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Contains fulltext : 290820.pdf (Publisher’s version ) (Open Access) Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34(+) CD19(-) ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS. 01 januari 2023

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units.Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 106/kg body weight) after lymphodepleting chemotherapy without cytokine boosting.Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with 4 T cells/kg and 5 B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly, in vivo HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential “off-the-shelf” translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107–18. ©2017 AACR.

Figure S2. Cy/Flu conditioning induces temporary lymphocytopenia.

Figure S3. HSPC-NK cells mature in vivo.

Figure S1. Functional activity of HSPC-NK cell products.

Supplementary materials and methods, Supplementary Tables and Supplementary Figures File Supplementary Table 1. Characteristics of HSPC-NK cell products. Supplementary Table 2. Functional activity of HSPC-NK cell products. Supplementary Table 3. Adverse events associated with Cy/Flu regimen.

Contains fulltext : 290818.pdf (Publisher’s version ) (Open Access) BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice. 01 januari 2023

Systemic inflammation (SI) plays a detrimental role in various conditions with high mortality rates1–4. SI manifests an acute hyperinflammation followed by long-lasting immunosuppression, increasing patients’ risks for secondary infections and impaired clinical outcomes5–7. Due to the extensive heterogeneity in SI etiology, the mechanisms governing these states are incompletely understood. Here, we characterized acute and late effects of lipopolysaccharide (LPS)-induced SI (LPS-SI8) on blood monocytes and bone marrow (BM) cells of healthy volunteers. Like clinical SI, LPS administration elicited a profound but transient acute response. Single-cell transcriptomic analysis of acute LPS-SI unveiled loss of BM monocytes and appearance of an inflammatory monocyte-like (i-Mono’s) population, expressing gene programs similar to early-stage sepsis patients9. In the ensuing late phase of LPS-SI, we observed reduced expression of interferon type I (IFN-I) responsive genes in monocytes and profound attenuation of in vivo response to a second LPS challenge. Furthermore, late LPS-SI led to impaired myelopoiesis with a loss of intermediate and non-classical monocytes. In accordance, we show compromised myelopoiesis also occurs in late-stage sepsis. Finally, IFNβ treatment reversed LPS-induced immunosuppression in monocytes. Our results reveal long-lasting effects of SI on myelopoiesis and substantiate the importance of IFN-I in the pathophysiology of SI-induced immunosuppression.

The 10-color panel consisting of 21 monoclonal antibodies (mAbs) is developed as a one-tube panel to detect leukemia and lymphoma cells in all hematopoietic cell lineages. In particular, this tube is mentioned for a fast screening to identify aberrant cells in samples suspected for malignant cell localization and to enable comprehensive immunophenotyping of samples with low cell counts. The panel contains mAbs for selection of the populations and mAbs against target antigens on the various hematopoietic maturation stages. Due to the limited number of PMTs in most used flow cytometers for clinical purposes, stacking of conjugates in one color is needed to include all relevant markers for simultaneous analysis of the aberrant cells. The 21-mAb panel is tested on peripheral blood (PB), and bone marrow (BM) samples and enables an efficient and correct identification of hematological malignancies. This panel improves the diagnostic potential.

Flow-cytometric detection of now termed measurable residual disease (MRD) in acute myeloid leukemia (AML) has proven to have an independent prognostic impact. In a previous multicenter study we developed protocols to accurately define leukemia-associated immunophenotypes (LAIPs) at diagnosis. It has, however, not been demonstrated whether the use of the defined LAIPs in the same multicenter setting results in a high concordance between centers in MRD assessment. In the present paper we evaluated whether interpretation of list-mode data (LMD) files, obtained from MRD assessment of previously determined LAIPs during and after treatment, could reliably be performed in a multicenter setting. The percentage of MRD positive cells was simultaneously determined in totally 173 LMD files from 77 AML patients by six participating centers. The quantitative concordance between the six participating centers was meanly 84%, with slight variation of 75%-89%. In addition our data showed that the type and number of LAIPs were of influence on the performance outcome. The highest concordance was observed for LAIPs with cross-lineage expression, followed by LAIPs with an asynchronous antigen expression. Our results imply that immunophenotypic MRD assessment in AML will only be feasible when fully standardized methods are used for reliable multicenter assessment. ispartof: LEUKEMIA RESEARCH vol:76 pages:39-47 ispartof: location:England status: published

Current risk algorithms are primarily based on pre-treatment factors and imperfectly predict outcome in acute myeloid leukemia (AML). We introduce and validate a post-treatment approach of leukemic stem cell (LSC) assessment for prediction of outcome. LSC containing CD34+CD38− fractions were measured using flow cytometry in an add-on study of the HOVON102/SAKK trial. Predefined cut-off levels were prospectively evaluated to assess CD34+CD38−LSC levels at diagnosis (n = 594), and, to identify LSClow/LSChigh (n = 302) and MRDlow/MRDhigh patients (n = 305) in bone marrow in morphological complete remission (CR). In 242 CR patients combined MRD and LSC results were available. At diagnosis the CD34+CD38− LSC frequency independently predicts overall survival (OS). After achieving CR, combining LSC and MRD showed reduced survival in MRDhigh/LSChigh patients (hazard ratio [HR] 3.62 for OS and 5.89 for cumulative incidence of relapse [CIR]) compared to MRDlow/LSChigh, MRDhigh/LSClow, and especially MRDlow/LSClow patients. Moreover, in the NPM1mutant positive sub-group, prognostic value of golden standard NPM1-MRD by qPCR can be improved by addition of flow cytometric approaches. This is the first prospective study demonstrating that LSC strongly improves prognostic impact of MRD detection, identifying a patient subgroup with an almost 100% treatment failure probability, warranting consideration of LSC measurement incorporation in future AML risk schemes.

A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.

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Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation of innate immune cells occurs at the level of myeloid progenitors, which adds exciting opportunities for novel treatment strategies.

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Acute myeloid leukemia (AML) is a heterogeneous clonal disease and in different patients, different combinations of mutations may be found that drive the disease. Also within patients, various leukemic clones with a different combination of mutations may co-exist, reflecting various evolutionary stages of the disease. At present, flowcytometry and mutational analyses are used to keep track of the malignant cells during and after treatment. Using panels of antibodies, abnormal cells can be identified when lineage markers are over- or underexpressed, or when abnormal differentiation leads to the co-expression of markers that normally are not expressed together on hematopoietic cells. These patient-specific leukemia-associated immunophenptypes (LAIPs) can be followed during and after treatment, and enable the early detection of resistant and recurring disease. Often, several LAIPs are present simultaneously within a patient, but their clonal relationship is not always clear. In addition, next-generation sequencing and mutation-specific PCR can be used to follow the leukemic cells. As the clinical consequences of MRD-positivity may consist of high-dose re-induction chemotherapy or stem cell transplantation, accurate results are of the highest importance. Although it has already been shown that flowcytometry and mutational analysis may have added value, in-depth information on the genetic make-up of immunophenotypically defined normal and leukemic subclones and their clonal relationship in AML is still scarce. To study the genetic similarities and differences between various normal and leukemic subclones defined by immunophenotyping, we performed an in-depth analysis of the genetic makeup of FACS-sorted immunophenotypically normal and aberrant subclones in 10 cases of AML. In total, 86 LAIPs and normal cell fractions were sorted and fully genotyped using error-corrected sequencing with a panel genes that are recurrently mutated in AML. In some cases, different aberrant populations from the same patient carried identical mutations, indicating that the leukemia was highly homogeneous, and that the phenotypically different LAIPs all were part of the same genetic clone. In these cases, monitoring the disease by following one of the present LAIPs would be sufficient, or, alternatively, following disease dynamics by molecularly monitoring one of the mutations would accurately show the dynamics of the disease. In other cases, different subclones harbored a different set of mutations. In addition, immunophenotypically normal populations frequently carried mutations, or, inversely, no mutations were found in immunophenotypically clearly aberrant clones. In these cases, following the disease by LAIPs or molecular markers would carry the risk that several subclones would be missed. In case these clones would be relevant for resistance or relapse, using either technique would be at risk to fail to detect MRD positivity. This work underscores that currently available, state-of-the-art immunophotyping and gene panels have added value and should be used simultaneously. We conclude that currently, MRD detection by molecular and immunophenotypic techniques are complementary, and should be used in parallel in order to obtain the most complete view on resistant disease and early detection of relapse. At the same time, both techniques can be further optimized with more monoclonal antibodies and genes that are screened for mutations, in order to increase their resolution. Disclosures No relevant conflicts of interest to declare.

Background: Myelodysplastic syndromes (MDS) are considered clonal diseases and are diagnosed according to WHO by cytomorphology and cytogenetics. The diagnostic potential of flow cytometric aberrancies has not yet been comprehensively evaluated. Aim: Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies predefined according to European LeukemiaNet (ELN). Methods: 1682 patients undergoing diagnostics for suspected MDS according to WHO 2016 criteria were analyzed in parallel by flow cytometry according to ELN recommendations. Results: Median age was 72 years (18-97). MDS, MPN-RS-T or CMML were confirmed by cytomophology in 1029 (61%) cases, 653 (39%) were non-MDS. IPSS-R data was available in 857 (51%). An overall flow cytometric readout was available in 1679 (99.8%). 1001 (60%) were in agreement with MDS while 678 (40%) were not. Flow cytometric readout significantly correlated with cytomorphologic diagnosis (p Non-MDS cases had a fewer myeloid progenitor cells (MPC) (mean±SD, 0.8±0.9%) compared to low-risk MDS (1.7±2.3%, p3% was strongly associated with MDS/CMML (286/293, 98%, p Neutrophil aberrancies were found more frequently in neoplastic cases than in non-MDS cases (table 1). Again, frequencies of aberrations were higher for high-risk MDS as compared to low-risk MDS while this was not the case for CMML showing frequencies rather similar to low-risk MDS. Frequencies of aberrancies in monocytes revealed a similar figure as in neutrophils with higher rates in neoplastic cases but clearly significant numbers positive in non-MDS cases. Interestingly, frequencies were not higher in high-risk MDS as compared to low-risk MDS. As anticipated, frequencies were highest in CMML (table 1). Regarding erythroid cells only an aberrant percentage of them and aberrant CD71 expression were found in a reasonable number of cases. Importantly, rates of positivity were rather high in non-MDS cases which did not differ from CMML cases (table 1). In order to identify the diagnostic value of each individual aberrancy multivariate analyses were performed in the three subgroups, low-risk MDS, high-risk MDS and CMML, as well as in the total cohort. In low-risk MDS ten aberrancies were independently related to MDS (table 2). Five of these aberrancies were found in MPCs, two each in neutrophils and monocytes and one in erythroid cells. In high-risk MDS 11 aberrancies were independently related to MDS (table 2). Eight were found in MPCs, two in neutrophils, none in monocytes and one in erythroid cells. In CMML 12 aberrancies were independently related to CMML (table 2). Four were found in MPCs, neutrophils and monocytes, respectively, and none in erythroid cells. Considering all these three groups together and all aberrancies identified significantly related to MDS/CMML in at least one group in univariate analysis, multivariate analysis identified 12 aberrancies independently related to MDS/CMML (table 2). Six were found in MPCs, two in neutrophils, three in monocytes and one in erythroid cells. Taking into consideration only aberrancies independently associated with MDS/CMML, three such aberrancies resulted in an 80% agreement with the cytomorphologic diagnosis of MDS/CMML, i.e. 20% concordantly negative and 60% concordantly positive. Importantly, this applies without need of at least two cell compartments being affected as specified in the ELN recommendations. Conclusions: This multicenter prospective evaluation confirms the diagnostic potential of flow cytometric aberrancies. A core set of 17 markers identified as independently related to a diagnosis of MDS/CMML is suggested mandatory for flow cytometric evaluation of suspected MDS. An MPC count >3% should be considered indicative of MDS/CMML. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Van de Loosdrecht: Celgene: Consultancy, Research Funding; Amgen: Consultancy; Roche: Consultancy; Novartis: Consultancy; Alexion: Consultancy.

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A subset of the red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) lacks GPI-anchored proteins. Some of these proteins, such as CD59, inhibit complement activation and protect against complement-mediated lysis. This pathology thus provides the possibility to explore the involvement of complement in red blood cell homeostasis and the role of GPI-anchored proteins in the generation of microvesicles (MVs) in vivo. Detailed analysis of morphology, volume, and density of red blood cells with various CD59 expression levels from patients with PNH did not provide indications for a major aberration of the red blood cell aging process in patients with PNH. However, our data indicate that the absence of GPI-anchored membrane proteins affects the composition of red blood cell-derived microvesicles, as well as the composition and concentration of platelet-derived vesicles. These data open the way toward a better understanding on the pathophysiological mechanism of PNH and thereby to the development of new treatment strategies.

Mutations in the transcription factor Growth Factor Independence 1B (GFI1B) and Runt-related transcription factor 1 (RUNX1) are causal to familial bleeding disorders. Mutant RUNX1 and GFI1B affect megakarocyte development, resulting in decreased numbers of platelets with significantly reduced α-granules and platelets that express the hematopoietic stem and progenitor cell marker CD34. A case that presented in our clinic with a bleeding disorder (Tosetto bleeding score of 11) was diagnosed with a δ-granule secretion defect. Platelet light transmission aggregometry revealed an aggregation defect upon stimulation with epinephrine and low concentrations of collagen and ADP. ATP release from δ-granules was normal after stimulation with a high concentration of collagen, however, no ATP was released upon stimulation with epinephrine and low levels were measured after stimulation with the thromboxane analogue U46619. Whole mount electron microscopy showed increased numbers of δ-granules in platelets derived from the index case compared to controls (average 5.84 δ-granules vs 3.66, per platelet respectively). To identify a genetic cause for the disease, a bleeding disorder gene panel was screened following whole exome sequencing. No disease-causing variants were called. Open exome analysis detected a ~100Kb heterozygous chromosome 16q22.1 deletion that was confirmed by SNP array. The observed deletion covers nine genes, including the last exon of CBFB. To date, none of the deleted genes have been implicated in inherited bleeding or platelet disorders. However, conditional knockout of Cbfb in mice results in a plethora of hematopoietic abnormalities including disturbed megakaryopoiesis and thrombocytopenia. CBFB is part of a heterodimeric transcription factor complex with either RUNX1, -2 or -3. Because of the CBFB-RUNX1 interaction and the fact that bleeding disorders caused by RUNX1 mutations result in platelet CD34 expression, we tested the platelets of the case reported here. Using flow cytometry, we clearly detected platelet CD34 expression compared to healthy controls, albeit to a lower level compared to RUNX1 and GFI1B affected cases. Mutations in RUNX2 may cause cleidocranial dysplasia and entire CBFB deletions have been reported in rare cases with congenital bone abnormalities. The medical record of the case presented here indicated that she had undergone surgery at the age of 13 for a congenital malformed collar bone. These data suggest a relation between the 16q/CBFB deletion and the observed abnormalities. To address this, we determined whether the abnormalities segregated with the 16q deletion. The mother and son of the index case had no history of clinical bleedings, no signs of bone malformations and did not have the 16q deletion. The father who was deceased was reported by the family not to have a bleeding propensity nor bone abnormalities. Platelets from the son did not express CD34. Remarkably, platelets from the son showed an aggregation defect upon stimulation with epinephrine and ADP, similar to the index patient. No ATP was released from δ-granules following stimulation with epinephrine. Thus, the 16q deletion associates with platelet CD34 expression and a congenital bone abnormality but is not responsible for the δ -granule secretion defect as the latter was observed in the son who did not have the 16q deletion, nor bleedings. Defective downmodulation of CD34 during platelet formation in the index case reported here suggests abnormal megakaryocyte development that might contribute in a multifactorial manner to bleedings. Recently, we reported CD34 expression on platelets from patients with rare GFI1B variants that may predispose to a bleeding propensity, but do not cause bleedings on their own. Thus, it will be important to determine whether the 16q deletion (and C-terminal truncation of CBFB) reported here affects megakaryocyte development and if so, contributes to the bleeding disorder in the index case. In contrast to Cbfb null mice, Cbfb heterozygous mice do not exhibit discernable hematopoietic phenotypes. Thus, if the C-terminal CBFB truncation contributes to the disease characteristics, it is likely that this occurs through a dominant-negative mechanism rather than haploinsufficiency. The findings reported here warrant further studies into the role of 16q (and CBFB) deletions in megakaryocyte development. Disclosures No relevant conflicts of interest to declare.

In April 2013, a symposium was organized to highlight different aspects of differentiation and activation of the monocyte-macrophage lineage as analyzed on the flow cytometer. Characterization of this lineage requires knowledge of the maturation process from their progenitors that are present in bone marrow up to the mature monocytic cells in peripheral blood, because each monocytic lineage cell with an aberrant phenotype refers to the corresponding maturation stage. A standardized quantitative analysis will facilitate the monitoring of the pathological processes and the clinical features, such as the outcome of treatment. However, changes in marker expression by variation in intensity, asynchronism, and lineage infidelity must be considered. The dynamics of normal marker expressions in early differentiation stages, e.g. molecules like HLA II, CD64 or CD14, give rise to a hypothesis on their possible role in monocyte ontogeny. Besides their usual role in tissue homeostasis, mature macrophages may also play a similar role in hematopoiesis. This meeting highlighted the large potential of flow cytometric tools available for monitoring of all these aspects in the monocytic and macrophage cell lineage. © 2015 International Clinical Cytometry Society.

The T cell compartment can form a powerful defense against extrinsic (e.g., pathogens) and intrinsic danger (e.g., malignant cells). At the same time, specific subsets of T cells control this process to keep the immune system in check and prevent autoimmunity. A wide variety in T cell functionalities exists, which is dependent on the differentiation and maturation state of the T cells. In this review, we report an overview for the identification of CD4+ T-αβ cells (T-helper (Th)1, Th2, Th9, Th17, Th22, and CD4+ regulatory T cells), CD8+ T-αβ cells (cytotoxic T lymphocyte (Tc)1, Tc2, Tc9, Tc17, and CD8+ regulatory T cells), and their additional effector memory status (naive, stem cell memory, central memory, effector memory, and effector) using flow cytometry. These different subsets can be discriminated based on selective extracellular markers, in combination with intracellular transcription factor and/or cytokine stainings. Additionally, identification of very small subsets, including antigen-specific T cells, and important technical considerations of flow cytometry are discussed. Together, this overview can be used for comprehensive phenotyping of a T cell subset of interest. © 2019 International Society for Advancement of Cytometry.

Current risk algorithms are primarily based on pre-treatment factors and imperfectly predict outcome in acute myeloid leukemia (AML). We introduce and validate a post-treatment approach of leukemic stem cell (LSC) assessment for prediction of outcome. LSC containing CD34+CD38- fractions were measured using flow cytometry in an add-on study of the HOVON102/SAKK trial. Predefined cut-off levels were prospectively evaluated to assess CD34+CD38-LSC levels at diagnosis (n = 594), and, to identify LSC

In 1985, external quality assurance was initiated in the Netherlands to reduce the between-laboratory variability of leukemia/lymphoma immunophenotyping and to improve diagnostic conclusions. This program consisted of regular distributions of test samples followed by biannual plenary participant meetings in which results were presented and discussed. A scoring system was developed in which the quality of results was rated by systematically reviewing the pre-analytical, analytical, and post-analytical assay stages using three scores, i.e., correct (A), minor fault (B), and major fault (C). Here, we report on 90 consecutive samples distributed to 40–61 participating laboratories between 1998 and 2012. Most samples contained >20% aberrant cells, mainly selected from mature lymphoid malignancies (B or T cell) and acute leukemias (myeloid or lymphoblastic). In 2002, minimally required monoclonal antibody (mAb) panels were introduced, whilst methodological guidelines for all three assay stages were implemented. Retrospectively, we divided the study into subsequent periods of 4 (“initial”), 4 (“learning”), and 7 years (“consolidation”) to detect “learning effects.” Uni- and multivariate models showed that analytical performance declined since 2002, but that post-analytical performance improved during the entire period. These results emphasized the need to improve technical aspects of the assay, and reflected improved interpretational skills of the participants. A strong effect of participant affiliation in all three assay stages was observed: laboratories in academic and large peripheral hospitals performed significantly better than those in small hospitals. V C 2015 International Clinical Cytometry Society Key terms: external quality control; flow cytometry; immunophenotyping; leukemia; lymphoma

Contains fulltext : 152789.pdf (Publisher’s version ) (Closed access) Early natural killer (NK)-cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease, indicating that donor NK cells have specific antileukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK-cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor antagonist StemRegenin1 (SR1) to our culture protocol potently enhances expansion of CD34(+) HSPCs and induces expression of NK-cell-associated transcription factors promoting NK-cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and interferon-gamma production toward acute myeloid leukemia and multiple myeloma cells. Importantly, we observed that NK-cell proliferation and function are not inhibited by cyclosporin A, an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF-mobilized CD34(+) HSPCs, providing great promise for effective NK-cell-based immunotherapy after allo-SCT.

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units. Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 106/kg body weight) after lymphodepleting chemotherapy without cytokine boosting. Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential “off-the-shelf” translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107–18. ©2017 AACR.

Item does not contain fulltext Although immunophenotyping (IPT) using flow cytometry is a routine technique that is applied in many laboratories as a diagnostic tool for lymphadenopathy, some diagnostic challenges persist. In this review, we will discuss pitfalls in the daily practice of lymph node diagnostics with the focus on general characteristics as lymphoid scatter patterns and lineage specific antigens that are used to define lymphoid populations. The absence of these characteristics on proliferating lymphoid cells can potentially lead to a wrong diagnosis. At the same time, this provides evidence for malignant transformation. Sporadic examples of reactive lymphoid proliferations with similar phenotypes are also discussed, illustrating the need for correlating IPT with morphology and clinical features.

The gray platelet syndrome is a hereditary, usually autosomal recessive bleeding disorder caused by a deficiency of alpha granules in platelets. We detected a nonsense mutation in the gene encoding the transcription factor GFI1B (growth factor independent 1B) that causes autosomal dominant gray platelet syndrome. Both gray platelets and megakaryocytes had abnormal marker expression. In addition, the megakaryocytes had dysplastic features, and they were abnormally distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant GFI1B transcriptional activity in a dominant-negative manner. Our studies show that GFI1B, in addition to being causally related to the gray platelet syndrome, is key to megakaryocyte and platelet development. A large family is described with gray platelet syndrome due to an autosomal dominant inheritance pattern related to a dominant-negative mutation in GFI1B. The mutation leads to a loss in gene repression during megakaryocyte development. Platelets are formed through fragmentation of megakaryocytes that reside in the bone marrow.(1),(2) Platelet alpha granules, which are by far the most abundant platelet organelles, store proteins that stimulate platelet adhesiveness, hemostasis, and wound healing.(3),(4) The gray platelet syndrome is an inherited bleeding disorder characterized by defective production of alpha granules.(5),(6) Patients with this syndrome have reduced numbers of larger-than-normal platelets, and on light microscopy these platelets have a typical gray appearance caused by the lack of alpha granules. For a final diagnosis, the lack of alpha granules must be confirmed by means of electron microscopy.(7) Clinically, ...

Contains fulltext : 138552.pdf (Publisher’s version ) (Open Access) We have recently shown that BCG (Bacillus Calmette-Guerin) vaccination in healthy volunteers induces epigenetic reprogramming of monocytes, leading to increased cytokine production in response to nonrelated pathogens for up to 3 months after vaccination. This phenomenon was named 'trained immunity'. In the present study we assessed whether BCG was able to induce long-lasting effects on both trained immunity and heterologous T helper 1 (Th1) and Th17 immune responses 1 year after vaccination. The production of TNFalpha and IL-1beta to mycobacteria or unrelated pathogens was higher after 2 weeks and 3 months postvaccination, but these effects were less pronounced 1 year after vaccination. However, monocytes recovered 1 year after vaccination had an increased expression of pattern recognition receptors such as CD14, Toll-like receptor 4 (TLR4) and mannose receptor, and this correlated with an increase in proinflammatory cytokine production after stimulation with the TLR4 ligand lipopolysaccharide. The heterologous production of Th1 (IFN-gamma) and Th17 (IL-17 and IL-22) immune responses to nonmycobacterial stimulation remained strongly elevated even 1 year after BCG vaccination. In conclusion, BCG induces sustained changes in the immune system associated with a nonspecific response to infections both at the level of innate trained immunity and at the level of heterologous Th1/Th17 responses. (c) 2013 S. Karger AG, Basel.

Item does not contain fulltext The introduction of autologous stem cell transplantation (SCT) and novel drugs has improved overall survival in multiple myeloma (MM) patients. However, minimal residual disease (MRD) remains and most patients eventually relapse. Myeloma plasma cells express tumor-associated antigens (TAA), which are interesting targets for immunotherapy. In this phase 1 study, we investigated the safety and immunological effects of TAA-mRNA-loaded dendritic cell (DC) vaccination for treatment for MRD in MM after SCT. Mature monocyte-derived DCs were pulsed with keyhole limpet hemocyanin (KLH) and electroporated with MAGE3, Survivin or B-cell maturation antigen (BCMA) mRNA. Twelve patients were vaccinated three times with intravenous (5-22 x 10(6) DCs) and intradermal vaccines (4-11 x 10(6) DCs), at biweekly intervals. Immunological responses were monitored in blood and delayed-type hypersensitivity (DTH) biopsies. All patients developed strong anti-KLH T-cell responses, but not KLH antibodies. In 2 patients, vaccine-specific T cells were detected in DTH biopsies. In one patient, we found MAGE3-specific CD4(+) and CD8(+) T cells, and CD3(+) T cells reactive against BCMA and Survivin. In the other patient, we detected low numbers of MAGE3 and BCMA-reactive CD8(+) T cells. Vaccination was well tolerated with limited toxicity. These findings illustrate that TAA-mRNA-electroporated mature DCs are capable of inducing TAA-T-cell responses in MM patients after SCT. 01 augustus 2013

We measured the vaccination response to the new H1N1 virus in relation to the lymphocyte count prior to vaccination in pediatric cancer patients. The absolute lymphocyte count above the lower normal limits (LNL) for age prior to vaccination predicts the response to influenza vaccination in pediatric cancer patients treated with chemotherapy.

Harmonemia: a universal strategy for flow cytometry immunophenotyping—A European LeukemiaNet WP10 study

Item does not contain fulltext RATIONALE: Reversal of sepsis-induced immunoparalysis may reduce the incidence of secondary infections and improve outcome. Although IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) restore immune competence of ex vivo stimulated leukocytes of patients with sepsis, effects on immunoparalysis in vivo are not known. OBJECTIVES: To investigate the effects of IFN-gamma and GM-CSF on immunoparalysis in vivo in humans. METHODS: We performed a double-blind, placebo-controlled, randomized study in 18 healthy male volunteers that received Escherichia coli endotoxin (LPS; 2 ng/kg, intravenously) on days 1 and 7 (visits 1 and 2). On days 2, 4, and 6, subjects received subcutaneous injections of IFN-gamma (100 mug/day; n = 6), GM-CSF (4 mug/kg/day; n = 6), or placebo (NaCl 0.9%; n = 6). MEASUREMENTS AND MAIN RESULts: In the placebo group, immunoparalysis was illustrated by a 60% (48-71%) reduction of LPS-induced tumor necrosis factor (TNF)-alpha plasma concentrations during visit 2 (P = 0.03), whereas the antiinflammatory IL-10 response was not significantly attenuated (39% [2-65%]; P = 0.15). In contrast, in the IFN-gamma group, TNF-alpha concentrations during visit 2 were not significantly attenuated (28% [1-47%]; P = 0.09), whereas the IL-10 response was significantly lower (reduction of 54% [47-66%]; P = 0.03). Compared with the placebo group, the reduction in the LPS-induced TNF-alpha response during visit 2 was significantly less pronounced in the IFN-gamma group (P = 0.01). Moreover, compared with placebo, treatment with IFN-gamma increased monocyte HLA-DR expression (P = 0.02). The effects of GM-CSF tended in the same direction as IFN-gamma, but were not statistically significant compared with placebo. CONCLUSIONS: IFN-gamma partially reverses immunoparalysis in vivo in humans. These results suggest that IFN-gamma is a promising treatment option to reverse sepsis-induced immunoparalysis.

Item does not contain fulltext Adaptive features of innate immunity, recently described as "trained immunity," have been documented in plants, invertebrate animals, and mice, but not yet in humans. Here we show that bacille Calmette-Guerin (BCG) vaccination in healthy volunteers led not only to a four- to sevenfold increase in the production of IFN-gamma, but also to a twofold enhanced release of monocyte-derived cytokines, such as TNF and IL-1beta, in response to unrelated bacterial and fungal pathogens. The enhanced function of circulating monocytes persisted for at least 3 mo after vaccination and was accompanied by increased expression of activation markers such as CD11b and Toll-like receptor 4. These training effects were induced through the NOD2 receptor and mediated by increased histone 3 lysine 4 trimethylation. In experimental studies, BCG vaccination induced T- and B-lymphocyte-independent protection of severe combined immunodeficiency SCID mice from disseminated candidiasis (100% survival in BCG-vaccinated mice vs. 30% in control mice). In conclusion, BCG induces trained immunity and nonspecific protection from infections through epigenetic reprogramming of innate immune cells.

Vitamin D3 is known to induce regulatory T (Treg) cells by rendering antigen-presenting cells tolerogenic, its direct effect on human naturally occurring Treg cells is unclear. Here, we investigated if and how 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] can directly affect the proliferation and function of human naturally occurring Treg cells in vitro. First, we demonstrated that these Treg cells express vitamin D receptors that were up-regulated following anti-CD3/CD28-bead stimulation. 1,25(OH)2D3 inhibited proliferation of Treg cells even when exogenous interleukin-2 was provided. Treg cells were more susceptible to the inhibitory effect of 1,25(OH)2D3 than conventional T cells(.) 1,25(OH)2D3 neither affected the anergic state nor the suppressive function of Treg cells but induced a subtle increase in interleukin-10-secreting cells. The cell-division-inhibiting effect of 1,25(OH)2D3 on Treg cells was also demonstrated in vivo by supplementing vitamin D-deficient HIV-1-infected patients with 2000 IU cholecalciferol (vitamin D3). Increased serum 1,25(OH)2D3 levels were associated with a drop in the number and percentage of Treg cells, which may be attributed to a decrease in the proliferating Foxp3+ Treg cell population. In conclusion, 1,25(OH)2D3 directly affects Treg cell growth and promotes interleukin-10 production without apparent effects on activation status and suppressive phenotype whereas in vivo, high serum 1,25(OH)2D3 levels are associated with reduced Treg cell proliferation and a reduced number of Treg cells.

The final quality control of cryopreserved progenitor cells is a successful and persistent three lineage engraftment after transplantation. Of course, the stem cell providing institution is obliged to have a program for controlling and monitoring the manufacturing of cellular therapy products before the patients’ conditioning therapy is started. The FACT-JACIE Standards [1] and the Netcord ⁄ FACT Stan- dards prescribe that the director of the institute shall define tests and procedures for measuring and assaying cellular therapy products to ensure their safety, viability and integ- rity and shall also ensure that products meet predetermined release specifications. This requires specifications of assays and the definition of thresholds to allow release.

Item does not contain fulltext BACKGROUND: Polychromatic immunophenotyping improves characterization of leukocyte subpopulations and their malignant counterparts. However, the lack of various fluorochrome-labeled monoclonal antibodies (MoAbs) hinders the formation of multi-color panels. CD45 appears to be an important MoAb for immunophenotyping of these cells. Plotted against the side scatter, CD45 provides immunological cell differentiation and the ability to recognize various normal and malignant leukocyte subpopulations. CD45 is commonly used and labeled with various fluorochromes and as a result, is incorporated in multi-color panels as a conjugate of less available fluorochromes, such as the violet laser dyes. However, these dyes (e.g. Pacific Orange/PO) often possess low fluorescence intensity, which may be too weak to differentiate between populations. The new organic dye Krome Orange (KO, emission at 528 nm) appears to be a more intense violet laser dye, serving as an alternative to PO. METHODS: Intensities of CD45 conjugated with FITC, PE, ECD, PE-Cy5, PE-Cy7, PO and KO were tested in different cell sources. Various lineage markers were sequentially back gated on CD45-KO to identify subpopulations. A 10-color MoAb panel for determination of aberrancies in small cell samples was composed to test specificity of CD45-KO. CONCLUSIONS: We showed in various fixed and unfixed cells from different sources that KO is a suitable fluorochrome with a significantly higher quantum yield than PO and is even brighter than other violet laser dyes (e.g. Pacific Blue). CD45-KO/SS enables us to distinguish and characterize various normal and malignant leukocyte subpopulations. By using a 10-color MoAb panel to screen on aberrancies, we showed that CD45-KO provides reliable immunophenotyping within small amounts of cells and thereby improves the quality of 10-color stainings.

Contains fulltext : 89505.pdf (Publisher’s version ) (Closed access) Cyclosporin A (CsA), rapamycin (Rapa) and mycophenolic acid (MPA) are frequently used for GVHD prophylaxis and treatment after allogeneic stem cell transplantation (SCT). As NK cells have received great interest for immunotherapeutic applications in SCT, we analyzed the effects of these drugs on human cytokine-stimulated NK cells in vitro. Growth-kinetics of CsA-treated cultures were marginally affected, whereas MPA and Rapa severely prevented the outgrowth of CD56(bright) NK cells. Single-cell analysis of NK cell receptors using 10-color flow cytometry, revealed that CsA-treated NK cells gained a similar expression profile as cytokine-stimulated control NK cells, mostly representing NKG2A(+) KIR(-) NCR(+) cells. In contrast, MPA and Rapa inhibited the acquisition of NKG2A and NCR expression and NK cells maintained an overall NKG2A(-) KIR(+) NCR(+/-) phenotype. This was reflected in the cytolytic activity, as MPA- and Rapa-treated NK cells, in contrast to CsA-treated NK cells, lost their cytotoxicity against K562 target cells. Upon target encounter, IFN-gamma production was not only impaired by MPA and Rapa, but also by CsA. Overall, these results demonstrate that CsA, MPA and Rapa each have distinct effects on NK cell phenotype and function, which may have important implications for NK cell function in vivo after transplantation. 01 september 2010