CD45 Gating Research Articles

Addition of CD14 improves discrimination of lymphocytes in the TBNK phenotyping panel.

Peripheral blood lymphocyte phenotyping panels typically include CD45 for discrimination of the lymphocyte population, and fluorophore-conjugated monoclonal antibodies to identify T, B, and Natural Killer (NK) cells. While CD45 combined with side scatter is generally sufficient to clearly distinguish lymphocytes from monocytes in the majority of peripheral blood samples, it is challenging to accurately gate lymphocytes in samples from patients with monocytosis or significant lymphopenia, or from very young infants. Addition of a monocyte marker to lymphocyte phenotyping panels for monocyte exclusion has previously been evaluated for improved discrimination of lymphocytes, albeit largely in healthy donor adult samples. Here we evaluate the effect of the addition of CD14 to a standard lymphocyte phenotyping panel on total lymphocyte, T, B, and NK cell percentages in a predominantly pediatric population of patients under evaluation chiefly for immunodeficiency, immune-depletion, or immune reconstitution. Addition of CD14 to the standard lymphocyte phenotyping improved discrimination of lymphocytes from monocytes, resulted in decreased NK cell percentages, likely because CD16+ and/or CD56+ monocytes were included in the CD56+CD16+ NK cell gate with conventional gating, and although less significant, resulted in an increased percentage of B cells, since relatively larger B cells were likely gated out by more restrictive light scatter gating used with the conventional gating approach. The change in NK and B cell percentages were more pronounced in samples from patients below a year of age, and in patients who were relatively lymphopenic. These data suggest that addition of CD14 to conventional lymphocyte phenotyping panels that utilize CD45 versus side scatter gating results in significant improvement in the accuracy of lymphocyte gating, and accurate quantification of NK and B cells particularly in samples from infants and lymphopenic individuals.

CD123 As a Therapeutic Target in Accelerated and Blast Phase Myeloproliferative Neoplasms (MPNs)

Background: CD123 expression in MPNs has been reported in case series and CD123 has been a therapeutic target in one phase I study of patients with chronic myelomonocytic leukemia or myelofibrosis in chronic phase. CD123 has also been described as a marker of leukemic stem cells and incorporated in chimeric antigen receptor T-cell products for treatment of acute myeloid leukemia in clinical trials. Herein, we describe the clinical phenotype of patients with accelerated (AP) or blast phase (BP) MPNs paired with CD123 co-expression on CD45(dim) myeloid blasts. Methods: This is a retrospective, single-institution study of patients with AP and BP MPNs. Subjects were identified by search for MPN diagnoses in the Ohio State University Comprehensive Cancer Center Leukemia Tissue Bank (OSUCCC-LTB). Relevant clinical data was collected by retrospective chart review with approval of Institutional Review Board. CD123 expression was measured by immunophenotyping of cryopreserved peripheral blood (PB) or marrow (BM) samples. Blasts were identified by CD45(dim) gating of lymphocytes. Lymphocytes were also gated on CD7, CD13, CD33, CD34, and CD117. CD123 expression was stratified by quartile and correlation with disease phase, symptoms, splenomegaly, transfusion-dependence, and molecular mutations measured by Chi-square test. Results: Forty-one PB or BM samples from 24 subjects were identified from the OSUCCC-LTB. Nine subjects had paired PB and BM specimens and 6 subjects had samples obtained at multiple timepoints in the course of their disease. Median age at time of sample collection was 65.8 years (range 35.2-79.7) and median time from initial diagnosis to sampling was 13.1 months (range 0-96.5). Constitutional symptoms were present at the time of collection in 24 specimens (58.5%) and splenomegaly present in 20 specimens (51.3%). At sample collection, 19.5% had never received treatment, 19.5% had been previously treated, and 61% were currently on treatment. Most patients on treatment (n=16, 64%) were receiving JAK inhibitor at sample collection. Nineteen samples were obtained in chronic phase (46.3%), nine in AP (22.0%), and thirteen in BP (31.7%). Median WBC count was 4.37K/uL (range 0.3-174.6), median peripheral blasts were 3.2% (range 0-76%), median hemoglobin was 8.8 g/dL (range 5.6-13.2), and median platelets were 42K/uL (range 5-564). Sixteen samples (39.0%) were from transfusion-dependent patients, of which, 68.8% were both red blood cell (RBC) and platelet transfusion-dependent, 25.0% were RBC transfusion-dependent only, and 6.2% were platelet transfusion-dependent only. Median lactate dehydrogenase at time of sampling was 300 U/L (range 116-2,142). Median bone marrow blasts at time of sampling were 3% (range 0-82%). Molecular sequencing and karyotype +/- 30 days of sample collection were available for 18 (43.9%) and 27 (65.9%) of specimens, respectively. The most common mutations identified were JAK2 (50%), ASXL1 (27.8%), SRSF2 (22.2%), CALR (16.7%), and TP53 (16.7%). Normal karyotype was reported in 37% of the samples (one was consistent with donor chimerism post-allogeneic SCT), 33.3% had complex karyotype, 11.1% had del 5q or 7q/-7, and 18.5% had del 13q or 20q. CD123 expression on the CD45(dim) gated blasts ranged from 0.3-95.6% with a median value of 11.02%. CD123 expression is presented in figure 1 box plot. Figure 2a shows correlation between CD123 expression and AP/BP compared to chronic phase disease (p < 0.01). Figure 2b shows CD123 expression correlation with ASXL1 compared to unmutated ASXL1 (p =0.023). There were no statistically significant associations between CD123 expression and any other tested variables. Conclusions: These single-institution data suggest that high CD123 expression on blasts is associated with leukemic transformation and high-risk disease. This is demonstrated by correlation of higher expression in AP and BP MPN as well as correlation with ASXL1 mutation. CD123 expression in MPNs has been previously reported and explored as a therapeutic target in a single phase I trial, though these data suggest that specifically targeting high risk disease, defined by blasts >10% or the presence of ASXL1 mutation, may be more effective with greater target availability. This is an especially important subpopulation of patients with MPNs in need of more effective and less toxic therapy than current standards of care that typically include cytotoxic chemotherapy.

Differential expression of CCR2 and CX3CR1 on CD16+ monocyte subsets is associated with asthma severity

BackgroundMonocytes play an important role in immune and inflammatory diseases and monocyte subsets are predictors of disease in certain conditions. Expression of the chemokine receptors, CCR2 and CX3CR1 on monocyte subsets relates to their function and can be used in their characterization. Our objective was to determine whether CD14, CD16, CCR2 and CX3CR1 on monocyte subsets are potential indicators of asthma severity.MethodsBlood samples were collected from Saudi Arabian patients with asthma and normal healthy individuals. Six-color flow-cytometry phenotypic analysis was used to identify human blood monocyte subsets, based on their expression of CD14 and CD16 following CD45 gating. Expression of CCR2 and CX3CR1 was analysed on classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) subsets and correlated with disease severity.ResultsWe demonstrated a significant increase in percentage of total CD45-positive monocytes in the blood of patients with severe asthma, but the proportion of the individual monocyte subsets was not significantly changed when patients with mild, moderate and severe asthma were compared with healthy individuals. CD16 expression (mean fluorescence intensity, MFI) was decreased on intermediate and non-classical subsets in patients with severe asthma compared to healthy controls. CX3CR1 expression was also lower, with a lower percentage of cells expressing CX3CR1 in the non-classical CD14+CD16++ subset in all patients with asthma and this was inversely related to the percentage of cells expressing CCR2.ConclusionsCCR2 expression on monocytes indicated a tendency toward more phagocytic monocytes in patients with asthma. The differential expression of CD16, CX3CR1 and CCR2 on monocyte subsets in peripheral blood indicates modulation of the inflammatory response and suggests a role for monocytes in asthma pathogenesis.

Evaluation of Clinical Usefulness of Monocyte Gating Using CD14 and CD64 for Detecting PNH Clone By Flow Cytometry

IntroductionFlow cytometry is the gold standard in diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) by detecting the absence of glycol-phosphatidyl inositol (GPI)-linked protein expression on red blood cell, granulocyte, and monocyte. The current assays are 4-color analyses of GPI-linked markers such as fluorescein-labeled proaerolysin (FLAER), CD24, CD14, CD59, and CD235a and the lineage markers for granulocyte (CD15) and monocyte (CD64) cells to detect PNH clones. We investigated the utility of CD14/CD64 monocyte gating by comparing with CD45/light scatter (LS) gating in PNH study of the patients with cytopenia and analyzed the types and cell lineages of PNH clone according to the disease groups.MethodTotal 138 cases were recruited in this study from July 2017 to February 2018 at Gachon University Gil Medical Center in Korea. Flow cytometric analysis was performed with EDTA blood by Beckman Coulter Cytomics FC500 cytometer using gating antibodies such as CD45, CD14, CD15, CD64, CD235a and GPI-linked antibodies such as CD59, CD14, CD24, FLAER. The proportion of monocyte was estimated by CD14/CD64 gating and compared with those using CD45/LS gating. The type of PNH clone was defined according to the size of PNH population. A PNH clone is defined as a PNH population exceeding 1% of the gated cells, a minor PNH clone as between 0.1 and 1%, and rare cells with GPI-deficiency defined as a PNH population less than 0.1%. The types and cell lineages of the PNH clone were analyzed according to the disease groups. Statistical analysis was done using SPSS 17.0 and MedCalc 15.2, and P<0.05 was considered statistically significant.ResultsOf the 138 cases, PNH clone was detected with 27 cases including 15 cases with a PNH clone and 12 cases with a minor PNH clone. PNH clone was observed in all 8 cases (100%) of PNH cases. Two PNH clone and 4 minor PNH clones were identified in 6 of 16 cases (38%) of acute myeloid leukemia. In 6 of 21 cases (29%) of aplastic anemia (AA) show 5 PNH clones and 1 minor PNH clone. In 5 of 78 cases (6%) of cytopenia(s) only minor PNH clone was observed. The CD45 plus LS gating in monocyte represents a sensitivity of 100%, a specificity of 40.2%, and 60% (73/89) false positive rate in detecting of PNH clone. McNemar test indicates a significant difference between CD14/CD64 and CD45/LS gating methods (P = 0.00). The Bland-Altman plot of monocyte proportion between the two gating methods revealed that CD45/LS gating method was tended to underestimate monocyte proportion and the larger the number of monocytes, the greater the difference in number of monocyte between the two gating methods. The trend of the size of PNH clone in each cell lineage was confirmed by follow-up in three patients with PNH clone. Two patients showed more abrupt changes of PNH clone in monocytes than in red blood cells or in granulocytes. However, in the other patient, a significant trend found in only PNH clone of RBC.ConclusionThe types of PNH clone observed in each disease group showed different characteristics. PNH clone was identified in 5 of 6 PNH population detected AA cases, whereas minor PNH clones were observed in all 5 PNH population detected cytopenia cases. Four minor PNH clones and two PNH clones were discovered in 6 PNH population detected AML cases. However, all observed PNH clones observed in AML cases were monocyte. Monocyte gating with CD45 and LS not only underestimated the proportion of monocyte in total WBCs but also showed a high false positive rate of 60% in detecting PNH clone. In contrast, the CD14/CD64 gating method can accurately measure the monocyte population and avoid making a false positive measurement of PNH clone. In addition, in monitoring PNH patients, the measurement of the PNH clone in monocyte tends to be more sensitive to change of PNH clone size than those measured in RBC or granulocytes. In conclusion, the gating using CD14 and CD64 is significantly valuable in flow cytometric diagnosis for detecting the PNH clone in diagnosing new patents as well as monitoring of PNH patients. DisclosuresNo relevant conflicts of interest to declare.

Phenotipic Heterogeneity Studied By Spade Algorithm in Relapsed/Refractory Acute Myeloid Leukemia

Background.Acute myeloid leukemia (AML) is a heterogeneous disease with respect to cell phenotypes and clinical outcomes.Recently, detailed genetic studies have highlighted the mutational spectrum of clonal variability and increased our knowledge of biologic features of this disease. Nonetheless, characterization of AML cell heterogeneity by traditional Flow Cytometry (FC) is still ineffective because the hierarchical two-dimensional gating analysis of FC data is unable to distinguish all different sub-populations, as defined by the combination of multiple cell parameters. Here, we applied the SPADE algorithm (Ref. Qiu P., Nat Biotechnol, 2011), a new computational approach for dimensional reduction of FC data, in order to represent the intratumoral heterogeneity of AML at single-cell resolution in leukemias at the diagnosis vs. relapsed/refractory AMLs. The purpose of our study was to investigate the clonal dissimilarities of cell subsets as identified by SPADE analysis between diagnosis and relapse/refractory disease status in AML and to define the cell markers mainly associated to the clonal variation of tumor before and after standard therapies.Methods. 30AML patients were investigated at diagnosis and at relapse/ refractory (R/R) disease status by FC analysis.The SPADE algorithm was applied using Cytosplore software (Ref. Hollt. T, 2016) in order to depict the immunophenotype of clonal structures in AML before and after treatment. Each subpopulation was represented by colored circles (nodes) and described the substantial differences between and within individual leukemia subsets. At least a threshold of 7% of events was used to define significant clusters (node) and up to 50 target nodes were used to define the complexity of cell population. All nodes were hierarchically separated by relatedness into a “minimal spanning tree” in relation to the expression levels of 8 Ab markers used for this experiment in the FC panel. The readouts of spanning-tree progression analysis of normalized events (SPADE) reflected the cell heterogeneity underlying the fluorescence intensity of markers used in the FC analysis and defined the relationships among hierarchical cell subsets of leukemia before and after treatment. T-tests and Pearson's coefficient were used to compare antigenic distribution between diagnosis and R/R status. Results.Based on CD45 gating strategy, leukemic population of AML patients at diagnosis showed a median number of nodes significantly lower than that found at R/R disease status (n=4 vs. n= 6 nodes, p=0.0129)(Figure). These data defined an increase of different subpopulations in the R/R subset. Following antigens resulted mainly espressed on the entire study population: CD117, CD34, CD33, CD13, HLADR. The distribution of these markers were further investigated in order to define which cell parameters were able to discriminate the different subpopulations by CD45 expression. Indeed, a comparison of the number of nodes with highest antigenic expression (>90% of intensity value) between AML samples at diagnosis and R/R status was showed. CD117 and CD34 distribution showed a significant difference (p=0.0041 vs p=0.0139, respectively)(Figure) while no statistically significant values were observed among the others. However, only CD34+ nodes showed a significant linear growth between diagnosis and R/R status (p=0.0227).Conclusions. SPADE analysis allowed us to find an increase of subpopulations in R/R AML when compared to samples at diagnosis. CD34 and CD117 antigens mainly characterized these subpopulations, thus defining the cell markers mainly associated to the clonal variation. CD34 antigen was the widest distributed antigen among the new subpopulations, denoting a great instability of more immature clones. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometric Analysis of Inflammatory Cells in Experimental Acute Pancreatitis

Background: Accumulating evidence indicates that inflammatory cells migrate into the pancreas tissue and play an important role in the pathogenesis of acute pancreatitis (AP). The aim of this study was to establish a flow cytometric method to enumerate these infiltrating cells in the pancreas of an experimental AP. Materials and Methods: Twelve hours after inducing of AP, mice pancreatic tissues were cut into small fragments and single cells were prepared by mechanical dissociation. The isolated cells were stained with either anti-mouse CD45-PerCP or isotype antibody and analyzed by flow cytometry. Using side scatter (SSC)/CD45 gating we were able to identify inflammatory cells from non-inflammatory cells. Results: The mean percentage of leukocytes was 5.9±1.6 in the control group whereas, it was 26.7±8.1 in the AP. Moreover, we found that the percentage of lymphocytes, monocytes and granulocytes were 1.1±0.2, 0.9±.04 and 2.9±1.8 of total pancreatic cells, respectively, in the control mice. In contrast to lymphocytes, the percentage of monocytes and granulocytes were significantly increased in the AP group and it was 3±1.3 and 18.2±3.2 for monocytes and granulocytes, respectively. Conclusion: Quantitative flow cytometric analysis is feasible and provides a reliable and rapid assay to determine the number and percentage of inflammatory cells in experimental AP. K e y w o r ds: Acute pancreatitis, flow cytometry, inflammatory cells

Comparison Of Lymphocytic Immune Profiles Of Aplastic Anemia and Hypocellular Myelodysplastic Syndrome

BackgroundAplastic anemia (AA) and hypocellular myelodysplastic syndrome (MDS) are two common acquired bone marrow failure diseases. AA is mostly an acquired bone marrow disease caused by cellular and humoral mediated immune attack of hematopoietic stem cells (HSC) due to dysregulation of lymphocytic system, which leads to hematopoietic progenitor cell apoptosis and bone marrow failure. MDS is a group of heterogeneous acquired clonal HSC disorders with ineffective hematopoiesis. Approximately 10% to 20% of MDS manifests a reduced bone marrow cellularity, which comprises hypocellular MDS. There is increasing experimental and clinical indication that an immune-mediated damage to hematopoietic HSCs and changes in the hematopoiesis-supporting microenvironment contribute to the pathogenesis of hypocellular MDS. Because of the similarity of their bone marrow manifestation, hypocellular MDS and AA are often hard to distinguish. Mounting evidence indicates that abnormal activation of cytotoxic T cells plays a crucial role in the pathophysiology of these diseases. One study showed that AA patients have an abnormally activated subpopulation of CD4+ helper cells and a decreased number and function of T regulatory cells in the bone marrow. GVHD mouse models further demonstrated that self-reactive T cells were capable of recognizing non-polymorphic tissue or commensally-derived antigens. Recent literature suggests that immune dysregulation plays a major role in pathogenesis of acquired bone marrow failure disease. However immune profiles of these two diseases have not been thoroughly studied, specially the role of B lymphocyte population. Our study aims to find lymphocytic surface marker expression patterns of hypocellular MDS and AA in both immature cell and lymphocyte populations. MethodsThis retrospective study analyzed flow cytometry lymphocytic antigen expression profiles from patients diagnosed as AA and hypocellular MDS as per standard criteria. A total of 31 AA and 26 hypocellular MDS patient cases were recruited. The bone marrow aspirate/biopsy data, bone marrow aspiration flow cytometry reports, and Complete Blood Counts (CBC)s from individual patients were analyzed. Using side scatter (SSC) vs. CD45 gating flow cytometry panels, we identified immature cell population (SSClow/CD45low) and lymphocyte population (SSClow/CD45high). We then quantitatively analyzed the expression patterns of 33 cluster differentiation (CD) molecules on individual sample. Finally, we compared the CD expression patterns between AA and hypocellular MDS in both cell populations respectively. ResultsCD19 expression was significantly higher in AA than in hypocellular MDS in both SSClow/CD45low cell population (P=0.001) and SSClow/CD45high cell population (P=0.003). Hypocellular MDS contains significantly higher CD34high cells than AA in SSClow/CD45low populations (mean:28.5% vs 8.5%; range; 1% to 94% vs 2% to 27%; P=0.04). However, patients with both diseases similarly contains very few CD34high cells in SSClow/CD45high cell population (mean: 0.6% vs 2.6%; range: 0.0% to 2% vs 0.0% to 32%; P=0.99). Conclusion1. In AA, B cells are highly proliferative in both immature stage and mature stage. This data indicates that B cells which may play a unique role in AA pathogenesis but not in hypocellular MDS.2. In both AA and hypocellular MDS, the majority of lymphocyte population are mature cells. This data suggests that the pathogeneses of both diseases caused by a persistently dysregulated immune microenvironment, not by an acute insult.CD19 expression pattern may be a useful marker to distinguish AA and hypocellular MDS. Disclosures:No relevant conflicts of interest to declare.

The basis examination of leukocyte–platelet aggregates with CD45 gating as a novel platelet activation marker

Platelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy-to-use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte-platelet aggregate formation associated with activated platelets in circulation. Whole blood was stained with PC5-conjugated anti-CD45 monoclonal antibody and fluorescent isothiocyanate-conjugated anti-CD41 monoclonal antibody for leukocyte-platelet aggregate analysis. For platelet activation, 5 μm thrombin receptor-activated peptide (TRAP) or 2 μg/mL collagen was added. Samples were analyzed by EPICS XL (Beckman Coulter, Miami, FL, USA). Monocytes, neutrophils, and lymphocytes were gated based on differences in CD45 fluorescence intensity and side scatter. For each gate, the percentage (%) of platelets expressing CD41 was analyzed. Same drawing sample was stained with anti-CD62P monoclonal antibody. Platelet CD62P expression was then analyzed with gating for platelet cell population. We analyzed leukocyte-platelet aggregates and platelet CD62P expression in 18 healthy individuals. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, increased when platelets were activated by platelet agonists. Monocyte-platelet aggregates and neutrophil-platelet aggregates also increased over time with mild platelet activation. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, appear to be a sensitive marker of platelet activation in circulation.

The application of CD45 gating flow cytometric analysis for diagnosis of lymphoid malignancies: especially in tissues and body cavity fluids

The application of CD45 gating flow cytometric analysis for diagnosis of lymphoid malignancies: especially in tissues and body cavity fluids

Flow Cytometric Minimal Residual Disease Levels After First Inducton Can Define T-Acute Lymphoblastic Leukemia Patients with High Risk of Relapse

AbstractAbstract 4817T-cell acute lymphoblastic leukemia (T-ALL) patients have increased risk for treatment resistance and early relapse. The precise bone marrow evaluation for the presence of minimal residual disease (MRD) is essential for guiding treatment options. This requires techniques more sensitive than the level of sensitivity of light microscopic technique such as multicolour flow cytometry (FCM). Immunophenotypic alterations called leukemia associated immunophenotypic patterns (LAIP) (i.e.aberrant myeloid markers) and ectopic phenotypic expression (i.e. appearance of immature phenotypes such as TdT, CD1a and CD3 outside their normal site in the thymus) are of benefit to track the residual leukemic cells in T-ALL.A retrospective data analysis of MRD was done comprising T-ALL patients diagnosed and followed-up at the Institute of Cancer Research/Royal Marsden Hospital by means of 3-colour flow cytometry (3C FCM).The aim was to answer a question whether the 3C FCM can reliably split patients into two groups (positive, MRD+ and negative, MRD-) and predict a subsequent relapse and to define a right time point for performing MRD tests. Eight patients were enrolled in the study following the inclusion criteria: (i) complete remission after 1st induction phase of chemotherapy, (ii) presence of LAIP or an ectopic phenotypic expression, and (iii) monitored at defined time points after initial treatment. MRD was measured during the first year of treatment as follows: at the end of phase 1 induction (day 29–35, MRD1), before the start of consolidation (3 months, MRD2), after consolidation (MRD3), during the maintenance therapy (12 months, MRD4). Immunophenotyping was performed on lysed-washed bone marrow samples using CD45 gating strategy and originally defined blast gates at diagnosis. The phenotypes to be followed-up included: TdT+/CytCD3+, CD34+/CYTCD3+, TdT+/CD2+, CD8+/CD10+, CD2+/CD10+, CD7+/CD10+, CD7+/CD33+, CD7+CD34+. Patients were divided into 2 groups in relation to subsequent relapse. Group 1 included 6 patients without relapse. Patient characteristics of the group were: male:female 5:1, mean age 17.7 years, overall survival (OS) 59 months, relapse free survival (RFS) 85 months. Group 2, relapsed patients, included 2 men, mean age 56 years, OS 13 months, RFS 8.5 months. According to the EGIL classification system the 2 men in Group 2 were with an early T-precursor phenotype, whilst Group 1 was heterogenous but cortical-T-ALL predominated. Cytogenetics/FISH and RQ-PCR studies were performed at diagnosis and showed normal karyotype in only one of the Group 2 patients.MRD results showed a difference between the two groups as regards MRD1 and MRD2 time points. Group 1 patients had negative or low MRD levels (below 0.18%) in their MRD1 bone marrow - MRD-, n=4 and MRD+,n=2 (0.18% and 0.12% respectively, sensitivity 0.04%). Those of them who were tested at MRD2 and MRD3 were negative. Both patients in Group 2 showed higher levels of MRD positivity at MRD1 (1% of total bone marrow cells), the first one of them also being positive at MRD2 and the second one becoming MRD+ at MRD4 time point. Although turning to MRD- at MRD3 time point both Group 2 patients relapsed 2.5 and 4.5 months, respectively, after the end of consolidation treatment. Additionally, Group 1 patients had a significantly longer RFS than Group 2 (median 58 months RFS vs. 8.5 months; P <0.001).Conclusions: Reliable detection of MRD in T-ALL is possible by 3C FCM using a combination of TdT and a T cell marker (cytCD3 or mCD3) as such a combination is normally found exclusively in the thymus. The higher MRD-positive levels in Group 2 reflect the more resistant disease in this group and higher probability of early relapse and shortened overall survival. Early T-cell precursor phenotype in these patients appeared to be a subtype at very high risk for treatment failure irrespective of the lack or the presence of genetic lesions. Based on MRD positivity above 0.18% at time points MRD1 or both MRD1 and MRD2 these patients need reassessment of treatment options and more intensive therapy has to be considered for relapse prevention. Finally, the results of our retrospective study suggest the usefulness of implementation of MRD testing by FCM for taking clinical decisions in the prospective clinical trials for novel therapies for T-ALL. Acknowledgments:The study was supported by the Union for International Cancer Control, Geneva, Switzerland (Grant ICRETT-080–2011) Disclosures:No relevant conflicts of interest to declare.

Increased natural killer cells and decreased CD3+CD8+CD62L+ T cells in CML patients who sustained complete molecular remission after discontinuation of imatinib

Increased natural killer cells and decreased CD3⁺CD8⁺CD62L⁺ T cells in CML patients who sustained complete molecular remission after discontinuation of imatinib

Leukemic T Cells Are Specifically Enriched In a Unique CD3dimCD7low Subpopulation of CD4+ T Cells In Acute-Type Adult T Cell Leukemia

AbstractAbstract 4144 [Background]Adult T-cell leukemia (ATL) is a malignant disorder caused by human T-cell leukemia virus type I (HTLV-I). Morphological discrimination of leukemic cells from non-leukemic T cells is often difficult in ATL since ATL cells reveal morphological diversity except for typical “flower cells”. Although a study using CD3 gating in flow cytometry reported that ATL cells were distinguishable as a CD3low population from normal lymphocytes, these cells were not well characterised as ATL cells. Considering that defective expression of CD7 as well as CD3 is common in ATL cells, we applied multi-color flow cytometry to detect a putative leukemia-specific cell population in the peripheral blood from ATL patients. [Methods and Results](1) In flow cytometry, after dead-cell and monocyte removal, CD4+ T lymphocytes were gated on the CD3 versus CD4 plot. Based on cell density and fluorescence intensity of CD3 and CD7 in this population, we designated three subpopulations on this plot: CD3highCD7high, CD3dimCD7dim and CD3dimCD7low(Results of a representative ATL and a control sample are shown in Figure). The proportion of the CD3dim/CD7low subpopulation was significantly higher in acute-type ATL CD4+ lymphocytes than in normal controls(Figure). (2) To extensively characterise this subpopulation, we next estimated the HTLV-I proviral load by quantitative real-time PCR after FACS sorting based on this CD3 versus CD7 plot. In all patient samples, HTLV-I proviral integration was detected in all subpopulations. However, the proviral load was significantly higher in the CD3dim/CD7low subpopulation compared to the CD3high/CD7high subpopulation. Almost all of the cells in the CD3dim/CD7low subpopulation were HTLV-I infected. (3) We next examined CCR4 and CD25 expression in each subpopulation. Both CCR4 and CD25 expression levels were maintained at very low and similar levels throughout all subpopulations in normal control cells and in the CD3high/CD7high subpopulation of patients with ATL as well. In contrast, CCR4 expression was significantly up-regulated in CD3dim/CD7low subpopulation of patients with ATL compared to the CD3high/CD7high subpopulation (MFI: 36.5±17.2 vs. 3.8±1.1). The expression of CD25 was also up-regulated in the subpopulation (MFI: 7.8±8.0 vs. 2.7±1.6). (4) Monoclonal expansion of HTLV-I-infected cells in the CD3dim/CD7low subpopulation was indicated by the genomic integration site analysis using a long inverse polymerase chain reaction (PCR) method. (5) We reviewed the glass-slide specimens of FACS-sorted samples to evaluate the morphology of each subpopulation on the CD3 versus CD7 plot. Atypical lymphocytes with morphology such as a notch in the nucleus were observed in all subpopulations. The majority of sorted cells from CD3dim/CD7low subpopulation showed “flower cell”-like morphology. (6) We also detected a small CD3dim/CD7dim subpopulation other than the CD3dim/CD7low and CD3high/CD7high subpopulations in all patients with acute-type ATL who were analysed(Figure). This subpopulation contained the same clone as the CD3dim/CD7low subpopulation, although a phenotypical difference existed between these subpopulations. [Display omitted] [Conclusion](1) Above findings indicate that leukemic T cells are specifically enriched in a unique CD3dim/CD7low subpopulation of CD4+ T cells in acute-type ATL. This multi-color FACS system may be useful for precisely monitoring disease during chemotherapy, detecting minimal residual disease and analysing ATL cells. (2) Previous reports have revealed that HTLV-I-infected cells transform through multi-step oncogenesis. Detailed analysis of these three subpopulations (CD3high/CD7high, CD3dim/CD7dim and CD3dim/CD7low) may give some insight into oncogenesis of HTLV-I-infected cells. Disclosures:No relevant conflicts of interest to declare.

Translational medicine as implementation science in the field of monitoring HIV and TB. New concepts emanating from resource‐poor countries

In the last decade, there has been generous support by Western nations to deal with the HIV/AIDS epidemic and accompanying plethora of infections, a significant portion of the aid is extended to resource-restricted countries (1). The wave of support has contributed to the development of a branch of biomedical activity, referred to as translational medicine. This new field covers two areas: (i) the ‘‘translational medicine proper’’ (T-1) to establish proofs of concepts mostly in animal models that lead to pilot studies in humans, and (ii) ‘‘the translational medicine-2’’ (T-2, also referred to as ‘‘implementation science’’) for optimizing already established medical concepts in rich and poor countries alike, for wider applications in communities (2). Fledgling journals which carry the ‘‘translational’’ title clearly favor T-1 publications; by contrast, on the pages of Clinical Cytometry mostly T-2 topics are featured as clinical implementations of diagnostically relevant developments. For two reasons, the T-2 field is subject to political scrutiny. First, In 2008, US $ 15.6 billion was spent on AIDS programs in low and middle income countries. Currently, a very high proportion (50%) of these are coming from external, including generous North American, sources and these may not be sustainable. Furthermore, the calculated trends reveal that this expenditure is to shoot up to 2–3 times higher levels, and that a major epidemic will still be with us in 2031 without a change in approach (3). Second, medical care in the West is, by tradition, expensive (4). The health care practiced in the US appears to be in the 37th position with respect to cost efficiency (5). Consequently, when suggesting that from now on the resource-restricted partners must contribute more to their HIV care/prevention (3), the possibilities of modifying diagnostics according to the local needs using local implementation science must be considered. This field in Africa has been revitalized since generic antiretroviral therapy (ART) became affordable. During the last decade requirements for simplifying diagnostics has frequently been advocated in the pages on Clinical Cytometry (6,7). When African health care workers are faced with acute crisis, they are often proven to be inventive. Evidence is gathering that the technologists in resource-poor regions are capable of developing and/or modifying methods in order that these are elegantly simplified for more affordable large scale testing formats. Some of these efforts represent improvements of precision, accuracy, quality assessment, and training (8–10). In his recent book, Nigel Crisp, the former Chief Executive of the National Health Service of the United Kingdom (11) explains that ‘‘poor countries do not need a diluted version of what rich countries have now, and may have an easier time building a better system from little.’’ Inspired by his approach, in this editorial, we would like to briefly review five recent papers (12–16). But first we need to consider two significant changes in the global outlook which influence the use of diagnostics. In 2006, the WHO recommended that all HIV positive patients start ART when their CD4 T-cell count fell to 200 cells/mm. In patients with CD4 counts between 200 and 350 cells/mm, the initiation of ART was optional on the basis of these patients showing some symptoms (Grade 2–4) or were still asymptomatic (Grade 1). However, in countries with poor resources this guideline was frequently interpreted as to start ART at 200/mm, reaching many patients too late (17,18) even at levels as low as 50–150 CD4 T-cells/mm. Recently, it has also been shown that earlier start of ART at higher CD4 T-cell counts reduces mortality and morbidity rates (17,18). Therefore WHO, in unison with other regulatory bodies, now recommends ART to be

Abstract 323: Early detection of cytogenetic switch from monoallelic to biallelic 13q14 deletion using BCL-2 index in CLL

Abstract Monoallelic deletion of 13q14.3 is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL). The recently described mir15a/16-1 mutation can result in increased BCL-2 expression reflecting 13q14 status. We present a case of CLL with a transient bimodal BCL-2 subclone. The patient is a 60 years old female, Rai stage IV, with a ten-year history of CLL. Prognostic markers included: mutated IgVH, Zap70, CD38 and CD49d negative, with 75 % monoallelic 13q14 deletion at the time of diagnosis. At year ten, seven-color flow cytometric evaluation was performed twice within a three month interval showed three subpopulations. BCL-2 mean fluorescence intensity (MFI), and BCL-2 index were estimated on each population. Based on forward scatter (FS) vs. side scatter (SS), and BCL-2 MFI, these cells were sorted. Interphase FISH was performed on sorted cells using D13S319 spectrum orange/LSI 13q34 spectrum green probe to define 13q14x1, 13q14x2, and diploid cells. A lymphoid population was identified by CD45 vs. SS gating. Using FS vs. SS 90.6% were small lymphoid cells (SL1) and 6.4 % were large lymphoid cell (LL1). The overall small lymphocyte population showed 1% diploid cell, 78% 13q14x1, 21% 13q14x2, with BCL-2 index 1.9. The CD19+CD5+ (SL1) population could be further sub-divided into two distinct malignant BCL-2 subclone. Each subclone had a different BCL-2 index, and different percentage of (13q14x1, 13q14x2). The first subclone (42.8%) brightly expressed BCL-2 (SL1B), and showed 59% 13x14x1, and 41% 13q14x2 with a BCL2 index of 3.1. The second subclone (57.2%) dimly expressed BCL-2 (SL1D), and showed 89% 13q14x1, 10.5 % 13q14x2, and 0.5 % diploid cell with a BCL2 index of 1.1. Both subclone were : kappa+, CD5+, CD19+,CD20+,CD27+,CD23+,CD45+, and they were negative for CD38, CD49d,and ZAP-70 . The 6.4 %(LL1) population showed 2% diploid cell, 59% 13q14x1, 39% 13q14x2, with BCL-2 index 3.5.A second sample three month later showed one uni-modal homogenous BCL-2 population within the small lymphoid cells (SL2), and a decreased in the percentage of large lymphoid cell population (LL2). Both SL2, and LL2 showed increased in the BCL-2 index compared to the first sample. SL2 (93.5 %) showed 0.5 % diploid cell, 61.5% 13q14x1, 38 % 13q14x2, with BCL-2 index 2.2, and LL2 (2.9 %) showed 1.5% diploid cell, 52.5% 13q14x1, 46% 13q14x2, and BCL-2 index 4.5. This case demonstrates that there may be a relationship between the levels of BCL-2 MFI in the CLL lymphocyte, and the relative proportions of leukemia cells having either 13q14x1, or 3q14x2. Our study is the first to explore the importance of identification of subclones within a patient harboring different BCL-2 MFI at any given time. This denotes a difference in 13q14 status, and might signal the start of a new phase of the disease. This type of analysis enabled us to time the occurrence of events if we have the BCL-2 index base line for the clone at the time of initial diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 323.

A fluorescent biomarker of the polyamine transport system to select patients with AML for F14512 treatment

The polyamine transport system (PTS), hyperactive in cancer cells, can constitute a gate to deliver F14512, a novel spermine epipodophyllotoxin conjugate recently selected for clinical development in AML phase I. We investigated in vitro the high antiproliferative effect of F14512 against 13 leukemia cell lines, and demonstrated a statistically significant correlation with the level of PTS activity, using a novel fluorescent marker F96982. This labelling protocol was then adapted for clinical applications for blood, bone marrow and AML samples with CD45 gating. Within the patient samples, the PTS activity varied significantly in AML cells, as compared to normal lymphocytes. In conclusion, the identification of PTS-positive AML with F98982 probe offers new perspectives to select patients prone to respond to F14512.

Comparison of interlaboratory variation in absolute T‐cell counts by single‐platform and optimized dual‐platform methods

Previous studies have reported that the adoption of a single-platform flow cytometry cell counting method resulted in lower interlaboratory variation in absolute T cell counts as compared to predicate dual-platform flow cytometry methods which incorporate independent automated lymphocyte counts (Schnizlein-Bick et al., Clin Diagn Lab Immunol 2000;7:336-343; Reimann et al., Clin Diagn Lab Immunol 2000;7:344-351). In the present study, we asked whether use of a single-platform method could reduce variation in absolute cell counts across the laboratories in the Multicenter AIDS Cohort Study (MACS) (n = 4), as suggested by the studies cited. Identical study samples were shipped overnight to the MACS laboratories either by the National Institute of Allergy and Infectious Diseases, Division of AIDS Immunology Quality Assessment (NIAID- IQA) proficiency-testing program (n = 14), or by the Los Angeles site of the MACS (n = 10). For each sample, two tubes of blood were received; one was used for an automated complete blood count and differential, and the other for flow cytometry. The latter was performed using both our current dual-platform method (three-color CD45 gating and automated hematology) and the single-platform method (with TruCOUNT beads to generate the absolute counts). The median percent coefficients of variation (%CVs) for the dual-platform and single-platform methods were 6.6 and 9.9, respectively, for CD4 T cell counts, and 5.9 and 8.5, respectively, for CD8 T cell counts (n = 24). These differences were not statistically significant. The differences in absolute T-cell counts between the MACS sites and the median of all laboratories participating in the NIAID-IQA were smaller for the dual-platform than for single-platform absolute count method. In contrast to previous reports, we did not observe lower interlaboratory variation across the MACS sites for single-platform absolute lymphocyte subset counting relative to dual-platform methods. This result may be at least partly explained by the lower interlaboratory variation with the optimized dual-platform method in this study relative to the previous reports.

PP-080 Application of CD3 gating to dection of HBV MHC-pentamer

PP-080 Application of CD3 gating to dection of HBV MHC-pentamer

A case of extranodal NK/T-cell lymphoma, nasal type mimicking typical manifestations of adult-onset Still’s disease (AOSD) with hemophagocytic syndrome: diagnostic consideration between malignant lymphoma without lymphadenopathy and AOSD

A 25-year-old Japanese man was suffering from high fever, sore throat, arthralgia, and macular salmon-pink eruption. The superficial lymph node was not palpable, and computed tomographic scans from the neck to pelvis demonstrated hepatosplenomegaly without apparent lymphadenopathy. Therefore, the possibility of malignant lymphoma was considered to be extremely low. Serology for Epstein Barr virus (EBV) and cytomegalovirus showed a postinfectious state, and blood culture was negative. Serum rheumatoid factor and antinuclear antibody were negative. Leukocytopenia (2.4 x 10(3)/mul) was observed, and thus a diagnosis of adult-onset Still's disease (AOSD) with hemophagocytic syndrome (HPS) was made. Fifty-five milligrams of prednisolone daily improved his symptoms and leukocytopenia promptly, but high fever with severe and progressive thrombocytopenia occurred 12 days later. Bone marrow aspiration revealed the presence of lymphoma cells and hemophagocytosis, and the CD45 gating analysis showed expanding population of CD2(+), CD3(-), and CD56(+) cells. Further, mucosal ulceration in the nasal cavity was detected. Therefore, a diagnosis of extranodal natural killer (NK)/T-cell lymphoma, nasal type, concomitant with HPS was made, and treatment with dexamethasone, etoposide, ifosfamide, carboplatin (DeVIC) regimen ameliorated his symptoms and platelet transfusion dependency. Later, a high titer of serum EBV-DNA was detected, which supported the diagnosis. Diagnosing AOSD, extranodal presentation of malignant lymphoma such as extranodal NK/T-cell lymphoma, nasal type, should be carefully considered.

Flow Cytometric Immunophenotyping of Peripheral T Cell Neoplasms Using CD3 Gating

Peripheral T cell neoplasms (PTCNs) such as peripheral T cell lymphoma, adult T cell leukemia/lymphoma, and mycosis fungoides are associated with poorer prognoses compared to B cell neoplasms. Hence, an accurate and early diagnosis is necessary for the successful treatment of PTCNs; however, this can be difficult to achieve. In this study, flow cytometric immunophenotyping using CD3 gating was performed retrospectively on 56 samples from 52 patients diagnosed with PTCNs, and the analytical data were compared with the immunohistochemical findings. Abnormal CD3 T cell populations were distinguishable from the normal T cell population, using this CD3 gating strategy.

Human leukocyte antigen-DR negative de novo acute myeloid leukemia with Philadelphia chromosome

It is well known that human leukocyte antigen (HLA)-DR is present in the majority of acute myeloid leukemia (AML) [1, 2]. However, acute promyelocytic leukemia (APL) with t(15;17)(q22;q12) usually lacks the HLA-DR expression as well as CD34 expression [2, 3]. Recently, such HLA-DR/CD34 phenotype has also been described in a subset of AML with cuplike nuclear invaginations (AML-cuplike) [4]. Cytogenetically or molecular genetically, AML-cuplike is characterized with normal karyotype and high frequency of FMS-like tyrosine kinase 3 (FLT3) gene internal tandem duplication (ITD) as well as nucleophosmin (NPM1) gene mutation [4, 5]. In this paper, we reported an extremely rare case of HLA-DR/CD34 AML, which was not categorized into either APL or AMLcuplike and showed Philadelphia chromosome (Ph) at diagnosis. A 64-year-old male was admitted to the hospital because of cellulitis of left leg in April 2008. He showed neither hepatosplenomegaly nor generalized lymph node swellings. There was no bleeding tendency. Laboratory findings were as follows: white blood cells 17.7 9 10/L, with 25% blasts and 20% basophils; hemoglobin, 14.0 g/dL; platelets, 129 9 10/L. Serum lactate dehydrogenase level and D-dimer was elevated to 386 IU/L and 50.2 lg/mL, respectively. Neutrophil alkaline phosphatase (NAP) score was within the normal range. Bone marrow showed hypercellularity with 39% blasts, 7% myelocytes, 4% metamyelocytes, 13% mature neutrophils, 1% eosinophils, 25% basophils, 8% erythroblasts and 3% lymphocytes. Morphologically, blasts were medium to large, and had round or sometimes slightly indented nuclei with dispersed chromatin. Fine azurophilic granules were sometimes found in their scant basophilic cytoplasm, but typical Auer-bodies were not detected (Fig. 1). They were cytochemically positive to myeloperoxidase (MPO) staining. Therefore, he was morphologically diagnosed as having AML-M2 according to the French–American–British (FAB) classification. There were no characteristic cuplike nuclear invaginations suggesting a diagnosis of AML-cuplike on either Wright–Giemsa or MPO staining [6]. Flow cytometric immunophenotyping using the CD45 gating method showed that leukemic blasts were CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD15, CD19, CD20, CD33, CD34, CD41, CD90, CD117 and HLA-DR, suggesting HLA-DR AML (Fig. 2). More than 85% of these cells also lacked the CD56 expression. Chromosomal analysis of bone marrow using the conventional G-banding method showed t(9;22)(q34;q11) in 14 of the 20 metaphases analyzed in addition to normal variance of inv(9)(p12q13) (Fig. 3). Fluorescence in situ hybridization (FISH) also showed BCR/ABL fusion signal in 92% of T. Inaba (&) N. Fujita Department of Infection Control and Laboratory Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan e-mail: inaba178@koto.kpu-m.ac.jp