International Clinical Cytometry Society Research Articles

Acceptable "Real-Life" Variability for Lymphocyte Counts by Flow Cytometry.

As part of quality assurance in laboratories (labs) offering clinical cell analysis by flow cytometry (FCM), cell counting precision and robustness are evaluated but international desirable ranges are still missing. The aim of this study was to provide desirable interlaboratory variability reference values. A prospective survey on monthly quality assessment was proposed to all French laboratories routinely performing lymphocyte subpopulation quantification, over one arbitrarily selected month (June 2017), regardless of instrument, counting system and quality controls used. Relative variabilities of the commercially available internal quality control (IQC) used locally were collected. Robust mean, standard deviation and CV were calculated on relative and absolute counts. Sixty-two labs participated, providing 91 sets of data on 82 instruments. All but three were enrolled in external quality assessment (EQA) and 46 in externalized IQA. The mean CV of five repeats ranged from 1.00 ± 0.33 for T cells to 4.78 ± 1.92 for NK cells and from 2.88 ± 1.46 to 5.87 ± 1.83 for relative and absolute counts, respectively. The precision correlated directly to the concentration of cells rather than the phenotype. Negligible differences were observed between IQC material: Multicheck™ (3.36 ± 1.30, n = 11); Immunotrol™ (3.62 ± 3.24, n = 21) and Statusflow™ (3.63 ± 1.87, n = 24) on CD4+ T cell, for example. Little difference was observed between counting systems such as Flowcount™ (4.55 ± 3.45, n = 19), Trucount™ (3.17 ± 1.40, n = 30) and the fully automated Aquios™ system (1.87 ± 0.75, n = 5) on single platforms, while dual platform CV was at 2.00 ± 0.58 (n = 2) on CD4+ T cell, as example. Robustness was measured on 21 ± 11 consecutive analyses of the same IQC material, providing CVs ranging from 4.45 ± 1.74 for T cells to 7.57 ± 2.19 for NK absolute counts. Average residuals calculated from the different low count IQC samples for CD4 T cells (median value below 200 cells/μl) were below 10 cells/μl, demonstrating their robustness for medical decisions. Real life variabilities in cell counting are directly related to the cell concentration and not to their phenotype. Desirable ranges within three SD are proposed according to different cell levels, based on 62 labs, different IQC material and systems. © 2018 International Clinical Cytometry Society.

Novel DRAQ5™/SYTOX® Blue Based Flow Cytometric Strategy to Identify and Characterize Stem Cells in Human Breast Milk.

Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples. Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5™ and SYTOX® blue dye as well as antibodies to characterize cells within the pellet fraction. Flow cytometry analysis identified viable DRAQ5™+ /SYTOX® Blue- cells and determined the content of CD11b+ monocytes and TRA-1-81+ putative stem cells in human breast milk samples. Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content. © 2018 International Clinical Cytometry Society.

Adult Bone Marrow Three-Dimensional Phenotypic Landscape of B-Cell Differentiation.

The different B-cell subsets in human bone marrow result from a dynamic equilibrium between endogenous production, B-cell bone marrow reentry and terminal plasma cell differentiation. Our aim was to define and quantify the different medullary B-cell subsets. A series of 32 normal adult bone marrows plus 15 normal adult blood samples was studied by nine color flow cytometry (CD10, CD19, CD24, CD27, CD34, CD38, CD45, IgM, and IgD). With the Kaluza software radar plots, two 2D triple parametric histograms (CD10/CD34/CD45 and CD27/IgM/IgD) were set-up to identify six progenitor and five mature B-cell subsets. Very early B-cell progenitors were CD19neg/CD10pos/CD34pos. B-cell progenitors were split into five subsets on the CD10/CD34/CD45 triple parametric histogram, sequentially ordered according to the loss of CD34 and CD10 and acquisition of surface IgM and IgD. CD19pos/CD38low mature B-cells were divided into four subsets on the CD27/IgM/IgD triple parametric histogram, with two stages of naïve B-cells and two CD27hi marginal zone and switched memory B-cell compartments. CD19pos/CD34neg/CD10low immature B-cells were the main bone marrow B-cell subset, accounting for one third of bone marrow B-cells. Transitional B-cells were the only immature bone marrow stage found in the blood. Compared to blood, the bone marrow was enriched in both marginal zone and switched B-cells. We provide the first analysis of 3D B-cell differentiation by multicolor flow cytometry leading to propose reference values for each bone marrow and blood B-cell compartment. This warrants further exploration of normal and pathological human B-cell maturation. © 2018 International Clinical Cytometry Society.

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping.

BackgroundWhile it is known that CD123 is normally strongly expressed on plasmacytoid dendritic cells and completely absent on nucleated red blood cells, detailed information regarding CD123 expression in acute leukemia is scarce and, if available, hard to compare due to different methodologies.MethodsCD123 expression was evaluated using standardized EuroFlow immunophenotyping in 139 pediatric AML, 316 adult AML, 193 pediatric BCP‐ALL, 69 adult BCP‐ALL, 101 pediatric T‐ALL, and 28 adult T‐ALL patients. Paired diagnosis‐relapse samples were available for 57 AML and 19 BCP‐ALL patients. Leukemic stem cell (LSC) data was available for 32 pediatric AML patients. CD123 expression was evaluated based on mean fluorescence intensity, median fluorescence intensity, and percentage CD123 positive cells.ResultsEuroFlow panels were stable over time and between laboratories. CD123 was expressed in the majority of AML and BCP‐ALL patients, but absent in most T‐ALL patients. Within AML, CD123 expression was lower in erythroid/megakaryocytic leukemia, higher in NPM1 mutated and FLT3‐ITD mutated leukemia, and comparable between LSC and leukemic blasts. Within BCP‐ALL, CD123 expression was higher in patients with (high) hyperdiploid karyotypes and the BCR‐ABL fusion gene. Interestingly, CD123 expression was increased in BCP‐ALL relapses while highly variable in AML relapses (compared to CD123 expression at diagnosis).ConclusionsAuthors evaluated CD123 expression in a large cohort of acute leukemia patients, based on standardized and reproducible methodology. Our results may facilitate stratification of patients most likely to respond to CD123 targeted therapies and serve as reference for CD123 expression (in health and disease). © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

Alteration of T Cell Phenotypes in HIV‐Neurotuberculosis Coinfection

Neurotuberculosis is one of the commonest HIV associated opportunistic infections of the central nervous system in India. HIV-TB coinfection may lead to altered frequencies of T cells, thereby influencing the course and progression of the disease. We examined the frequencies of T cell subsets in HIV infected individuals with neurotuberculosis (HIV+nTB+) as compared to individuals with HIV associated systemic TB (HIV+sTB+), asymptomatic HIV (HIV+TB-), non-HIV neuro TB (HIV-nTB+), non-HIV systemic TB (HIV-sTB+), and healthy controls (HIV-TB-). Activation and senescence profiles of CD4 and CD8 T cells and memory subsets in peripheral blood mononuclear cells were studied by flow cytometry. The significant observations among the T cell subsets in HIV+nTB+ were: (1) Naïve T cells: decreased CD4 T cells compared to HIV-sTB+ (P = 0.005); decreased CD8 T cells compared to HIV-nTB+ and HIV-TB- (P ≤ 0.007), (2) Memory T cells: expanded CD4 TEMRA cells compared to HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.003); expanded CD8 TEMRA cells compared to HIV-nTB+ and HIV-TB- (P ≤ 0.005), (3) Activated T cells: higher CD4 T cells compared to HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.004); higher CD8 T cells compared to HIV + TB-, HIV-nTB+, HIV-sTB+, and HIV-TB- (P ≤ 0.001), and (4) Senescent T cells: increased CD8 T cells compared to HIV-nTB+ and HIV-TB- groups (P = 0.000). Increased activation compared to HIV+TB-, HIV-nTB+, HIV-sTB+, and HIV-TB- groups and increased senescence compared to HIV-nTB+ and HIV-TB- groups were observed in CD8 T cells in HIV+nTB+, suggesting that the frequencies of these T cell subsets are altered to a greater extent in these individuals. © 2018 International Clinical Cytometry Society.

Identification of functionally primitive and immunophenotypically distinct subpopulations in secondary acute myeloid leukemia by mass cytometry.

Background: Mass cytometry (CyTOF) is a powerful tool for analyzing cellular networks at the single cell level. Due to the high-dimensional nature of this approach, analysis algorithms have been developed to visualize and interpret mass cytometry data. In this study, we applied these approaches to a cohort of patients with secondary acute myeloid leukemia (sAML). We utilized mass cytometry to interrogate localization and intensity of thrombopoietin-mediated intracellular signaling in sAML. Extracellular and intracellular phenotypes were dissected using SPADE, viSNE, and PhenoGraph. Healthy controls exhibited highly localized signaling responses largely restricted to the hematopoietic stem/progenitor cell (HSPC) compartment. In contrast, sAML samples contained subpopulations outside the HSPC compartment exhibiting thrombopoietin (TPO) sensitivity comparable to or greater than immunophenotypically defined HSPCs. We employed unsupervised clustering by PhenoGraph to elucidate distinct subpopulations within these heterogeneous samples. One metacluster composed almost exclusively of Lin- CD61+ CD34- CD38- CD45low cells was identified. This subpopulation was not readily identified by established manual gating approaches, and generally exhibited greater STAT phosphorylation in response to TPO stimulation than did Lin- CD61- CD34+ CD38- cells. Lin- CD61+ CD34- CD38- CD45low cells were identified in three additional sAML patients analyzed independently using a manual gating approach based upon PhenoGraph results. Each patient exhibited a similar TPO hypersensitivity to the PhenoGraph metacluster. The identification of this cellular subpopulation highlights the limitations of manual gating in sAML. Our study demonstrates the potential for mass cytometry to elucidate rare subpopulations in highly heterogeneous tumors by utilizing unsupervised high dimensional analysis. © 2018 International Clinical Cytometry Society.

Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This unit will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry. In addition, the unit will provide practical procedures for three different types of validation. The first is a limited validation protocol that is applicable for research settings and non-regulated laboratories. The second is validation protocol that presents the minimum validation requirements in regulated laboratories. The third is a transfer validation protocol to be used when methods are transferred between laboratories. The recommendations presented in this unit are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (currently in preparation). © 2018 by John Wiley & Sons, Inc.

BrdU incorporation in multiparameter flow cytometry: A new cell cycle assessment approach in multiple myeloma.

Mutiple myeloma (MM) is a neoplasia characterized by the accumulation of malignant plasma cells (PC) in the bone marrow. Although proliferation markers have been studied in MM, none of the current staging systems include them. Moreover, approaches used to analyze proliferation do not separate MM cells (MMCs) from normal PC. In this study, we combined multiparameter flow cytometry and BrdU incorporation or Ki67 staining to analyze MM cell proliferation in 44 monoclonal gammopathy of undetermined significance (MGUS), 153 newly diagnosed MM patients and 69 MM patients at relapse. The prognostic value of proliferation assessment was analyzed in 60 newly diagnosed patients treated with high-dose chemotherapy supported by autologous hematopoietic stem cell transplantation. The median number of proliferating malignant PC significantly increases during MM disease progression. MM patients with a percentage of proliferating MMCs greater than 1.42% using BrdU/DAPI or greater than 1.1% using ki67/DAPI, are associated with a significantly shorter event free survival compared with patients with a lower percentage of proliferating MMCs. Combination of flow cytometry with BrdU or ki67/DAPI staining could become a standard for the determination of MM cell proliferation. Furthermore, in the context of new effective myeloma treatment options, assessment of MM cell proliferation may be valuable, in clinical trials, to identify novel agents that could significantly affect the small proliferative compartment of MM cells. © 2018 International Clinical Cytometry Society.

Pattern associated leukemia immunophenotypes and measurable disease detection in acute myeloid leukemia or myelodysplastic syndrome with mutated NPM1.

The presence of measurable residual disease after therapy is a significant risk factor of relapse in patients with acute myeloid leukemia (AML). By detecting cells with leukemia-associated immunophenotype (LAIP), multiparameter flow cytometry (MFC) can detect residual leukemia at a level significantly lower than that detected by morphology. However, changes in LAIPs during or after therapy may pose a challenge to MRD testing. AML with mutated NPM1 represents the largest subtype of AML sharing a common leukemogenic mechanism and similar LAIPs. Here, we identified a common pattern of LAIPs in myeloid blasts with mutated NPM1, and studied its stability and limit of detection after therapy. We summarized aberrancies of leukemic blasts with mutated NPM1 at diagnosis in 61 patients and paired relapse in 25 patients. In addition, we examined the detection of leukemic blasts in 590 specimens collected from 152 patients in complete remission after induction for AML/MDS-EB with mutated NPM1. Our findings demonstrate myeloid blasts with mutated NPM1 have a characteristic pattern of LAIPs that is present in nearly all cases of AML/MDS-EB with mutated NPM1 at initial diagnosis and relapse, regardless of morphologic variations, FLT3 ITD status, or karyotype abnormality. The myeloid blasts with mutated NPM1 can be detected at an approximate level of 0.1% of total leukocytes in morphologic remission with high specificity validated by clinical outcome. The characteristic pattern of LAIPs of myeloid blasts with mutated NPM1 is common and stable, and allows sensitive and specific detection of AML or MDS with mutated NPM1 after therapy. © 2018 International Clinical Cytometry Society.

Determination of Reference Values of MDR-ABC Transporter Activities in CD3+ Lymphocytes of Healthy Volunteers Using a Flow Cytometry Based Method.

MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined. The Solvo MDQ Kit™ was used for measurements. In this assay, fluorescent reporter substrates (Calcein-AM for MDR1 and MRP1 and mitoxantrone for BCRP, respectively) are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively), allowing for quantitative, standardized assessment. Cell surface staining was applied to select CD3+ cells. MAF values of MRP1 and BCRP are independent from age. MAFC and MAF of MDR1 show negative correlation with the age of the studied subjects (P = 0.003, r = -0.27 and P = 0.0001, r = -0.34, respectively). No difference was detected in any of the four MAF values between men and women. Gender does not affect the presence or lack of correlation between MAF values and age. The determination of the functional activity of MDR-ABC transporters is achievable using a flow cytometry based standardized method. Having established the normal range of MAF values on CD3+ lymphocytes of a healthy population, our results allow for the development of novel flow cytometry based diagnostic tools. © 2018 International Clinical Cytometry Society.

Use of flow cytometry in the phenotypic diagnosis of hodgkin's lymphoma.

Hodgkin's lymphoma (HL) has a unique immunophenotype derived from immunohistochemistry (positive for CD15, CD30, and Pax-5; negative for CD3, CD20 in most cases, and CD45). The knowledge gained over recent years enables better diagnosis, prognosis, and treatment of HL. Flow cytometry as a tool for the diagnosis of classic HL has not been useful in the past due to the difficulty in isolating Reed-Sternberg cells as they are admixed in a rich inflammatory background which consists mainly of T cells, B cells, eosinophils, histiocytes, and plasma cells. However, in the recent past, several studies have tried to identify Reed-Sternberg cells using flow cytometry on fine needle aspiration or tissue biopsy of lymph nodes to confirm or supplement immunohistochemistry staining in diagnosis. Newer and more sensitive tools such as flow cytometry can be used for diagnosis, technology that may have been difficult in the past for diagnosis of this lymphoma subtype. Using flow cytometry, diagnosis is faster and could lead to point-of-care technology especially where we have typical immunophenotype signatures. © 2018 International Clinical Cytometry Society.

Flow Cytometric Analysis of Mechanically Disaggregated Bone Marrow Trephine Biopsies.

Bone marrow (BM) aspirate analysis by flow cytometry (FC) is a key hematopathologic technique but dry taps hinder diagnosis. We describe the utility and limitations of a method of mechanically disaggregating BM trephine biopsies for FC. Trephine biopsies mechanically disaggregated for FC between 2010 and 2016 were reviewed. We recorded cell yield, pathological findings, and turnaround time. Where available, results of trephine FC were compared with aspirate FC. Eighty BM trephine samples were processed, representing 4.3% of BM biopsies. Mechanical disaggregation yielded cellular samples in 98% of cases (median yield 3 × 106 nucleated cells). The most frequent diagnoses were B cell lymphoproliferative disorders (n = 26) and acute leukemia (n = 18). Trephine FC correlated with histochemistry findings in 94% of cases, but two Hodgkin lymphoma infiltrates were missed by FC, and trephine FC underestimated B cell infiltrate burden compared to immunohistochemistry (IHC). Fluorescence intensity of CD34 and CD45 on disease populations was similar in processed trephine and contemporaneous aspirate samples. Trephine FC results were available 2 days earlier than stained IHC slides. Mechanical disaggregation of BM trephine samples provided a cellular suspension suitable for diagnostic FC in most cases. Limitations were similar to aspirate FC: disease burden was underestimated and some infiltrates were missed. Trephine FC results were available earlier than trephine IHC. We conclude that trephine FC is a useful technique to complement trephine IHC in the event of a failed aspirate, providing rapid diagnostic immunophenotyping. © 2018 International Clinical Cytometry Society.

Immunophenotypic Profile of CD34+ Subpopulations and Their Role in the Diagnosis and Prognosis of Patients with De-Novo, Particularly Low-Grade Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders with unknown aetiology. Multiparameter flow cytometry (MFC) is being evaluated for the diagnosis and prognosis of MDS. In the present study, five-color MFC was performed on bone marrow aspirates of 50 untreated patients, newly diagnosed with MDS and 27 age matched control samples. Patients were classified according to World Health Organization 2016, International Prognostic Scoring System (IPSS), and Revised IPSS (IPSS-R). Significantly higher CD133+/CD90-CD45weak, CD117+/TdT-CD45weak, and CD33+/MPO-neutrophil precursor percentages on CD34+ cells, as well as a significant decrease of lymphoid and erythroid precursors were observed in the group of MDS patients in comparison to controls. A new scoring system was based on these findings, which can be helpful in discriminating lower risk MDS patients, including those with normal karyotype (a subgroup of MDS with diagnostic challenges). In addition, an increased level of apoptosis of CD34+/CD117+ cells was identified as an independent favorable prognostic factor both for the risk of transformation to acute myeloid leukemia and for overall survival. A new scoring system based on the expression of immature cell antigens on CD34+ cells (by itself or in combination with the Ogata score) can discriminate lower risk MDS patients, including those with normal karyotype, from the normal control group. © 2018 International Clinical Cytometry Society.

Extended immunophenotyping reference values in a healthy pediatric population.

For the accurate diagnosis of immunodeficiencies is crucial to compare patients' immunology laboratory values with age-sex matched controls, yet there is a paucity of normal values for most populations. To define appropriate reference values of extended lymphocyte subpopulations and T-cell receptor excision circle (TRECs) levels in healthy pediatric donors between 1 month and 18 years of age. Extended immunophenotyping values were obtained by analysis of multiparameter flow cytometry panels for the following subpopulations: CD4+ and CD8+ Naive, Effector, Effector Memory and Central Memory, T helper subpopulations and their degrees of activation, T Regulatory cells, Recent Thymic Emigrants (RTE), B Lymphocyte subpopulations (Transitional, Naive, Preswitch-Memory, Switch-Memory, Plasmablasts, CD21low, and Exhausted), and subpopulations for Monocytes, NK cells and Dendritic Cells. Median values and the 10th and 90th percentiles were obtained for 32 lymphocyte and monocyte subpopulations, and for TRECs levels in each age group of children. Naive CD4+ and CD8+ T-cell populations tended to decrease with age, with significant difference between the groups, in parallel with the reduction in thymic function assessed by TRECs counts and the recent thymic emigrant population. Relative numbers of Th cell populations tended to increase with age. The percentage of class-switched B cell populations showed a significant increase between the youngest group and the others. This study provides essential data for interpreting extended immunophenotyping profiles in the pediatric and young adult populations, which could be of value for the diagnosis of PIDs and immune-mediated diseases, particularly those associated with subtle immunological abnormalities. © 2018 International Clinical Cytometry Society.

Evidence of a role for functional heterogeneity in multidrug resistance transporters in clinical trials of P-glycoprotein modulation in acute myeloid leukemia.

Multidrug resistance (MDR) transporter proteins such as P-glycoprotein (P-gp) efflux a variety of chemotherapeutic drugs from acute myeloid leukemia (AML) blasts leading to clinical drug resistance. This study examined heterogeneity of MDR functional efflux by AML blasts using two flow cytometry bioassays. Bone marrow specimens (N = 50) from elderly patients with newly diagnosed AML were analyzed for CD34+ blasts with MDR efflux function. Efflux was measured with a fluorescent dye (DiOC2 ) as a surrogate for oncology drugs that are substrates for MDR efflux. P-gp-mediated efflux was differentiated from non-P-gp MDR activities using zosuquidar, a highly selective P-gp modulator. The bioassays included a zosuquidar-dependent DiOC2 accumulation bioassay that measured only P-gp. The second method, termed the efflux bioassay, could detect P-gp and other non-P-gp efflux depending on bioassay culture conditions. Sixty-two percent of the specimens were considered positive for blasts with P-gp function, and 26% of such P-gp-positive specimens also exhibited zosuquidar-resistant (i.e., non-P-gp) MDR efflux activity; 37% of P-gp-negative AML blast specimens displayed zosuquidar-resistant MDR function in the efflux bioassay. These results confirm the heterogeneous nature of MDR efflux pumps in AML blasts, and provide support for the hypothesis that non-P-gp MDR contributed to negative results with zosuquidar in AML trials like ECOG-ACRIN E3999. © 2018 International Clinical Cytometry Society.

Application of immunophenotypic analysis in distinguishing chronic myelomonocytic leukemia from reactive monocytosis.

The purpose of this study was to determine whether immunophenotypic profiles detected by flow cytometry are useful in differentiating chronic myelomonocytic leukemia (CMML) from reactive monocytosis, and between CMML subtypes. Eight-color flow cytometry was used to immunophenotype blasts, monocytes, and granulocytes in the bone marrow of 34 patients with CMML and 12 patients with reactive monocytosis. Bone marrow myeloblast, promonocyte, and monocyte counts by flow cytometry were significantly higher in the CMML group than in the reactive monocytosis group. Myeloblast aberrancies were present in all CMML patients as compared with 2 of 12 (16.7%) reactive monocytosis patients (P < 0.001). The number of blast aberrancies ranged from one to nine (median, four) in CMML patients and 94.1% of CMML cases exhibited ≥ two aberrancies. In contrast, two reactive monocytosis cases showed only one phenotypic abnormality of blasts. Monocyte and granulocyte aberrancies were present in 26 of 34 (76.5%) and in 31 of 34 (91.2%) CMML patients, respectively. Decreased side scatter (SSC) and abnormal CD11b/CD13/CD16 maturation pattern in granulocytes were more frequent in CMML than in reactive monocytosis. No significant differences in antigen expression were detected between the CMML subtypes except that altered CD45/SSC pattern on the blasts was more commonly observed in CMML-0/1 than in CMML-2. CMML has phenotypic aberrancies in monocytes, granulocytes, and more frequently in myeloblasts. Aberrant expression of two or more antigens in myeloblasts by flow cytometry has a high sensitivity (94.1%) and a high specificity (100%) to differentiate CMML from reactive monocytosis. © 2018 International Clinical Cytometry Society.

Flow Cytometric Analysis of Erythrocytes Osmotic Fragility in Hereditary Spherocytosis: A Case-Controlled Study Evaluating the Best Anticoagulant, Sample Pre-Treatment and NaCl Concentration for Reliable Screening of this Red Blood Cell Membrane Disorder.

The cytometric flow osmotic fragility test (FC-OFT) was recently introduced. However, the test is still under development and some variables have not yet been fully tested. The osmotic fragility of hereditary spherocytosis (HS) cases and healthy controls were evaluated by FC-OFT using a series of tubes containing decreasing concentrations of NaCl. The analyses were executed in fresh and incubated (37°C for 24 h) blood samples anticoagulated with EDTA and heparin. The percentages of residual red blood cells were used to plot the osmotic fragility curves. The OF curves of each tested condition were compared using the median corpuscular fragility (MCF). ROC curve analyses identified the most accurate NaCl concentrations for differentiation between HS cases and healthy controls. FC-OFT curves assumed a sigmoidal dose-response shape and the MCF of cases and controls were different in all instances. MCF comparisons revealed that incubation and anticoagulant have major and minor effects on the FC-OFT, respectively. One hundred percent of sensitivity and specificity was obtained from 5.5 to 6.0 g/L of NaCl in EDTA-treated fresh blood, from 6.0 to 8.0 g/L of NaCl in EDTA-treated incubated blood, and in none of the tested NaCl concentration in heparinized blood. EDTA is the anticoagulant of choice for the assay. Incubation at 37°C for 24 h increased its diagnostic capability. The most reliable NaCl concentration for the discrimination of HS case from controls was 6.0 g/L of NaCL in fresh EDTA-treated blood, and was 7.5 g/L of NaCl in incubated EDTA-treated blood. © 2018 International Clinical Cytometry Society.

Naïve/memory T-cell phenotypes in leukemic cutaneous T-cell lymphoma: Putative cell of origin overlaps disease classification.

Mycosis fungoides (MF) and Sézary Syndrome (SS) are clinically distinct cutaneous T-cell lymphomas with strikingly similar morphologic and phenotypic features. Prior studies have suggested phenotypic differences based on markers of antigen experience, suggesting a different cell of origin. Seventy-nine involved peripheral blood or bone marrow samples from 33 patients with SS and 19 patients with MF were studied by 10-color flow cytometry, including CD62L, CD45RA, CCR4, and PD-1. Gated tumor events were classified as naïve (TN ), central memory (TCM ), effector memory (TEM ), or effector memory with reacquired CD45RA (TEMRA ); based on CD62L+ /CD45RA+ , CD62L+ /CD45RA- , CD62L- /CD45RA- , or CD62L- /CD45RA+ phenotype, respectively. Sequential specimens were compared to assess for phenotypic stability. The naïve/memory phenotype of the neoplastic T-cells was markedly heterogeneous, with a dominant TN , TCM , TEM , or TEMRA subset on 11 (14%), 32 (41%), 30 (38%), and 6 (8%) cases, respectively. There was no correlation between the diagnosis of MF or SS and putative cell of origin (P = 0.4). Overexpression of CCR4 and PD1 was observed in most cases, with higher intensity in SS compared to MF. The naïve/memory phenotype remained the same for 10 patients up to 273 days after the initial analysis; while on six patients, the naïve/memory phenotype was different from the original phenotype. Both SS and MF can have phenotypic features of any of the major naïve/memory T-cell subsets, which questions the current principle of "cell-of-origin" distinction between SS and MF. Phenotypic shifts within these subsets are common, suggesting a functional state rather than a cell-of-origin surrogate. © 2018 International Clinical Cytometry Society.

CD200 is a useful marker in the diagnosis of chronic lymphocytic leukemia.

The diagnosis of CLL is supported by a typical morphology and immunophenotype and usually does not present difficulties. Nevertheless, some patients with CLL can show an atypical phenotype, this raising the possibility of a lymphoproliferative disorder other than CLL. It has been recently shown that the expression of CD200 could be a rather consistent marker for CLL. The expression of CD200 was investigated in 120 consecutive patients with B-cell chronic lymphoproliferative disorders (B-CLPD) (65 cases diagnosed as typical CLL, 16 atypical CLL, and 39 non-CLL before entering the study) by using multiparametric flow cytometry with four color combinations. CD200 was analyzed as percentage of positive cells (≥30%) and MFIR expression. ROC curves were used to determine the cut-off for the CD200 MFIR. Matutes score (MS) was used as comparator. All 81 (100%) patients classified as CLL and 25 of 39 (64.1%) classified as non-CLL expressed high CD200 expression (≥30%). CD200 expression showed a high sensitivity (100%) and a low specificity (35.9%), and the accuracy was similar to that of Matutes score markers (range: 79.2%-86.7%); except SmIg that was 59.1%. The addition of CD200 to the Matutes score correctly identified 74 of 81 (91.4%) CLL cases including 9 of 16 atypical CLL cases. As per non CLL cases, 37 of 39 (94.9%) were correctly diagnosed by the modified system. Altogether, CD200 improved the diagnostic accuracy of Matutes score from 86.7% to 92.5% (P < .01). These results show that CD200 is a valuable, albeit not specific, CLL diagnostic marker. © 2018 International Clinical Cytometry Society.

CD133 expression in circulating hematopoietic progenitor cells.

Circulating hematopoietic progenitors (HPCs) are involved in inflammatory diseases such as atherosclerosis, the immune response to cancer, and disorders of the hematopoietic system. HPC characterization by flow cytometry typically utilizes CD34 in combination with other cell surface markers to identify cell populations that give rise to specific hematopoietic lineages. CD133, also known as prominin-1, is a cell surface protein found in HPCs that has a similar but not interchangeable expression pattern with CD34 for characterization of HPC populations. Our goal was to determine the distribution of CD133 expression within HPC sub-types amongst circulating CD34+ cells. The quantity of different HPC populations within the CD34+ CD133+ and CD34+ CD133- fractions of venous blood obtained from healthy human subjects was measured using multicolor flow cytometry. The majority of circulating CD34+ cells is CD133-. CD133+ CD34+ cells express low levels of CD38, contain cell populations bearing cell surface markers of hematopoietic stem cells, multipotent progenitors, and multilymphoid progenitors, and are largely devoid of CD38 expressing lineage specified progenitors. These findings clarify the composition of circulating CD133+ CD34+ cell types in adult human subjects. © 2018 International Clinical Cytometry Society.