Multiparameter Flow Cytometry Approach Research Articles

A Novel 10-Fluorochrome Multiparameter Flow Cytometry (MFC) Panel for Precise Plasma Cell (PC) and MRD Assessment in Clinical Routine of Multiple Myeloma (MM) Patients and throughout the Disease Course

Introduction: Since the introduction of novel agents into the treatment of Multiple Myeloma (MM), much deeper remissions can be achieved. This leads to a demand for disease evaluation tools with higher sensitivity and clinical practicability. In various clinical trials the assessment of the MRD status has been proven to have strongest prognostic value for MM patients (pts). The Euroflow Consortium has pioneered a highly standardized next generation flow (NGF) approach with the establishment of two 8-color tubes tests (Flores-Montero 2017). However, due to cost-intensive and time-consuming preconditions, this MRD approach might not be feasible in every center. Thus, other readily available, validated MRD tools may also guarantee highly sensitive MRD evaluation which allows for early relapse detection and to steer treatment, such as decision upon need for consolidation, duration of maintenance, therapy pauses or even earlier FDA/EMA approval of novel antimyeloma agents. Methods: Our 10 color single tube MFC panel consisting of the antigens CD38, CD138, CD19, CD45, CD27, CD56, CD28, CD81, CD117 and CD200, was systematically validated using Fluorescence-minus-one controls, consecutive spike-in-controls, consistency analyses and measurement of 9 MM cell lines. Thereafter, we assessed 131 Bone Marrow (BM), peripheral blood (PB), and leukapheresis (LA) samples from 97 different MM, MGUS and SMM pts and 13 healthy individuals (HI) as controls. Samples were processed within 6-20 hours of aspiration and a bulk lysis was performed. Subsequently, >3x106 viable nucleated cells were acquired on a BD LRSFortessa Cytometer. Data analysis was performed using the BeckmanCoulter software Kaluza. BM was sampled at initial diagnosis (ID) or at progression (PD), after standard therapy (ST) or stem cell transplantation (SCT). The study was performed with written consent from all pts and approval from the ethics committee. Results: Our MFC approach confirmed the antigen expression in all MM cell lines (RPMI 8226; U266; IM-9; MM1.S; MM1.R; L363; Karpas620; NCI-H929; OPM-2) as previously reported. The spike-in-controls determined the limit of detection (LOD) at 10-5. This LOD was achieved in 89% of our MRD samples. An easy-to-adapt gating strategy to identify abnormal plasma cells (aPC) vs. normal plasma cells (nPC) was also established in our MM pt samples. In this cohort, we identified 10 distinct aPC and nPC subpopulations that differed in their expression pattern, suggesting clonal subtypes of MM. Moreover, in 19% of BM samples, two malignant subpopulations coexisting in the same pt, were identified. Since our BM cohort consisted of 46% samples at ID or PD vs. 54% posttreatment samples after ST and SCT, clonality variation in the former, differences of aPCs vs. nPCs ratio and to the latter group could be determined. Moreover, significant differences of much higher percentage of aPC in symptomatic MM vs SMM/MGUS pts were readily verified with our panel (p=0.0087). With a median time of 51 days from treatment to MRD evaluation, 24% of post treatment samples were MRD negative (MRD-) at the 10-5 level (<0.001% aPC). In line, MRD negativity induced an improved PFS, with OS analyses needing a longer follow-up. Since we also assessed pre- and post-treatment samples, antibody expression levels on PC from ID to post treatment demonstrated a significant normalization in Median Fluorescence Intensity (MFI) of 5 antibodies (CD56, CD81, CD45, CD200, CD27). These may thus serve as informative markers for the MRD assessment of these samples. Conversely, with progression from remission to PD, CD81 and CD45 antibodies showed significant decreases (p=0.0007 and p=0.0026, respectively) in expression, which suggest that both may serve as early relapse markers. Of the assessed LA samples at harvest before ASCT, 44% were MRD-, warranting future investigations into possible implications for pts' prognosis, treatment and PFS/OS data. Conclusion: Here we present a readily available, cost-effective, quick and highly validated MFC panel with an easy to adapt gating strategy that allows for precise aPC vs. nPC assessment in- and outside clinical trials. Our 10 color MFC panel is applicable in BM samples of MM pts and precursor diseases, as well as in LA samples. With the additional MFC information, ideally at ID and repeatedly after treatment, readily available individual treatment decisions seem possible to obtain for every MM patient. Disclosures Wäsch: Amgen: Other: travel, Research Funding; Sanofi: Other: Travel, Research Funding; Jazz: Other: travel, Research Funding; Celgene: Other: travel, Research Funding; Gilead: Other: travel, Research Funding; Takeda: Consultancy; Gilead: Consultancy; Sanofi: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy.

Comparison of Minimal Residual Disease Detection in Autografts of Patients with Multiple Myeloma between 8-Color Multiparameter Flow Cytometry (EuroFlow) and Next-Generation Sequencing

Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper responses are required. Next-generation sequencing (NGS) and multiparameter flow cytometry (MFC) methods have been used to assess MRD. However, the lack of standardization of conventional MFC approaches has had a negative impact on its reproducibility. Recently, a next-generation MFC method (EuroFlow, NGF) has been developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for a highly sensitive and standardized detection of MRD in MM.Aims: To compare the prognostic value of MRD detection in autografts in MM between NGS (Adaptive) and 8-color MFC method (EuroFlow, NGF), and also MRD levels between fresh and cryopreserved autografts.Methods: A total of 39 newly-diagnosed MM patients who underwent ASCT were enrolled in this study. Median age 60 at ASCT (range 41-69); males 22, females 17; ISS 1 (n=10), 2 (n=19), 3 (n=10). 10 patients showed high-risk chromosomal abnormalities (t(4;14) (n=9), del17p & t(4;14) (n=1)). The induction regimen was bortezomib-based chemotherapy. All patients received melphalan 200 mg/sqm as conditioning regimen before ASCT. 34 of 39 (87%) patients received maintenance therapy until progressive disease. The best response post-ASCT was as follows: 23sCR, 2CR, 12VGPR, 2PR. 39 autografts, one from each MM patient, were analyzed using NGF and NGS methods. The NGF method was based on a standardized lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of cells and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC) (J Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). To assess the correlation of MRD levels between fresh and cryopreserved autografts using NGF, 6 additional MM patients' autografts were used.Results: MRD levels in all 39 autografts were assessed using EuroFlow, while those in 32 of 39 (82%) were assessed with NGS due to limited availability of material for calibration. We identified abnormal plasma cells (aPC) in autografts based on multivariate analysis of individual cells from each patient (e.g. CD56+, CD19-, CyIgκ+, CD117+). Since there was a good correlation in MRD levels between fresh and thawed frozen autografts detected by EuroFlow (R=0.943, P=0.02), we assessed the MRD levels in thawed frozen autografts. For the MM MRD in autografts, the events from tube 1 and tube 2 were combined and a median of 7.3×106 (range: 2.2×106-37.6×106) events was acquired. The sensitivity of EuroFlow was 1×10-5-2×10-6 while that of NGS was 10-7 due to the high number of DNA derived from autografts (Takamatsu et al., Ann Oncol 2017). 21 of 39 (54%) cases were MRD positive by 8-color MFC while 22 of 32 (69%) cases were MRD positive by NGS. The correlation of MRD levels between 8-color MFC and NGS was relatively high (Fig. 1A). MRD negative by NGF (MRDMFC (-)) cases tended to show better PFS than MRDMFC (+) cases (P=0.145) (Fig. 1B) while MRD negative by NGS (MRDNGS (-)) cases showed significantly better PFS than MRDNGS (+) cases (P=0.03) (Fig. 1C). Furthermore, MRDMFC (-) MRDNGS (-) cases showed significantly better PFS than MRDMFC (-) MRDNGS (+) cases (P=0.01), but the PFS of MRDMFC (-) MRDNGS (+) cases was not different from that of MRDMFC (+) MRDNGS (+) cases (P=0.70). MRDMFC (-) and MRDNGS (-) cases showed better OS than MRDMFC (+) (P=0.14) and MRDNGS (+) (P=0.08) cases, respectively.Conclusions: Although EuroFlow is a fast and accurate method for detecting MRD of MM in autografts, in this study the NGS platform had a higher sensitivity and prognostic value than EuroFlow. The homogenous nature of the mobilized autograft relative to the focal nature of myeloma in bone marrow might provide a better sample to assess MRD. [Display omitted] DisclosuresTakamatsu:Celgene: Honoraria, Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

High frequency of CD34+CD38-/low immature leukemia cells is correlated with unfavorable prognosis in acute myeloid leukemia.

AIMTo evaluate the importance of the CD34+CD38- cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival.METHODSTwo hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia (AML) were studied between September 2008 and December 2010 at our Institution (Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8C panels and FACS CANTO and Diva software (BD Bioscience).RESULTSWe analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38- population. Using a cut-off value of 1% of CD34+CD38- from total “bulk leukemic cells” we found that a high (> 1%) level of CD34+CD38- blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38- leukemic cells > 1% was an independent predictor of DFS [HR = 2.8 (1.02-7.73), P = 0.04] and OS [HR = 2.65 (1.09-6.43), P = 0.03].CONCLUSIONTaken together, these results show that a CD34/CD38 “backbone” for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.

CD180 overexpression in follicular lymphoma is restricted to the lymph node compartment.

Altered Toll-like receptor (TLR) expression levels and/or mutations in its signaling pathway (such as MyD88 mutation) contribute to the pathogenesis of lymphoproliferative disorders (LPD). CD180 is an orphan member of the TLR family that modulates the signaling of several TLRs, but only limited studies have evaluated its expression by flow cytometry (FCM) in LPD. Using a multiparameter FCM approach, we have assessed CD180 mean fluorescence intensity (MFI) in lymph nodes (LNs) and peripheral blood (PB) samples obtained from patients with follicular lymphoma (FL; LN/PB, n = 44/n = 15), chronic lymphocytic leukemia (CLL, n = 26/n = 21), mantle cell lymphoma (MCL, n = 13/n = 17), and marginal zone lymphoma (MZL, n = 16/n = 12). Specimens from non-tumoral PB and LN (n = 8/n = 12) were used as controls. In the LN specimens, FL and control B-cells showed similar CD180 expression (MFI = 1,049 vs. 1,381, P > 0.05; Mann-Whitney U-test). This level was markedly lower in the other LPDs, MCL (MFI = 396, P < 0.05), or CLL (MFI = 502 P < 0.05), and similar to MZL (MFI = 858, P > 0.05). However, the CD180 expression of FL B-cells assessed in PB was dim and/or negative, in the same range as MCL and CLL (FL MFI = 453, MCL MFI = 305, CLL MFI = 420, P > 0.05) but lower than in MZL (MFI = 895, P < 0.05). Therefore, these results suggest a modulation of CD180 expression by neoplastic FL B-cells based on the anatomical compartment. These FCM data confirm the usefulness of CD180 in the accurate diagnosis of LPDs and emphasize the need to interpret this marker according to the origin of the sample. © 2015 Clinical Cytometry Society.

Issue Highlights—March 2015

In their review Damuzzo et al. 1 focus on the correct identification of circulating and tumour-associated so-called myeloid-derived suppressor cells (MDSCs) in humans and mouse models. The reason to do this is because MDSCs are endowed with suppressive activity and because their expansion has been associated with disease progression and reduced survival. Because of their heterogeneous composition, accurate phenotyping of these cells will almost invariantly require a multicolour approach in order to allow appreciation of MDSC subsets. Correct minimal residual disease (MRD) detection is crucial for therapy response assessment in a considerable number of hematologic disorders including mantle cell lymphoma (MCL) 2. Multiparameter flow cytometry (MFC) immunophenotyping is currently increasingly used to assess MRD in malignant disorders. Recently, a FACSCantoll (DB Biosciences, San Jose, CA)-based 7-color/8-antbody MFC approach has been published, allowing for the determination of MRD in multiple myeloma patients 3. Chovancova et al.4 found that, although MCL presents high immunophenotypic variability, the combination of CD20/23/5/19/200/62 L/45 is very favourable in flow cytometric MRD measurement in MCL reaching a sensitivity of up to 2×10 −4. In another study, Mathis et al. 5 show that this protocol is transposable between harmonized ≥ 7-color instruments and that a homogeneous rapid MRD evaluation can be performed in most MFC platforms. Although not all immunohistochemical techniques can easily be automated by image analytical systems, in a remarkable study Qin, Y et al. 6 concluded that a computerized delineation of nuclei in re-stained PAP smears with p16/ki67 can result in an automated high-throughput profiling method achieving accurate data. Because cigarette smoke is a powerful producer of reactive oxygen species 7, hypothesized that spermatozoa of smokers would be at increased risk of having DNA fragmentation as compared to spermatozoa of non-smoking men. However, by applying a novel TUNEL assay coupled to a vitality marker (LIVE/DEAD®) the authors found no deleterious effect, i.e., increased DNA fragmentation, of smoking on spermatozoa. More studies concerning the potential mutagenic capacities of cigarette smoke on spermatozoa are mandatory. Eidenschink Brodersen et al. 8, utilizing a NH4Cl lysis protocol have defined various immunophenotypic abnormalities that indicate dyserythropoiesis in myelodysplastic syndromes. Preliminary studies also indicate strong correlation between phenotypic erythroid dysplasia and poor prognosis, as classified cytogenetically. It is well-known that the epidermal growth factor receptor (EGFR) is overexpressed in carcinoma and can be used as a therapeutic target. Levi et al. 9 developed a Bio-Ferrography method enabling isolating EGFR overexpressing carcinoma cells from human blood. Recovery values as high of 8% for 1 mL PBS and 53% for 1 mL whole blood, with a limit of detection of 30 and 100 target cells, respectively, were achieved. Further studies are warranted to assess whether this technique allows early diagnosis of EGFR overexpressing tumour types. In MRD flow cytometry results can easily be confounded by (unknown) expression of markers on nearby cells. In their article Soma et al. 10 focuses on the expression of CD19 on NK cells. They conclude that it is an apparent but infrequent finding, not a consequence of technical error. More research to optimizing panels, combining techniques or using other specimens like bronchoalveolar lavage is needed. Perspectives from Didier G. Ebo. Didier G. Ebo* Department of Immunology - AllergologyUniversity of Antwerp,Belgium

Blood Flow Cytometry in Sézary Syndrome

Sézary syndrome (SS) is characterized by erythroderma, generalized lymphadenopathy, and the presence of circulating atypical lymphocytes, which are difficult to identify by morphologic data. We revised our series of 107 patients in an attempt to better define the phenotypic aberrancies in blood at diagnosis and the immunophenotypic stability over time detected by flow cytometry. Polymerase chain reaction assay was also used to study CD26/dipeptidyl peptidase IV (DPPIV) gene methylation. The most common aberrancies were represented by the lack of CD26 (96/107) or CD38 (101/107) expression and the presence of a "dim" CD3, CD4, or CD2 population. There was a high variability in CD7 expression. In total, 31% of the patients had phenotypical heterogeneity in CD26 and CD7 expression at diagnosis. The phenotype was stable over time in 73 of 95 patients with available follow-up data, while 22 of 95 patients developed changes in CD26, CD7, or CD2 expression. CD4+CD26- SS showed hypermethylation of the CpG islands for the promoter region of CD26/DPPIV. Multivariate analysis showed that CD26 expression is a favorable prognostic factor (hazard ratio, 2.94; P = .045). We confirm the relevance of CD26 negativity in SS diagnosis and monitoring. Nevertheless, the presence of rare CD26+ cases suggests that a multiparameter flow cytometry approach should be used. Changes in methylation profile could account for phenotypical heterogeneity.

Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for “prime time”?

Abstract The past 40 years have witnessed major advances in defining the cytogenetic aberrations, mutational landscape, epigenetic profiles, and expression changes underlying hematological malignancies. Although it has become apparent that acute myeloid leukemia (AML) is highly heterogeneous at the molecular level, the standard framework for risk stratification guiding transplant practice in this disease remains largely based on pretreatment assessment of cytogenetics and a limited panel of molecular genetic markers, coupled with morphological assessment of bone marrow (BM) blast percentage after induction. However, application of more objective methodology such as multiparameter flow cytometry (MFC) has highlighted the limitations of morphology for reliable determination of remission status. Moreover, there is a growing body of evidence that detection of subclinical levels of leukemia (ie, minimal residual disease, MRD) using MFC or molecular-based approaches provides powerful independent prognostic information. Consequently, there is increasing interest in the use of MRD detection to provide early end points in clinical trials and to inform patient management. However, implementation of MRD assessment into clinical practice remains a major challenge, hampered by differences in the assays and preferred analytical methods employed between routine laboratories. Although this should be addressed through adoption of standardized assays with external quality control, it is clear that the molecular heterogeneity of AML coupled with increasing understanding of its clonal architecture dictates that a “one size fits all” approach to MRD detection in this disease is not feasible. However, with the range of platforms now available, there is considerable scope to realistically track treatment response in every patient.

Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for "prime time"?

The past 40 years have witnessed major advances in defining the cytogenetic aberrations, mutational landscape, epigenetic profiles, and expression changes underlying hematological malignancies. Although it has become apparent that acute myeloid leukemia (AML) is highly heterogeneous at the molecular level, the standard framework for risk stratification guiding transplant practice in this disease remains largely based on pretreatment assessment of cytogenetics and a limited panel of molecular genetic markers, coupled with morphological assessment of bone marrow (BM) blast percentage after induction. However, application of more objective methodology such as multiparameter flow cytometry (MFC) has highlighted the limitations of morphology for reliable determination of remission status. Moreover, there is a growing body of evidence that detection of subclinical levels of leukemia (ie, minimal residual disease, MRD) using MFC or molecular-based approaches provides powerful independent prognostic information. Consequently, there is increasing interest in the use of MRD detection to provide early end points in clinical trials and to inform patient management. However, implementation of MRD assessment into clinical practice remains a major challenge, hampered by differences in the assays and preferred analytical methods employed between routine laboratories. Although this should be addressed through adoption of standardized assays with external quality control, it is clear that the molecular heterogeneity of AML coupled with increasing understanding of its clonal architecture dictates that a "one size fits all" approach to MRD detection in this disease is not feasible. However, with the range of platforms now available, there is considerable scope to realistically track treatment response in every patient.

OMIP-023: 10-color, 13 antibody panel for in-depth phenotyping of human peripheral blood leukocytes.

THIS panel was developed and optimized to determine the phenotype and activation of 15 different leukocyte subtypes in one run. Leukocytes are identified by expression of CD45 (leu-1) pan leukocyte antigen. Neutrophil (CD16), monocyte (CD14), T(CD3), B-lymphocyte (CD19), and NK-cell (CD16 and CD56) markers are employed. Special gating strategy is used for subtyping of granulocytes (e.g., eosinophils, neutrophils, basophils). For further T-cell phenotyping, CD4/CD8 markers are used for differentiation of four T-cell subtypes, CD25/CD127 for regulatory T cell identification and CD3/CD16/CD56 for NKT-cells, additionally. Special gating strategies have been developed for B-cell, NK-cell, and monocyte subtyping. For detection and analysis of activation also further activation markers (HLA-DR, CD38, CD25, CD127, and CD69) are analyzed. This panel has been established for analysis of human RBC-lysed EDTAtreated whole blood samples and for cord blood (Table 1). Since the starting material is fresh EDTA-treated blood, dead cells are probably not an issue. Thus to save one channel for specific staining the vitality staining was not used in the panel.

Use of Multi-parameter Flow Cytometry as Tool to Monitor the Impact of Formic Acid on Saccharomyces carlsbergensis Batch Ethanol Fermentations

The use of lignocellulosic materials as substrate for bioethanol production is considered a cost-effective approach to make the biofuel production process economically sustainable. However, lignocellulosic hydrolysis releases toxic compounds such as weak acids which inhibit microorganism growth and ethanol production. In order to understand the physiological response of Saccharomyces carlsbergensis when fermenting glucose in the presence of formic acid (HF), the yeast growth was monitored by multi-parameter flow cytometry. Cytoplasmic membrane potential decreased as the HF concentration increased and as the yeast culture reached the stationary phase. However, the proportion of cells with permeabilized membrane did not increase with the HF concentration increase. The accumulation of reactive oxygen species was also monitored. Control and fermentations at low HF concentrations (<1 g/L) resulted in a high proportion of highly oxidized cells at the stationary phase. The multi-parameter flow cytometry approach proved to be a useful tool to monitor the physiological stress response of S. carlsbergensis growth and ethanol production in the presence of HF, an inhibitor present in lignocellulosic hydrolysates. The information here obtained at near real time can be used to enhance second-generation bioethanol production process efficiency.

Interlaboratory Validation of a 3-Tube 10-Color Antibody Flow Cytometry Panel for Diagnosis of Myelodysplastic Syndromes

AbstractAbstract 4941 Background:Multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of patients with suspected myelodysplastic syndromes (MDS). Modern MFC allows the simultaneous detection of 10 fluorochrome-conjugated antibodies recognizing different antigens and thus provides the basis for a refined detection of MDS-related aberrant antigen expression. While efforts are made to harmonize MFC approaches to diagnose MDS, especially on the markers useful to investigate (Westers et al., Leukemia 2012), there has been no interlaboratory evaluation yet of uniform procedures including a common antibody panel and thus reproducibility remains to be proven. Aims:Prove the interlaboratory comparability of diagnostic read-out using a uniform MFC approach to diagnose MDS. Methods:A 10-color/3-tube panel was designed using the fluorochromes FITC, PE, ECD, PC5. 5, PC7, APC, APCA700, APCA750, Pacific Blue, and Krome Orange conjugated to the following antibodies: tube 1) CD14, CD13, CD38, CD123, CD117, CD11b, CD34, CD33, CD16, CD45; tube 2) CD71, CD4, CD64, CD56, CD117, CD36, CD34, CD33, HLA-DR, CD45; tube 3) CD7, CD10, CD8, CD5, CD2, CD3, CD34, CD19, CD15, CD45. Tube 1 was designed for the assessment of myeloid progenitor cells and granulocytes, tube 2 for monocytes and erythroid cells, tube 3 for lymphoid cells and granulocytes. Data acquisition was performed using Navios flow cytometers (Beckman Coulter, Miami, FL) in each of three laboratories from Germany, France, and Canada. Instrument settings were adjusted at all sites to match pre-defined target channels using beads as controls. During the pilot phase, data of a total of 10 MDS cases acquired at the different sites were analyzed by all of the 3 sites following systematic procedures agreed upon. The parameters evaluated included the percentages of respective cell compartments, expression patterns of CD13/CD16/CD11b in granulocytes, aberrant expression of myeloid markers, coexpression of lymphoid markers in granulocytes, monocytes, myeloid progenitor cells and erythroid cells. Coefficients of variation (CV) were calculated for each analyzed parameter for each patient. Results:The quantification of cell compartments was homogeneously performed by the different sites with median CVs amounting to 5. 7% for granulocytes, 10. 5% for monocytes, 7. 1% for lymphocytes, and 5. 0% for CD34 positive progenitor cells. The mean side scatter signal for granulocytes was consistently determined with a median CV of only 3. 4%. Mean fluorescent intensities (MFIs) for distinct markers in general were homogeneously determined between the three sites with the following median CVs: CD33 1. 8% in granulocytes; CD33 2. 6%, CD14 15. 6%, CD13 4. 8%, CD11b 3. 6%, CD56 23. 8%, HLA-DR 5. 1%, CD2 16. 7% in monocytes; CD71 6. 9%, CD36 2. 9% in erythroid cells; CD34 4. 8% in progenitor cells. This provides an important basis for the harmonization of rating aberrantly expressed antigens in MDS which often are identified by only relatively small changes in the respective MFIs. Importantly, also the percentages of positivity for cross-lineage expression of lymphatic antigens in myeloid progenitor cells, which have previously been shown to carry prognostic significance, were determined homogeneously between the three sites with median CVs of 22. 0%, 13. 4% and 33. 9% for the very small subsets of CD2, CD7 and CD5 positive cells. Another aim of the present pilot study has been the objective read-out of parameters which in general have been judged on subjectively. In this regard, the expression patterns of CD11b and CD16 as well as of CD13 and CD16 in granulocytes were subjectively judged aberrant or not aberrant with complete agreement between the three sites. Importantly, the separation of the giraffe-like pattern of CD11b/CD16 expression in granulocytes into 4 subpopulations was performed homogeneously (median CVs 4. 8%, 22. 1%, 18. 0%, 13. 6%) and the resulting MFIs for CD11b (median CVs 7. 2%, 12. 8%, 10. 8%, 11. 5%) in these 4 subpopulations also were consistently quantified between the three sites. Conclusions:This data indicates that harmonization of procedures across different sites in the flow cytometric evaluation of patients with MDS is feasible. While agreement for judgement on antigen expression patterns is possible by experienced scientists it may be complemented or even substituted by standardized data analysis procedures following testing rounds. Disclosures:Kern:MLL Munich Leukemia Laboratory: Equity Ownership; Beckman Coulter, Miami, Florida: Research Funding. Béné:Beckman Coulter, Miami, Florida: Research Funding. Porwit:Beckman Coulter, Miami, Florida: Research Funding.

In Vivo Dioxin Favors Interleukin-22 Production by Human CD4+ T Cells in an Aryl Hydrocarbon Receptor (AhR)-Dependent Manner

BackgroundThe transcription factor aryl hydrocarbon receptor (AhR) mediates the effects of a group of chemicals known as dioxins, ubiquitously present in our environment. However, it is poorly known how the in vivo exposure to these chemicals affects in humans the adaptive immune response. We therefore assessed the functional phenotype of T cells from an individual who developed a severe cutaneous and systemic syndrome after having been exposed to an extremely high dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).Methodology/Principal FindingsT cells of the TCDD-exposed individual were studied for their capacity to produce cytokines in response to polyclonal and superantigenic stimulation, and for the expression of chemokine receptors involved in skin homing. The supernatants from T cells of the exposed individual contained a substantially increased amount of interleukin (IL)-22 but not of IL-17A, interferon (IFN)-γ or IL-10 when compared to nine healthy controls. In vitro experiments confirmed a direct, AhR-dependent, enhancing effect of TCDD on IL-22 production by CD4+ T cells. The increased production of IL-22 was not dependent on AhR occupancy by residual TCDD molecules, as demonstrated in competition experiments with the specific AhR antagonist CH-223191. In contrast, it was due to an increased frequency of IL-22 single producing cells accompanied by an increased percentage of cells expressing the skin-homing chemokine receptors CCR6 and CCR4, identified through a multiparameter flow cytometry approach. Of interest, the frequency of CD4+CD25hiFoxP3+ T regulatory cells was similar in the TCDD-exposed and healthy individuals.Conclusions/SignificanceThis case strongly supports the contention that human exposure to persistent AhR ligands in vivo induce a long-lasting effect on the human adaptive immune system and specifically polarizes CD4+ T cells to produce IL-22 and not other T cell cytokines with no effect on T regulatory cells.

Multiparameter flow cytometry quantification of bone marrow plasma cells at diagnosis provides more prognostic information than morphological assessment in myeloma patients

Quantification of bone marrow plasma cells in multiple myeloma patients using conventional morphology is of limited prognostic value, while the merit of multiparameter flow cytometry immunophenotyping is still considered unproven. Here we compare the bone marrow plasma cell counts obtained by morphology and multiparameter flow cytometry and explore the potential prognostic impact of both techniques in 765 newly diagnosed, uniformly treated multiple myeloma patients. Although multiparameter flow cytometry generally yields lower plasma cell counts (median percentage of 11% vs. 40%, respectively; p<0.001), there is a significant positive correlation between the two techniques (R =0.46, p<0.001). Regarding prognosis, multivariate analysis selected the bone marrow plasma cell counts obtained by multiparameter flow cytometry as an independent prognostic factor for overall survival (p=0.007), supporting the incorporation of multiparameter flow cytometry immunophenotyping into the routine diagnostic evaluation of multiple myeloma patients and validating the clinical utility of bone marrow plasma cell counting by multiparameter flow cytometry approaches. (clinicaltrials.gov identifier: NCT00560053).

Differential response of human basophil activation markers: a multi-parameter flow cytometry approach.

BackgroundBasophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions. MethodsCell suspensions were prepared from leukocyte buffy coat of K2-EDTA anticoagulated blood specimens; about 1500-2500 cellular events for each tested sample, gated in the lymphocyte CD45dim area and then electronically purified as HLADRnon expressing/CD123bright, were identified as basophilic cells. Basophil activation with fMLP, anti-IgE and calcium ionophore A23187 was evaluated by studying up-regulation of the indicated membrane markers with a two-laser six-color PFC protocol.ResultsFollowing stimulation, CD63, CD13, CD45 and the ectoenzyme CD203c up-regulated their membrane expression, while CD69 did not; CD63 expression occurred immediately (within 60 sec) but only in a minority of basophils, even at optimal agonist doses (in 33% and 14% of basophils, following fMLP and anti-IgE stimulation respectively). CD203c up-regulation occurred in the whole basophil population, even in CD63non expressing cells. Dose-dependence curves revealed CD203c as a more sensitive marker than CD63, in response to fMLP but not in response to anti-IgE and to calcium ionophore.ConclusionUse of polychromatic flow cytometry allowed efficient basophil electronic purification and identification of different behaviors of the major activation markers. The simultaneous use of two markers of activation and careful choice of activator are essential steps for reliable assessment of human basophil functions.

Relationship between CD38 expression on peripheral blood T‐cells and monocytes, and response to antiretroviral therapy: A one‐year longitudinal study of a cohort of chronically infected ART‐naive HIV‐1+ patients

HIV-1 infection has been associated with high expression of CD38 on peripheral blood (PB) CD8+ and CD4+ T-cells, which has been related with poor prognosis in untreated HIV-1+ patients. In turn, CD38 expression on PB monocytes from HIV-1+ individuals and its behavior after starting antiretroviral therapy (ART) have been poorly studied. CD38 expression on PB CD8+ and CD4+ T-lymphocytes and monocytes was prospectively analyzed in 30 ART-naive HIV-1+ patients, using a quantitative multiparameter flow cytometry approach. Patients were tested prior to therapy, and at weeks +2, +4, +8, +12, and +52 after ART. Prior to ART, CD38 expression was significantly increased on PB CD8+ and CD4+ T-cells and monocytes; despite a significant decrease after ART, CD38 expression remained abnormally high on PB CD8+ T-cells and monocytes, even after one year of therapy, in the absence of detectable plasma viral load. The ART-induced early changes on CD38 expression by PB T-cells and monocytes differed among the cell subsets analyzed and patient groups, probably reflecting an interaction between the direct effects of therapy and a redistribution of the PB compartments of T-cells and monocytes. Hierarchical clustering analysis showed that the overall pattern of changes in CD38 expression observed early after starting ART was predictive of a better response to therapy, not only for PB CD8+ T-cells, but also for CD4+ T-cells and monocytes. Accordingly, those HIV-1+ patients, who experienced a more pronounced increase in CD38 expression on both PB CD4+ T-cells and monocytes after 2 weeks of ART, showed a more rapid viral clearance, which might reflect decreased HIV-1 replication in lymph nodes and other tissues, and a partial restoration of hematopoiesis. Combined quantitative measurement of CD38 expression on PB monocytes, and CD8+ and CD4+ T-cells is a more useful tool for monitoring HIV-1+ patients under ART, rather than quantitation of CD38 expression on PB CD8+ T-lymphocytes alone.

Affinity of single S. cerevisiae cells to 2-NBDglucose under changing substrate concentrations.

Saccharomyces cerevisiae is a widely employed microorganism in biotechnological processes. Since proliferation and product formation depend on the capacity of the cell to access and metabolize a carbon source, a technique was developed to enable for analyzing the S. cerevisiae H155 cells' affinity to extracellular glucose concentrations. The fluorescent glucose analogue 2-NBDglucose was employed as a functional parameter to analyze the cells' affinity to glucose. Structural parameters (proliferation, neutral lipid content, granularity, and cell size) were also investigated. Cells were grown both in batches and in chemostat regimes. The 2-NBDglucose uptake in individual cells proceeds in a time- and concentration-dependent manner and is affected by respiratory and respirofermentative modes of growth. The process is inhibited by D-glucose, D-fructose, D-mannose, and sucrose, but not L-glucose, D-galactose or lactose; maltose is a weak inhibitor. The affinity of the individual cells to 2-NBDglucose was found to be high at low extracellular glucose concentrations, and weak at high concentrations. An additional, underlying pattern in the cells' affinity to glucose was detected, illustrated by the recurrent appearance of two subpopulations showing distinctly differing quantities of this substrate. A multiparameter flow cytometry approach is presented that enables, for the first time, for analysis of the affinity of individual S. cerevisiae cells to glucose. Besides the adjustment of the yeast cell metabolism to extracellular glucose concentrations by altering their affinity to glucose, at least one further mechanism is clearly involved. Two subpopulations of cells were resolved, with different affinities not correlated with other cellular parameters measured.

Differential constitutive and activation-dependent expression of prion protein in human peripheral blood leucocytes.

The cellular isoform of the prion protein (PrPC) is a cell surface glycoprotein that has recently been shown to play a role in haemopoietic cell activation and proliferation. We have characterized the constitutive expression of PrPC on human peripheral blood (pB) cell populations, using PrP-specific antibodies in a multiparameter flow cytometry approach. We found that T cells, NK cells and monocytes exhibit similar PrPC levels, whereas PrPC surface staining on B cells was significantly lower and was virtually absent on granulocytes. Within the T-cell compartment, CD8+ cells showed a significantly higher PrPC expression than CD4+ cells. Similarly, CD3+ cells co-expressing the activation marker CD56 (N-CAM) exhibited significantly higher PrPC expression levels than their CD56- counterparts. Culture of CD14+ pB monocytes for 12-48 h in the presence of interferon gamma (IFN-gamma) resulted in a significant increase in PrPC expression in a time- and concentration-dependent manner. This effect was partially abrogated by the addition of the metabolic inhibitor cycloheximide, indicating the role of protein synthesis in this process. Our results show that PrPC expression on human haemopoietic cells correlates with the activation and developmental status of these cells, suggesting an important functional role of PrPC in the haemopoietic system.