FACSCanto II and LSRFortessa flow cytometer instruments can be synchronized utilizing single-fluorochrome-conjugated surface-dyed beads for standardized immunophenotyping.

BACKGROUND: Multiparameter flow cytometry is the preferred method to determine immunophenotypic features of cells present in a wide variety of sample types. Standardization is key to avoid inconsistencies and subjectivity of interpretations between clinical diagnostic laboratories. Among these standardization requirements, synchronization between different flow cytometer instruments is indispensable to obtain comparable results. This study aimed to investigate whether two widely used flow cytometers, the FACSCanto II and LSRFortessa, can be effectively synchronized utilizing calibration bead-based synchronization. METHOD: Two FACSCanto II and two LSRFortessa flow cytometers were synchronized with both multicolor hard-dyed and single-fluorochrome-conjugated surface-dyed beads according to the manufacturer's instructions. Cell staining was performed on five whole-blood samples obtained from healthy controls and were analyzed upon synchronization with the respective synchronization protocols. RESULTS: Comparability criteria (defined as <15% deviation from the reference instrument) were met with both bead sets when synchronizing different FACSCanto II or LSRFortessa instruments. However, we observed that the criteria could not be met when synchronizing FACSCanto II with LSRFortessa instruments with multicolor hard-dyed beads. By utilizing single-fluorochrome-conjugated surface-dyed beads to determine and adjust PMT voltages, the accepted comparability criteria were successfully met. The protocol has been validated using five different eight-parameter stained samples. CONCLUSION: We show that FACSCanto II and LSRFortessa instruments can effectively be synchronized using single-fluorochrome-conjugated surface-dyed beads in case deviation criteria cannot be met using multicolor hard-dyed beads. Synchronization with single-fluorochrome-conjugated surface-dyed beads results in decreased deviations between instruments, allowing comparability criteria to become stricter.

A "No-Touch" Antibody-Staining Method of Adherent Cells for High-Throughput Flow Cytometry in 384-Well Microplate Format for Cell-Based Drug Library Screening.

In the last decade, screening compound libraries on live cells has become an important step in drug discovery. The abundance of compounds in these libraries requires effective high-throughput (HT) analyzing methods. Although current cell-based assay protocols are suitable for HT analyses, the analysis itself is often restrained to simple, singular outcomes. Incorporation of HT samplers on flow cytometers has provided an interesting approach to increase the number of measurable parameters and increase the sensitivity and specificity of analyses. Nonetheless, to date, the labor intensive and time-consuming strategies to detach and stain adherent cells before flow cytometric analysis has restricted use of HT flow cytometry (HTFC) to suspension cells. We have developed a universal "no-touch" HTFC antibody staining protocol in 384-well microplates to bypass washing and centrifuging steps of conventional flow cytometry protocols. Optimizing culture conditions, cell-detachment and staining strategies in 384-well microplates resulted in an HTFC protocol with an optimal stain index with minimal background staining. The method has been validated using six adherent cell lines and simultaneous staining of four parameters. This HT screening protocol allows for effective monitoring of multiple cellular markers simultaneously, thereby increasing informativity and cost-effectiveness of drug screening. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Monocytes and Granulocytes Reduce CD38 Expression Levels on Myeloma Cells in Patients Treated with Daratumumab.

Purpose: Daratumumab treatment results in a marked reduction of CD38 expression on multiple myeloma cells. The aim of this study was to investigate the clinical implications and the underlying mechanisms of daratumumab-mediated CD38 reduction.Experimental Design: We evaluated the effect of daratumumab alone or in combination with lenalidomide-dexamethasone, on CD38 levels of multiple myeloma cells and nontumor immune cells in the GEN501 study (daratumumab monotherapy) and the GEN503 study (daratumumab combined with lenalidomide-dexamethasone). In vitro assays were also performed.Results: In both trials, daratumumab reduced CD38 expression on multiple myeloma cells within hours after starting the first infusion, regardless of depth and duration of the response. In addition, CD38 expression on nontumor immune cells, including natural killer cells, T cells, B cells, and monocytes, was also reduced irrespective of alterations in their absolute numbers during therapy. In-depth analyses revealed that CD38 levels of multiple myeloma cells were only reduced in the presence of complement or effector cells, suggesting that the rapid elimination of CD38high multiple myeloma cells can contribute to CD38 reduction. In addition, we discovered that daratumumab-CD38 complexes and accompanying cell membrane were actively transferred from multiple myeloma cells to monocytes and granulocytes. This process of trogocytosis was also associated with reduced surface levels of some other membrane proteins, including CD49d, CD56, and CD138.Conclusions: Daratumumab rapidly reduced CD38 expression levels, at least in part, through trogocytosis. Importantly, all these effects also occurred in patients with deep and durable responses, thus excluding CD38 reduction alone as a mechanism of daratumumab resistance.The trials were registered at www.clinicaltrials.gov as NCT00574288 (GEN501) and NCT1615029 (GEN503). Clin Cancer Res; 23(24); 7498-511. ©2017 AACR.

Aberrant hepatic lipid storage and metabolism in canine portosystemic shunts.

Non-alcoholic fatty liver disease (NAFLD) is a poorly understood multifactorial pandemic disorder. One of the hallmarks of NAFLD, hepatic steatosis, is a common feature in canine congenital portosystemic shunts. The aim of this study was to gain detailed insight into the pathogenesis of steatosis in this large animal model. Hepatic lipid accumulation, gene-expression analysis and HPLC-MS of neutral lipids and phospholipids in extrahepatic (EHPSS) and intrahepatic portosystemic shunts (IHPSS) was compared to healthy control dogs. Liver organoids of diseased dogs and healthy control dogs were incubated with palmitic- and oleic-acid, and lipid accumulation was quantified using LD540. In histological slides of shunt livers, a 12-fold increase of lipid content was detected compared to the control dogs (EHPSS P<0.01; IHPSS P = 0.042). Involvement of lipid-related genes to steatosis in portosystemic shunting was corroborated using gene-expression profiling. Lipid analysis demonstrated different triglyceride composition and a shift towards short chain and omega-3 fatty acids in shunt versus healthy dogs, with no difference in lipid species composition between shunt types. All organoids showed a similar increase in triacylglycerols after free fatty acids enrichment. This study demonstrates that steatosis is probably secondary to canine portosystemic shunts. Unravelling the pathogenesis of this hepatic steatosis might contribute to a better understanding of steatosis in NAFLD.

Establishing human leukemia xenograft mouse models by implanting human bone marrow-like scaffold-based niches.

To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches.

Lymphocyte maintenance during healthy aging requires no substantial alterations in cellular turnover.

In healthy humans, lymphocyte populations are maintained at a relatively constant size throughout life, reflecting a balance between lymphocyte production and loss. Given the profound immunological changes that occur during healthy aging, including a significant decline in T-cell production by the thymus, lymphocyte maintenance in the elderly is generally thought to require homeostatic alterations in lymphocyte dynamics. Surprisingly, using in vivo (2) H2 O labeling, we find similar dynamics of most lymphocyte subsets between young adult and elderly healthy individuals. As the contribution of thymic output to T-cell production is only minor from young adulthood onward, compensatory increases in peripheral T-cell division rates are not required to maintain the T-cell pool, despite a tenfold decline in thymic output. These fundamental insights will aid the interpretation of further research into aging and clinical conditions related to disturbed lymphocyte dynamics.

Inability of monoclonal anti-light chain antibody to detect clonal B-cells in a patient with follicular lymphoma by multicolor flow cytometry.

BACKGROUND: Our recent publication "Inability of a monoclonal anti-light chain antibody to detect clonal plasma cells in a patient with multiple myeloma by multicolor flow cytometry," underlined the importance of choice of antibodies to detect cytoplasmic light chains. Our present study extends this finding for detection of surface immunoglobulin (SIg) light chains on clonal B-cells. METHODS: Multicolor flow cytometry was used for analyzing bone marrow (BM) from a patient with a CD10-positive follicular lymphoma for infiltrating clonal B-cells. RESULTS: In the BM aspirate, B cells could be identified expressing CD19, CD10, and high levels of CD20. No SIg light chain expression was found on this population of B cells employing monoclonal antibodies. Re-analysis using polyclonal antibodies against SIg light chains, revealed presence of lambda light chains on the CD10positive B-cells. CONCLUSIONS: These data illustrate when antibodies against SIg light chains are employed for B-cell clonality assessment, polyclonal antibodies are preferred over monoclonal antibodies.

Inability of a monoclonal anti-light chain antibody to detect clonal plasma cells in a patient with multiple myeloma by multicolor flow cytometry.

BACKGROUND: Multicolor flow cytometry (MFC) is increasingly important for the diagnosis and minimal residual disease (MRD) assessment of patients with plasma cells (PC) dyscrasias, like multiple myeloma. Recently published information shows that immunophenotype of myeloma PC can change over time and normal PC are heterogeneous in the expression of CD19 and CD56. This implies that for a sensitive, reliable detection of MRD clonality assessment by the detection of cytoplasmic kappa and lambda light chains is advisable. METHODS: Eight-color MFC was used to detect normal and myeloma PC by the expression of CD38 and CD138. Analysis of additional surface antigens like CD45, CD19, CD56, CD27, and the intracellular immunoglobulin light chain distribution were used to differentiate polyclonal from clonal PC. RESULTS: Absence of cytoplasmic light chains expression in a PC subpopulation with an abnormal phenotype suggested the presence of non-secretory plasma cells in the bone marrow (BM) of this patient. This observation however, was contradicted by the presence of free lambda light chains in the patient's serum. After repeating the analysis with polyclonal antibodies against intracellular immunoglobulin light chains instead of monoclonal antibodies, the abnormal PC subpopulation appeared to express lambda light chains. CONCLUSION: These data illustrate that if clonality assessment of PC is included in disease monitoring, the use of polyclonal over monoclonal antibodies is preferred for the detection of intracellular immunoglobulin light chains.

Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma.

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies.

Multicolor flow cytometry for evaluation of platelet surface antigens and activation markers.

INTRODUCTION: Flow cytometry allows the analysis of multiple antigens in a single tube at a single cell level. We present a rapid and sensitive two tube flow cytometric protocol for the detection of multiple platelet antigens and activation markers gated on a pure platelet population. MATERIALS AND METHODS: The presence of platelet specific antigens was analyzed in citrated whole blood of normal platelets and from patients diagnosed with platelet abnormalities. Quiescent platelets as well as stimulated platelets were analyzed using a gating strategy based on ubiquitously expressed platelet membrane markers. A ubiquitously expressed platelet marker was combined with antibodies against the activated alpha2b-beta3 (PAC-1), Lysosomal Activated Membrane Protein (CD63) and P-selectin (CD62P). RESULTS: We were able to detect the platelet antigens CD36, CD41, CD42a, CD42b and CD61 in one single tube. Our approach allowed the single tube determination of PAC-1, CD63 and CD62P after activation of platelets by thrombin, collagen, ADP and PAR-1, and determination of platelet abnormalities. CONCLUSIONS: Our two tube multi-parameter screening protocol is suited for the analysis of platelet antigens expressed on quiescent and activated platelets and allows the detection of aberrancies as found in blood of patients with thrombocytopathy such as Glanzmann Thrombasthenia, storage pool disease with diminished granule content and patients treated with clopidogrel and acetylsalicylic acid.

The inflammasome and caspase-1 activation: a new mechanism underlying increased inflammatory activity in human visceral adipose tissue.

The immune competent abdominal adipose tissue, either stored viscerally [visceral adipose tissue (VAT)] or sc [sc adipose tissue (SAT)], has been identified as a source of IL-1ß and IL-18. To become active, the proforms of these cytokines require processing by caspase-1, which itself is mediated by the inflammasome. In this descriptive study, we investigate the expression of inflammasome components and caspase-1 in human fat and determine whether caspase-1 activity contributes to the enhanced inflammatory status of VAT. Paired SAT and VAT biopsies from 10 overweight subjects (body mass index, 25-28 kg/m(2)) were used to study the cellular composition and the intrinsic inflammatory capacity of both adipose tissue depots. The percentage of CD8(+) T cells within the lymphocyte fraction was significantly higher in VAT compared with SAT (41.6 vs. 30.4%; P < 0.05). Adipose tissue cultures showed a higher release of IL-1ß (10-fold; P < 0.05), IL-18 (3-fold; P < 0.05), and IL-6 and IL-8 (3-fold, P < 0.05; and 4-fold, P < 0.05, respectively) from VAT compared with SAT that was significantly reduced by inhibiting caspase-1 activity. In addition, caspase-1 activity was 3-fold (P < 0.05) higher in VAT compared with SAT, together with an increase in the protein levels of the inflammasome members apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain (2-fold; P < 0.05) and nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (2-fold; nonsignificant). Finally, caspase-1 activity levels were positively correlated with the percentage of CD8(+) T cells present in adipose tissue. Our results show that caspase-1 and nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 inflammasome members are abundantly present in human VAT. The increased intrinsic caspase-1 activity in VAT represents a novel and specific inflammatory pathway that may determine the proinflammatory character of this specific depot.

Thrombocytopenia in early malaria is associated with GP1b shedding in absence of systemic platelet activation and consumptive coagulopathy.

Thrombocytopenia develops early in malaria, but the underlying mechanisms remain incompletely understood. We studied the aetiology of malaria-associated thrombocytopenia in volunteers experimentally infected with Plasmodium falciparum malaria, in Indonesian malaria patients and in ex vivo studies. In experimental human malaria, the decrease in platelet counts was associated with a concurrent rise in young platelets (immature platelet fraction) and thrombopoietin. D-dimer concentrations were moderately elevated without a prolongation in the activated partial thromboplastin time or decrease in fibrinogen. There was no increase in expression of the platelet surface markers CD62P, PAC-1 and CD63 and in plasma concentrations of the platelet factors P-selectin, CXCR4, CXCL7, RANTES and CD40L. In contrast, concentrations of soluble glycoprotein-1b (sGP1b), the external domain of the platelet receptor for von Willebrand factor (VWF), increased early. Indonesian malaria patients also had elevated concentrations of sGP1b, which correlated with VWF concentrations. Finally, incubation of platelets with parasitized erythrocytes in vitro failed to induce platelet aggregation or activation. We concluded that neither compromised platelet production nor platelet activation or consumptive coagulopathy were responsible for the early thrombocytopenia in malaria. We hypothesize that the increase in sGP1b concentrations results from VWF-mediated GP1b shedding; a process that may prevent excessive adhesion of platelets and parasitized erythrocytes.

GTS-21 inhibits pro-inflammatory cytokine release independent of the Toll-like receptor stimulated via a transcriptional mechanism involving JAK2 activation.

The vagus nerve can limit inflammation via the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). Selective pharmacological stimulation of the alpha7nAChR may have therapeutic potential for the treatment of inflammatory conditions. We determined the anti-inflammatory potential of GTS-21, an alpha7nAChR-selective partial agonist, on primary human leukocytes and compared it with nicotine, the nAChR agonist widely used for research into the anti-inflammatory effects of alpha7nAChR stimulation. Furthermore, we investigated whether the effects of both nicotinic agonists were restricted to specific Toll-like receptors (TLRs) stimulated and explored the mechanism behind the anti-inflammatory effect of GTS-21. GTS-21 and nicotine inhibited the release of pro-inflammatory cytokines in peripheral blood mononuclear cells (PBMCs), monocytes and whole blood independent of the TLR stimulated, with higher potency/efficacy for GTS-21 compared to nicotine. The anti-inflammatory cytokine IL-10 was relatively unaffected by both nicotinic agonists. The effects of GTS-21 and nicotine could not be reversed by nAChR antagonists, while the JAK2 inhibitor AG490 abolished the anti-inflammatory effects. GTS-21 downregulated monocyte cell-surface expression of TLR2, TLR4 and CD14. qPCR analysis demonstrated that the anti-inflammatory effect of GTS-21 is mediated at the transcriptional level and involves JAK2-STAT3 activation. In conclusion, GTS-21 has a profound anti-inflammatory effect in human leukocytes and that GTS-21 is more potent/efficacious than nicotine. The absence of a blocking effect of nAChR antagonists in human leukocytes might indicate different pharmacological properties of the alpha7nAChR in human leukocytes compared to other cell types. GTS-21 may be promising from a therapeutic perspective because of its suitability for human use.