residual disease (MRD), in which high rates of MRD negative can be achieved, allowing a bridge to transplantation and longer survival. However, some patients relapse with a phenotype lacking CD19 or CD22 expression can be found in blast cells. Those escape variants have also been described under CAR-T cells therapy, and different mechanism of escape have been proposed. This resistance mechanism has to be considered in flow cytometry immunophenotyping (FCM) analysis when monitoring MRD. Aims: Review the MRD monitoring by FCM in patients diagnosed of B-LLA treated with TI in our center that experience relapse. Methods: Two B-ALL patients treated in our center with TI experienced relapse. The FCM monitoring were performed in BMA samples, using 8-colour combinations of monoclonal antibodies adapted to the TI received and diagnosis phenotype. Stained cells were measured in FACSCanto II cytometer equipped with the FACSDiva software and analyzed with the Infinicyt software with a sensitivity (S) 10-4. Results: Between 2015 and 2018, 2 B-LLA patients relapsed after TI. One of them relapsed with the same phenotype previously detected, and the other patient experienced two relapses, the first one after Bln and then after IO. Interestingly, both relapses showed immunophenotypic changes (figure). The patient was a 72 years old woman that initially received treatment according to PETHEMA-LAL-OLD-07 protocol. Bone marrow aspiration (BMA) at the end of induction was in morphological complete response (CR) with MRD positive (0.12%). After 3rd consolidation cycle, MRD persisted (0.04%), so she started Bln and achieved MRD negative (S 10-4) after 1st cycle. However, after the 3rd cycle, a new CD19- blast cell population was detected (0.07%), with a lower expression of CD20, being the remaining markers similar to the diagnostic. In normal B cells (0.05%) the intensity of CD19 expression was preserved. Blast cells increased after 4th cycle (2.9%) with an associated morphological relapse (21%). She started IO treatment and reached CR and MRD negative (S 10-4) after 1st cycle. Treatment was stopped after the 4th cycle due to upper gastrointestinal bleeding, and in the BMA previous to the next cycle programmed, CD19- CD20dim CD34+ blast cells were detected again (0.55%). All blasts lost CD22 expression, and only 9% maintained the CD10 expression detected previously, being the majority CD10 negative. The patient was not candidate to any other therapy, she started palliative treatment and died 2 months later.Summary/Conclusion: In our experience, changes in immunophenotype of B-ALL blast cells are frequent after TI, due to loss of CD19 and CD22 expression. Therefore, FCM analysis in patients treated with TI demands a high degree of expertise and attention, to avoid the loss of detection of new populations that could emerge with different phenotype.

Follicular lymphoma with hyaline-vascular Castleman-like features: analysis of 6 cases and review of the literature

Follicular lymphoma (FL) with features reminiscent of hyaline-vascular Castleman disease (CD) is an unusual morphologic variant that may create diagnostic difficulties. To our knowledge, only 5 cases of this variant have been reported. We describe the clinicopathologic features of 6 cases including 2 men and 4 women with a median age of 63 years (range, 41-77). Morphologically, all lymph node biopsy specimens showed at least a focal area of conventional FL; 4 cases showed neoplastic follicles with hyalinized blood vessels penetrating into germinal centers (lollipop-like lesions); 4 cases had interfollicular areas with increased vascular stroma; 2 cases showed small neoplastic follicles with prominent, onionskin-like mantle zones; and 1 case showed 2 or more germinal centers within follicles (twinning). The small neoplastic follicles were more cellular than lymphocyte-depleted follicles of true hyaline-vascular CD, and the interface between germinal centers and mantle zones was ill defined. No cases showed dysplastic follicular dendritic cells. Immunohistochemistry for BCL-2 was positive in all 6 cases. Flow cytometry immunophenotypic analysis showed a monotypic B-cell population in 2 of 3 cases assessed. Conventional cytogenetic or fluorescence in situ hybridization studies performed in 3 cases showed t(14;18)(q32;q21) or IGH-BCL2, supporting the diagnosis of FL. The cases presented here add clinicopathologic data to the few cases of FL with hyaline-vascular CD-like features reported previously in the literature. Distinguishing this variant of FL from hyaline-vascular CD is important given the differences in treatment and prognosis of patients with each disease.

CD30 Expression vs. Serum Soluble CD30 (sCD30) Level: Role in Prognosis and Treatment of Acute Myeloid Leukaemia

Objectives: As we noted that CD30 is a valuable molecule in regulation of growth and death of lymphocytes in malignant lymphomas, we analyzed CD30 expression and serum soluble CD30 (sCD30) molecule level in patients with acute myeloid leukemia (AML) to assess their role as a prognostic markers and to examine the possibility of anti-CD30 to be a targeted therapy in these patients.Methods: We studied CD30 expression by Multicolor flow cytometry immunophenotypic analysis on bone marrow aspirates of 50 AML patients. Serum sCD30 level was measured by Enzyme Linked Immunosrbent Assay (ELSA). We correlate CD30 and sCD30 values with all of white blood cell counts, Hemoglobin, platelets, bone marrow blasts and cytogenetics. The Fisher’s exact test or chi-square was used for comparison of categorical variables and the t-test or one-way analysis of variance (ANOVA) was applied for numerical comparisons using SPSS version 20. A p value of <0.05 was considered to be statistically significant.Results: Our study conducted on 50 AML patients, the mean patients’ age was 47.4 ± 18.1 years (range, 17-77), 11 (22%) were males and 39 (78%) were females. 16 (32%) patients have high CD30-expression and 11 (22%) have elevated serum sCD30. We found that there was a significant correlation between both CD30 expression and sCD30 level with WBCs count, BM blasts, Adverse risk cytogenetics, FLT3/ITD and with relapse for CD30 expression, complete remission failure with elevated serum sCD30 level.Conclusions: CD30 is expressed by myeloblasts in AML patients. We found that high CD30 expression and elevated sCD30 level can be used as prognostic markers for relapse and complete remission failure respectively. Furthermore, these patients with adverse risk cytogenetics have not too many treatment options, so the use anti- CD30 targeted therapy may be a possible alternative for this patient group which need further studies.

Novel immunophenotyping marker in acute myeloid leukemia: does it implicate prognosis?

Introduction AML is a heterogeneous group of neoplasms that affect hematopoietic cells responsible for the production of myeloid lineages in bone marrow (BM). Novel immunophenotyping marker in AML (CD30) is a 120 kDa cell membrane glycoprotein that shares sequence homology with tumor necrosis factor (TNF) receptors; it is expressed by myeloblasts (CD34+ and/or CD117+) in a substantial number of cases of non Monocytic AML, either de novo or arising from MDS. CD30 expression is more common in AML patients with unfavorable chromosomal abnormality. It is suggested that CD30 could be a target for therapy by using anti-CD30 antibodies in a subset of patients with non Monocytic AML; also it might provide useful information for patient prognosis and stage of disease. Aim of work Is to assess novel immunophenotyping marker in AML (CD30) and cytogenetic abnormality and its correlation with prognosis. Materials and methods This prospective cohort study included 120 patients with newly diagnosed acute myeloid leukemia. Their ages ranged from (2 to 75) years with a mean of (40.15±23.55) years. Chromosomal analysis by karyotyping was done in all AML patients and Multicolor flow-cytometry immunophenotypic analysis was performed on bone marrow aspirates of the AML patients using fluroisothiocyanate (FITC) conjugated antibodies to assess CD30 expression on BM myeloblasts. Results The previous study showed that CD30 was expressed in all AML cases rather than M4 and M5 cases that showed negative expression. In addition, this study showed that there is more CD30 expression in myeloblasts with unfavorable chromosomal abnormalities. A significant association between platelets counts and CD30 expression was also observed. There was a higher degree of thrombocytopenia and a greater tendency to have higher leucocytic counts in patients having +ve CD30 expression than those with –ve CD30 expression. Conclusion The analysis of CD30 expression has a potential role to be used as a prognostic marker in AML.

De Novo MYC and BCL2 Double-hit B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) in Pediatric and Young Adult Patients Associated With Poor Prognosis

MYC and BCL2 translocations in B-cell lymphomas are defined as “double-hit” associated with poor prognosis in adult patients. Such double-hit events are extremely rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), especially in pediatric patients or young adults. This study is to investigate the clinical manifestation of de novo MYCyBCL2 double-hit BCP-ALL in young patients. Two pediatric and one young adult patients were identified after a retrospective data review and all without previous history of lymphoma. There were two females and one male aged 15, 18, and 24, respectively. All patients had an unremarkable medical history before presenting with extensive bone marrow and central nervous system involvement at diagnosis. Flow cytometry immunophenotypic analysis showed an immature B-cell immunophenotype (CD10+, CD19+, TdT+, surface Ig-) and immunohistochemistry showed high expression of MYC and BCL2 in all cases. All patients showed complex karyotypes associated with 8q24 abnormalities in the form of t(8;9)(q24;p13) or t(8;14)(q24;q32) and t(14;18)(q32;q21) and fluorescence in situ hybridization confirmed MYC and BCL2 rearrangements. Two patients died of refractory disease or disease progression 7 and 13 months after initial diagnosis, respectively, and the third patient was treated with protocol AALL0232 under the Children's Oncology Group study, achieved complete remission and remained in remission for 53 months at last follow-up. Our study showed that De novo MYCyBCL2 double-hit BCP-ALL is a rare disease that also occurs in pediatric and young adult patients and associated with complex karyotypes and poor prognosis. Younger patients may benefit from intensified chemotherapy.

Flow cytometry immunophenotypic analysis of Philadelphia-negative myeloproliferative neoplasms: Correlation with histopathologic features.

Compared with the proven utility of flow cytometry immunophenotyping (FCI) analysis in the workup of myelodysplastic syndromes (MDS), immunophenotypic alterations in myeloproliferative neoplasms (MPN) have been less studied and the potential utility of FCI is not defined. Bone marrow (BM) samples of 83 Philadelphia-negative MPN patients were assessed by multicolor FCI including 27 with essential thrombocythemia (ET); 17 polycythemia vera (PV); 33 primary myelofibrosis (PMF) and 6 MPN-unclassifiable (MPN-U). The time interval from initial diagnosis of MPN to FCI analysis was 18 months (0-370). Ninety-five age-matched MDS patients with a similar BM blast count were included for comparison. Immunophenotypic alterations, either in CD34(+) cells or myelomonocytic cells, were detected in 82 of 83 (99%) MPN cases. FCI abnormalities were more frequently observed in cases with substantial myelofibrosis but not different between PMF and fibrotic stage of ET/PV. Furthermore, FCI abnormalities were more frequent in cases with ≥5% BM blasts and/or circulating blasts (P = 0.006); as well as cases with an abnormal karyotype (P = 0.036); but not associated with morphologic dysplasia or JAK2 mutation status. Comparing with MDS, FCI abnormalities were overall less pronounced in MPN cases (P = 0.001). MPNs exhibit frequent immunophenotypic alterations, more pronounced in cases with adverse histopathologic features. These findings illustrate that immunophenotypic alterations are a part of constellational findings in MPN, and correlate progressively with disease stage. The study results also suggest a role of FCI in diagnosis of MPN and monitoring disease over time and after therapy.

Flow cytometry immunophenotypic analysis of Philadelphia-negative myeloproliferative neoplasms: Correlation with histopathologic features.

Background: Compared with the proven utility of flow cytometry immunophenotyping (FCI) analysis in the workup of myelodysplastic syndromes (MDS), immunophenotypic alterations in myeloproliferative neoplasms (MPN) have been less studied and the potential utility of FCI is not defined. Methods: Bone marrow (BM) samples of 83 Philadelphia-negative MPN patients were assessed by multicolor FCI including 27 with essential thrombocythemia (ET); 17 polycythemia vera (PV); 33 primary myelofibrosis (PMF) and 6 MPN-unclassifiable (MPN-U). The time interval from initial diagnosis of MPN to FCI analysis was 18 months (0-370). Ninety-five age-matched MDS patients with a similar BM blast count were included for comparison. Results: Immunophenotypic alterations, either in CD34+ cells or myelomonocytic cells, were detected in 82 of 83 (99%) MPN cases. FCI abnormalities were more frequently observed in cases with substantial myelofibrosis but not different between PMF and fibrotic stage of ET/PV. Furthermore, FCI abnormalities were more frequent in cases with ≥5% BM blasts and/or circulating blasts (p=0.006); as well as cases with an abnormal karyotype (p=0.036); but not associated with morphologic dysplasia or JAK2 mutation status. Comparing with MDS, FCI abnormalities were overall less pronounced in MPN cases (p=0.001). Conclusions: MPNs exhibit frequent immunophenotypic alterations, more pronounced in cases with adverse histopathologic features. These findings illustrate that immunophenotypic alterations are a part of constellational findings in MPN, and correlate progressively with disease stage. The study results also suggest a role of FCI in diagnosis of MPN and monitoring disease over time and after therapy. This article is protected by copyright. All rights reserved.

Utility of quantitative flow cytometry immunophenotypic analysis of CD5 expression in small B-cell neoplasms.

The value of assessing CD5 expression in the differential diagnosis of small B-cell neoplasms is well established. Assessment is usually done qualitatively. To assess CD5 expression levels by quantitative flow cytometry immunophenotyping and to determine possible differences among various small B-cell neoplasms. We performed 4-color flow cytometry analysis on specimens of peripheral blood and bone marrow aspirate and quantified CD5 expression in various small B-cell lymphomas and leukemias. We also assessed CD5 levels in peripheral blood samples of healthy blood donors. Cases of chronic lymphocytic leukemia and mantle cell lymphoma had higher levels of CD5 compared with control B cells (P < .001). Cases of marginal zone lymphoma and hairy cell leukemia had CD5 levels similar to control B cells (P = .35 and P = .14, respectively), whereas cases of follicular lymphoma and lymphoplasmacytic lymphoma had significantly lower CD5 levels than control B cells (P < .001 and P = .04, respectively). In B-cell neoplasms, a high level of CD5 expression was correlated with a homogeneous pattern of positive events, whereas lower CD5 levels were correlated with heterogeneous patterns of positive events. Using flow cytometric immunophenotypic analysis to quantify CD5 levels can aid in diagnosis. CD5 expression levels are higher in patients with chronic lymphocytic leukemia and mantle cell lymphoma, and expression is observed in a homogeneous pattern, as compared with other B-cell neoplasms that are either negative for CD5 or express CD5 at lower levels with a heterogeneous pattern. However, there is some overlap in CD5 expression levels between a subset of atypical chronic lymphocytic leukemia and marginal zone lymphoma cases.

CD30 expression in acute lymphoblastic leukemia as assessed by flow cytometry analysis

We assessed CD30 expression in patients with acute lymphoblastic leukemia (ALL) of either T-cell or B-cell lineage to examine the potential benefit of anti-CD30-targeted therapy in this group of patients. Bone marrow specimens of 34 patients with T- and 44 with B-ALL were assessed for CD30 expression by multicolor flow cytometry immunophenotyping analysis. Of these 78 patients, 75 (96%) were adults; and 63 (81%) had refractory/relapsed disease. Using an arbitrary 20% cut-off, 13/34 (38%) cases of T-ALL and 6/44 (13%) cases of B-ALL were considered to express CD30. In five patients with T-ALL with sequential bone marrow tested, increased CD30 expression was observed during the course of high-dose chemotherapy (p = 0.025). Philadelphia chromosome/BCR–ABL1 fusion was positive in 14/44 cases of B-ALL and 2/32 cases of T-ALL, which showed no significant correlation with CD30 expression. In summary, we detected CD30 expression in approximately one-third of patients with T-ALL, and less frequently in B-ALL (p = 0.017). In T-ALL, CD30 expression is up-regulated during high-dose chemotherapy. These data indicate that anti-CD30-targeted therapy may be a potential option for patients with T-ALL with refractory/relapsed disease.

CD30 Expression in High-Risk Acute Myeloid Leukemia and Myelodysplastic Syndromes

We assessed for CD30 expression in patients with acute myeloid leukemia (AML) or high-grade myelodysplastic syndrome (MDS) to examine the possibility that anti-CD30 could be targeted therapy in these patients. Multicolor flow cytometry immunophenotypic analysis was performed on bone marrow aspirates of 135 patients with AML or MDS and peripheral blood samples in a subset of 33 patients. Immunohistochemistry was performed on bone marrow aspirate clot specimens of 84 patients. The median patient age was 63 years (range, 13-92 years); 102 (75%) patients had refractory or recurrent disease, and 68 (50%) had high-risk cytogenetics. Overall, the median percentage of blasts positive for CD30 was 14% (range, 0%-91%). By using an arbitrary 20% cutoff, 49 (36%) patients were considered to have CD30 expression. Monocytic cells, either mature or immature, were consistently negative for CD30. Therefore, CD30 expression was less in AML with monocytic differentiation (P = .006). The patients with persistent disease who had been actively treated had a higher level of CD30 expression than the patients who were untreated (P = .031). In paired samples, CD30 expression was consistently higher in bone marrow blasts than in peripheral blood blasts (P = .002). Immunohistochemistry demonstrated CD30 expression by myeloblasts in a subset of patients, but reactivity was generally weaker and focally compared. CD30 is expressed by myeloblasts in a substantial subset of patients with AML or MDS. Because the study group was composed mostly of patients with high-risk AML or MDS in whom very few treatment options are available, these data raise the possibility that anti-CD30-targeted therapy could be a potential option for this patient group.

Simultaneous cytomorphological and multiparameter flow cytometric analysis of ALK-positive anaplastic large cell lymphoma in children

The aim of this study was to investigate the pathological features of anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) in children and to establish the effectiveness of screening and diagnosing ALCL with multiparameter flow cytometry immunophenotyping (FCI) of lymphoid tissue samples. A total of 121 lymph node tissue specimens obtained from 121 patients with a suspected diagnosis of lymphoma were analyzed with cytomorphological and FCI analysis. Fifteen cases were diagnosed as ALK-positive ALCL based on the pathological features and immunohistochemical results. Of these, there were 3 different types, common type (10 cases), lymphohistiocytic type (4 cases) and neutrophil-rich type (1 case). Thirteen cases (10 common, 2 lymphohistiocytic and 1 neutrophil-rich type) were diagnosed as ALCL using FCI. These cases were CD30-positive and aberrantly expressed at least two T-cell antigens, including CD4 (84.6%), CD2 (76.9%), CD7 (61.5%), CD3 (53.8%) and CD5 (38.4%). Neoplastic cells accounted for only a small proportion of the total cells in FCI, with a median of 19.3% (range, 7.9–31.8%), which was significantly higher than those in the control groups (all <1.0%). The sensitivity of FCI for diagnosing ALCL in lymph node samples was 86.7% with a specificity of 100%. The majority of neoplastic cells demonstrated high light forward and high light side scatter, similar to monocytes or granulocytes in dot plots. FCI may be used as an adjunct to histopathological examination for rapid and reliable diagnosis of pediatric ALCL. Flexible gating strategies and careful analysis are required to identify neoplastic cells with FCI.

Role of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Refractory Anemias

AbstractAbstract 4406 Background:The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in the setting of aplastic anemia (AA) and myelodysplastic syndrome (MDS) highlights the pathogenetic link between these disorders. The awareness that small clones of PNH cells could be detected by flow cytometry in aplastic anaemia patients who had negative Ham test has led to classifying PNH into two broad groups such as Haemolytic PNH which can have 50% presenting with thrombosis and a hypo plastic group which behaves like aplastic anaemia. Even if there is a small PNH clone in the setting of aplastic anaemia, then the patients respond better to immunosuppressive therapy.Flowcytometry is a relatively new advanced technique in many parts of India and there is no widespread consensus among methodology. Further, the clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is yet to be established here. Objective:To validate the role of flowcytometry in the diagnosis of PNH, in the setting of aplastic anaemia, myelodysplastic syndrome or abdominal or cerebral vein thrombosis in the setting of PNH which may help to prognosticate the course and treatment. Material and methods:The study was both retrospective and prospective. The prospective arm of the study was conducted from September 2009 to April 2010. Ham's test and Sucrose lysis test were done as screening tests and were reported as positive or negative. Other laboratory studies including hemogram and bone marrow analysis were done as part of standard care. In the retrospective group there were 32 patients who were diagnosed as PNH based on Ham's and Sucrose lysis test followed up from 1990.Four color flowcytometry immunophenotypic analysis with CD55 and CD59 was used to detect PNH clones in RBCs. EDTA-anticoagulated whole blood was stained with anti–CD 55-Phycoerythrin (PE) and anti–CD 59-PE.A minimum of 10,000 events were acquired. For analysis, RBCs were identified by light-scatter properties. For the purpose of the study, 3% of type II and/or type III cells were considered as threshold to diagnose PNH clones in both CD55 and CD 59 gated population. The results of laboratory studies including transfusion history and flow cytometry were correlated. Institutional ethical committee approval was obtained for the conduct of the study. Results:33 patients were included as part of the prospective group. The number of males was more compared to females (19:14). 78% of patients had hemoglobin of less than 7gm%. The presentation varied from unexplained anemia to thrombosis. Based on flow cytometry, 3 patients were found to have PNH clones in the RBC lineage by both CD 55 and CD 59. CD 55 alone picked up one more patient. In the retrospective group there were 32 patients who were followed up from 1990. Interestingly, two of them who had earlier presented with aplastic anemia had transformed into PNH in a span of 3years and 6 months. All patients were treated with Stanazolol and cyclosporine. The median survival was 12.14. months with Kaplan-Meier survival estimates of 30 and 20 percent at 121 months, and 143 months after diagnosis, respectively. The patients in the prospective group are being followed up. Discussion:The Ham test, sucrose lysis test, and modified Ham tests rely on the differential sensitivity of PNH red cells to haemolysis. These tests, although suitable for haemolytic PNH, cannot reliably detect small populations of PNH red cells nor differentiate partially and completely deficient cells. One patient who presented with DVT in whom increase in clone size was demonstrated by serial flowcytometry. Testing for the presence of PNH RBC clones provides additional diagnostic information and the thrombotic event may probably directly related to the size of the PNH clone.Conclusions: Our data demonstrate the utility of RBC assay in flow cytometry in the primary screening of PNH, AA, and MDS patients. The clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is relatively new and could be demonstrated in one patient. Disclosures:No relevant conflicts of interest to declare.

Multi-color CD34+ progenitor-focused flow cytometric assay in evaluation of myelodysplastic syndromes in patients with post cancer therapy cytopenia

Bone marrow assessment for myelodysplastic syndrome (MDS) in a patient who develops cytopenia(s) following cancer therapy is challenging. With recent advances in multi-color flow cytometry immunophenotypic analysis, a CD34+ progenitor-focused 7-color assay was developed and tested in this clinical setting. This assay was first performed in 73 MDS patients and 53 non-MDS patients (developmental set). A number of immunophenotypic changes were differentially observed in these two groups. Based on the sensitivity, specificity and reproducibility, a core panel of markers was selected for final assessment that included increased total CD34+ myeloblasts; decreased stage I hematogones; altered CD45/side scatter; altered expression of CD13, CD33, CD34, CD38, CD117, and CD123; aberrant expression of lymphoid or mature myelomonocytic antigens on CD34+ myeloblasts; and several marked alterations in maturing myelomonocytic cells. The data were translated into a simplified scoring system which was then used in 120 patients with cytopenia(s) secondary to cancer therapy over a 2-year period (validation set). With a median follow-up of 11 months, this assay demonstrated 89% sensitivity, 94% specificity, and 92% accuracy in establishing or excluding a diagnosis of MDS.