Ficoll Density Gradient Separation Research Articles

Cryo-PRO facilitates whole blood cryopreservation for single-cell RNA sequencing of immune cells from clinical samples.

Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, PBMC isolation from blood requires technical expertise, training, and approximately two hours of onsite processing using Ficoll density gradient separation ('Ficoll') for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of PBMC isolation from cryopreserved whole blood that allows immediate onsite sample cryopreservation and subsequent PBMC isolation in a central laboratory prior to sequencing. We compared scRNA-seq results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical scRNA-seq outputs including cell substate fractions and marker genes were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis (Pearson correlation 0.78, p<0.001; 70% of top marker genes shared). Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding scRNA-seq in multicenter studies of sepsis and other diseases.

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients.

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients.

Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro.

Simple SummaryKnowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, will allow the development of targeted nutrition strategies, prevent infectious diseases and the usage of antimicrobials, and promote the health of animals. To investigate the mechanisms of action of immunomodulating compounds, controlled in vitro systems using freshly isolated immune cells from blood represent a promising alternative to animal experiments. Immune cell isolation from the blood of chickens is a complex and difficult process since the immune cell fractions are significantly contaminated with red blood cells and platelets. To our knowledge, a robust protocol for immune cell isolation from chicken blood and the subsequent cultivation of immune cells is not available. Here, we established a protocol for blood sampling and immune cell isolation and cultivation from chicken blood, which could be applied for the investigation of direct effects of immunomodulating compounds. This protocol, combining different techniques of immune cell isolation, cultivation, and differentiation of distinct immune cell populations, will serve as a potential alternative to animal testing in vivo. By gaining knowledge about the mechanisms of action of immunomodulating compounds, this in vitro model will contribute to promote health and welfare in chicken farming.Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na–citrate tubes revealed the highest count of vital cells compared to commercial Li–heparin (p < 0.01) and K3EDTA (p < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (p < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (p < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.

A Novel High-Throughput Microfluidic Technology for Isolating Lymphocytes from Buffy Coats for Allogeneic Cellular Therapies

Introduction: Adoptive immunotherapies involving infusion of third-party donor-derived T lymphocytes (T-cells) to treat life-threatening viral infections (including BK virus, adenovirus, cytomegalovirus, and Epstein-Barr virus) in immunosuppressed recipients have shown potentially curative efficacy in recent clinical trials. Currently, allogeneic T-cells are typically obtained by extracting mononuclear cells via Ficoll density gradient centrifugation of peripheral blood, or via leukapheresis collected from an HLA-matched, virus-seropositive donor. A library of cryopreserved HLA-typed allogeneic T-cell concentrates would provide a much more sustainable source of T-cells, and enable producing T-cell based therapies on-demand quickly. An abundant source of T-cells for generating such a library could be the ‘buffy coat’ (BC); the layer of white blood cells (WBCs) between the red blood cells (RBCs) and the platelet-rich plasma, which is produced when donated whole blood is processed into components via the 'buffy coat method.' However, the U.S. standard favors the alternative 'PRP method,' therefore WBCs (including T-cells) are typically trapped in leukoreduction filters and subsequently discarded. Here we present the initial proof-of-concept for applying our recently-developed microfluidic technique (controlled incremental filtration, CIF) to purification and concentration of lymphocytes from BC units, at practical flowrates.Materials and Methods: BC units were obtained from MD Anderson Blood Bank (Houston, TX). A preliminary study was performed to determine the optimal dilution factor and device design parameters to achieve the highest lymphocyte recovery with minimal RBC contamination from our CIF approach. Several individual CIF-based microfluidic modules were multiplexed in parallel to achieve high throughput. The microfluidic devices were fabricated via soft-lithography techniques. Diluted BC samples were passed through the CIF-based microfluidic cell separator (Run 1) - the lymphocyte-rich product was collected, re-diluted to its original volume, and passed through the same device again (Run 2), using a flow rate of 6 mL/min for both runs (Fig. 1). A CBC with 5-part differential was performed on all blood samples using a hematology analyzer to quantify the recovery of lymphocytes and removal of RBCs and platelets.Results and Discussion: Our preliminary study revealed that a starting WBC count of ~10-15×103/µL gives the best balance between lymphocyte recovery and RBC removal at a flow rate of 6 mL/min. BC units were typically 35-50 mL, with initial WBC count between 20-75×103/µL, therefore units required 2-5-fold dilution. In a subsequent separation study, Run 1 through the CIF-based microfluidic device recovered 92.6±0.6% of lymphocytes while removing 92.0±0.5% of RBCs from the diluted BC suspensions. During Run 2, the CIF device recovered 93.0±0.4% of remaining lymphocytes, and removed 83.3±0.3% of residual RBCs. Overall, processing a BC unit through the CIF-based microfluidic device took approximately 1-1.5 hours. Combined, the two runs achieved a total lymphocyte recovery of 86.1±0.9% (final concentration of 51.7±5.2×103/µL) and a total RBC removal of 98.6±0.1% (reducing RBC count from 2.6±0.1×106/µL to 0.13±0.01×106/µL), making the final product cryopreservation-ready without the need for further purification or concentration. These results are on par with those typically achieved via Ficoll density gradient separation, while avoiding the associated drawbacks of laborious workflow, reagent cost, and reliance on large-footprint machinery (e.g. a centrifuge).Conclusion: In this study we demonstrated the feasibility of using a simple, disposable, high-throughput microfluidic device for rapidly purifying and concentrating lymphocytes from buffy coat units. This capability could simplify the creation of cryopreserved HLA-typed lymphocyte lines from donated blood, thereby substantially improving treatment options for patients who need allogeneic T-cell therapy by providing clinicians with HLA-compatible cells, on-demand. [Display omitted] DisclosuresGifford:Halcyon Biomedical Incorporated: Employment, Equity Ownership. Shevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

Towards a Closed System for Extracting Lymphocytes from Whole Blood

Introduction: Cellular therapies promise new treatment options and potentially curative efficacy for a wide array of acute and chronic diseases. The ability to obtain viable, uncontaminated lymphocyte concentrates from peripheral blood is in high demand. A major barrier for translating lymphocyte-based therapies into clinical practice is the poor scalability of manual, labor- and time-consuming ‘open system’ methods (e.g. Ficoll density gradient centrifugation) currently being used for isolating lymphocytes. To address the well-known limitations of existing separation methods, we developed a two-stage cell isolation approach that enables highly-efficient extraction of lymphocytes from whole blood (WB) without the use of centrifugation or any open-air manipulations by (1) depleting ~99% of red blood cells (RBCs) from the sample via rapid sedimentation induced by HES (6% hydroxyethyl starch in 0.9% sodium chloride), and then (2) passing the leukocyte-rich supernatant through a high-throughput microfluidic device (based on our newly-developed approach of ‘controlled incremental filtration,‘ CIF) to separate lymphocytes from platelets and residual RBCs.Materials and Methods: WB was collected from healthy consenting volunteers under an IRB approved protocol. An optimization study was performed to identify parameters of the sedimentation protocol with the highest lymphocyte yield and minimal contamination. This optimal protocol was then used to compare performance of (i) the standard Ficoll density gradient separation method, and (ii) our new two-stage separation approach. Each WB sample was divided into two equal parts - one half was processed via the conventional density gradient method, and the other half was first mixed with HES, allowed to sediment, and then the supernatant was passed through a CIF-based microfluidic device (designed for high-precision separation of lymphocytes from smaller cells, and ~15-fold reduction of the sample volume) at a flow rate of 0.5 mL/min. For both separation methods, product yield and purity were quantified by performing CBC with a 5-part differential on all samples. Flow cytometry was used to measure viability of isolated lymphocytes, and activation level of residual platelets via P-selectin and phosphatidylserine (PS) exposure. Separated lymphocytes were cultured for 4 days using standard proliferation techniques. Total number of viable cells was determined using Trypan Blue exclusion dye and manual counting with a hemocytometer.Results and Discussion: The optimized sedimentation protocol, which involved mixing WB with 10% HES (v/v) and waiting 30 min to complete, yielded the best combination of lymphocyte recovery (72.1±2.2%) and RBC depletion (99.48±0.07%) for the first stage of our separation approach. In the split-sample study, our two-stage method overall recovered 70.4±0.7% of lymphocytes from the initial WB sample, with 98.2±0.6% viability, while depleting 92.4±0.4% of platelets and 99.62±0.1% of RBCs. In comparison, the Ficoll density gradient centrifugation method removed 99.95±0.2% of RBCs and 94.2±0.1% of platelets, while recovering 74.5±2.40% of lymphocytes, with 98.3±0.7% viability. Lymphocytes produced by our method showed a significnatly higher prolifiration capacity than cells separated via density gradient centrifugation (Fig. 1). Markers of activation for residual platelets in the lymphocyte concentrate produced by our two-stage method were several fold lower than for Ficoll density gradient separation: 1.9±0.1% (our method) vs. 8.4±7.0% (Ficoll) for P-selectin, and 2.0±1.5% (our method) vs. 4.4±1.5% (Ficoll) for PS exposure. Importantly, lymphocyte isolation using our two-stage method was 3-fold faster than the Ficoll density graduate separation for the same amount of WB sample: <60 min (our method) vs. >180 min (Ficoll).Conclusion: We developed an easy-to-use approach for extracting lymphocytes from peripheral blood with purity, viability and yield meeting the current standard for cellular therapies. Since our new approach is scalable, compatible with ‘closed system’ operation, produces lymphocytes with higher proliferation capacity, and is much faster than conventional density gradient separation, it could have a potentially transformative impact by accelerating research into new cellular therapies, and simplifying translation of these treatments into clinical practice. [Display omitted] DisclosuresGifford:Halcyon Biomedical Incorporated: Employment, Equity Ownership. Shevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

Detection of Antibodies in Serum and Lymphocytes of Patients with Hemorraghic Fever with Renal Syndrome

Hemorrhagic Fever with Renal Syndrome (HFRS) is zoonotic disease, endemic in the republic of Tatarstan. Increased endothelial barrier permeability, resulting in tissue edema, leukocyte migration and hemorrhages, is characteristic pathological finding in HFRS. Cytokine storm hypothesis was suggested to explain hantavirus pathogenesis, where over activation of pro-inflammatory cytokines was suggested to cause endothelial cell damage and increased leukocyte infiltration of target tissue. Hantavirus nucleocapsid (N) protein and glycoprotein proteins (G1 and G2) were suggested to be the most immunogenic viral proteins. However, information on immunogenic epitopes triggering humoral and cellular immune response is limited.Materials and methods. Blood and serum samples were collected from HFRS patients admitted to Republican Clinical Hospital for Infectious Disease named after Agafonov, Republic of Tatarstan along with controls matched for the region. The Institutional Review Board of the Kazan Federal University approved this study and informed consent was obtained from each study subject according to the guidelines approved under this protocol (article 20, Federal Law “Protection of Health Rights of Citizens of Russian Federation” N323- FZ, 11.21.2011).Peptide library consisting of overlapping peptides (15 aa; 5 aa overlap) of N, G1 and G2 of Puumala virus (PPUV) were generated. Total of 150 peptides were tested. PUUV was selected because it is endemic in Republic of Tatarstan causing HFRS.Immunogenic epitopes inducing antibody response in HFRS were analyzed using direct ELISA. Peptides were used to coat the plate (1 µg/well), followed by incubation with HFRS serum. Control serum samples were used to determine peptide non-specific reactivity. Statistical analysis was conducted using Minitab software; differences between medians of HFRS and control groups were analyzed using the Mann-Whitney test for non-parametric data. Data was considered significant at p ≤ 0.05.T-lymphocyte epitopes were analyzed using ELISpot assay. Blood from HFRS cases was collected in vacuum tubes containing sodium citrate (Vacuutest, Apexmed). Confirmation of PUUV infection was done by PCR (n=10). Peripheral blood mononuclear cells (PBMC) were isolated using ficoll density gradient separation (p=1,077 g/l). Isolated mononuclear leukocytes were cultured in 96-well plates coated with anti-IFNγ antibody. Leukocytes were incubated with each peptide for 48 hours. PBMC treated with phytohaemoagglutinine (PHA) served as a positive control. Spots were visualized using AEC substrate and counted by direct microscopy.Results. PUUV RNA was detected in all blood samples. Serum samples from 10 HFRS cases and 10 controls were analyzed on reactivity to the overlapping peptides coding nucleocapsid and G1 and G2 glycoproteins. Reactivity of eleven peptides (150 total) was statistically significant in HFRS serum as compared to controls (Figures 1a, 1b). Interestingly, the intensity of reactivity between HFRS serum and glycoprotein peptides was 1.5±0.2 times higher than that of N protein.Several G and N protein peptides were identified as inducing IFN-γ production by leukocytes. These peptides are M82 (YTRKACIQLGTEQTC), M69 (TDLELDFSLPSSASY), N3 (ARQKLKDAERAVEVY), N2 (ITRHEQQLVVARQKL), N1 (MSDLTDIQEEITRHE) and M104 (NLVRGQNTVHIVGKG) (Figure 1c).Collected data revealed that the most immunogenic peptides were M104 (NLVRGQNTVHIVGKG) and N3 (ARQKLKDAERAVEVY) from hantavirus G and N proteins respectively. These peptides revealed strong reactivity in ELISA and ELISpot assays suggesting that they play role in activation of humoral and immune responses.Conclusion. Data confirms that both N and G proteins of PUUV are immunogenic. New epitopes were identified on N and G proteins which can stimulate humoral and cellular immunity.This work was supported by Russian Science Foundation grant №15-14-00016. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Blocking HDAC3 in Bone Marrow Stromal Cells Has Direct Anti-Multiple Myeloma Effect and Modulates T Cell Function

IntroductionHistone deacetylases (HDAC) are therapeutic targets in Multiple Myeloma (MM) and the pan-HDAC inhibitor Panobinostat is FDA approved. However, clinical trials showed that non-selective HDAC inhibitors have a narrow therapeutic index, prompting the development of isoform-specific HDAC inhibitors such as the HDAC6 inhibitor Ricolinostat. Preclinical studies have shown that HDAC3 downregulation/inhibition exerts an anti-myeloma effect, suggesting that HDAC3 may be a novel molecular target in MM. However, the effect of targeting HDAC3 in the bone marrow (BM) microenvironment has not been investigated. The BM microenvironment, particularly stromal and immune cells, have been shown to be key in supporting MM growth, immune escape, and resistance to therapy. We sought to study whether targeting HDAC3 in BM stroma cells (BMSC) has direct cytotoxic effects against stromal cells and/or impacts MM cell and T cell function through secreted factors and/or cell-to-cell contact mechanisms.MethodsWe targeted HDAC3 in human BMSC line HS-5 and primary-derived BMSC from MM patients (MM-BMSC) via siRNA or via the HDAC3-selective inhibitor BG45. MTT assay and flow cytometry with annexin V/PI staining were used to assess viability and apoptosis post-HDAC3 KD/pharmacologic inhibition. Downregulation of HDAC3 was confirmed by immunoblotting. To assess the effect of HDAC3 silencing in MM-BMSC on MM cells, we performed siRNA-mediated HDAC3 KD in HS-5 and MM-BMSC, removed transfection media after 48 hours, and performed co-culture experiments with MM1S.Luc and H929.Luc MM cell lines. After 96 hours MM cell proliferation was measured using the Luciferase Assay. Scrambled siRNA transfected HS-5 and MM-BMSC were used as control. To assess the effect of HDAC3 silencing in BMSC on T cells, we isolated peripheral blood mononuclear cells from healthy donors by Ficoll density gradient separation. CD3+ T cells were isolated using EasySep™ Human T Cell Isolation Kit and activated using Dynabeads Human T-Activator CD3/CD28. Cytokine array profiling was performed on the cell-culture supernatant using the Raybiotech C-series Human Cytokine Array according to manufacturer's protocol.ResultsFirst, we showed that pharmacological inhibition of HDAC3 via BG45 does not have direct anti-proliferative or cytotoxic effect on the HS-5 stromal cell line at doses up to 5-fold higher than the EC50 of MM cell lines. Consistent with this data, siRNA-mediated HDAC3 KD does not have direct anti-proliferative and/or cytotoxic effects on HS-5 or MM-BMSC. However, when MM cell lines were co-cultured with HDAC3-silenced HS-5 or MM-BMSC, there was a statistically significant decrease in BMSC-induced MM proliferation compared to control (HS-5: 38% decrease in MM proliferation in HDAC3 KD, p < 0.05; MM-BMSC: 24.1-27.5% decrease in MM proliferation in HDAC3 KD, p < 0.05). To assess whether cell-to-cell contact is necessary to mediate this growth arrest, we cultured MM cell lines in conditioned media from HDAC3 or scrambled siRNA transfected BMSC and discovered that the former was sufficient to induce growth/proliferation arrest (HS-5: 31.5% decrease in MM proliferation in HDAC3 KD, p < 0.05; MM-BMSC: 24.3% decrease in MM proliferation in HDAC3 KD, p < 0.05), suggesting a paracrine effect.We next showed that conditioned supernatant from HDAC3-KD HS-5, cultured alone or in combination with MM cell lines, significantly inhibited the proliferation of activated T-cells (HS-5 supernatant: 47.8% decrease in MM proliferation in HDAC3 KD; HS-5 + MM co-culture supernatant: 32.6% decrease in MM proliferation in HDAC3 KD). To identify the soluble factors responsible for this paracrine effect, we subjected HDAC3-KD and scrambled-transfected HS-5 supernatant to cytokine profiling. We identified a number of cytokines modulated by HDAC3 KD, which we are currently analyzing. Among these, we identified a 1.37-fold increase in soluble PD-L1 when HDAC3 was silenced in HS-5 cells.ConclusionsOur results show that inhibiting HDAC3 in BMSC mediates anti-MM and anti-T cell effect via soluble factors. Gene expression profiling and proteomic studies are currently underway to define the molecular mechanisms supporting these effects. Our preliminary data suggest that targeting HDAC3 in BMSC may alter the checkpoint repertoire in the BM milieu, suggesting potential utility of therapeutic strategies combining HDAC3 and checkpoint inhibitors. DisclosuresHideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Mazitschek:Acetylon: Equity Ownership. Anderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees.

The Hematopoietic Reconstitution through Intra-Bone-Marrow Injection Using Murine Fetal Liver Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) were considered an effective treatment option for many blood diseases and autoimmune diseases. HSCs can currently be isolated from bone marrow and umbilical cord blood. Due to the shortage of these resources, an alternative source of HSCs preparation were sorted out in this study and the ability of hematopoietic reconstruction were examined using such HSCs obtained from fetal liver.Fetal liver hematopoietic stem cells (FL-HSCs) were isolated from embryos of transgenic mice with an erythoid-specific GFP expression that was driven by a human β-globin gene promoter (HS23-GFP mice). Thus, GFP expression could be used as tracer of marker genes of red blood cells derived from the donor FL-HSCs visually. Single-cell suspension of E13.5 HS23-GFP murine liver was used for Ficoll density gradient separation, and the FL-HSC enriched portion was collected for intra-bone-marrow injection (IBMI) into 6-8 week-old recipient mice post 137Cs γ irradiation treatment. Three doses were tested for injection, i.e. 1×105, 1×106 and 1×107 cells, respectively.Hematopoietic reconstruction ability was observed every week after injection using blood smear and routine blood panel such as RBC, HGB and HCT. Bone marrow smear and pathological sections of liver and spleen of recipient mice were analyzed 8 weeks post FL-HSC transplantation. GFP expressed erythrocyte was captured in the peripheral blood starting one week after HL-HSC transplantation. The ability of hematopoietic reconstruction continued to rise until week 7, and became stable thereafter. Among the three treatment groups, the highest cell number group (1×107 ) had the best hematopoietic restoration, according to the RBC, HGB and HCT data. No abnormality such as lymphocytic infiltration in pathological sections of liver and spleen of recipients were noted. Bone marrow smear of recipient mice showed no difference when comparing to that of the wide type mice. This demonstrated HSC obtained from fetal liver, are capable of reconstructing hematopoietic function after transplantation using IBMI. This could provide an alternative source of HSC for future study of hematopoietic function and for future therapeutic treatment for blood diseases. DisclosuresNo relevant conflicts of interest to declare.

Detailed Immunophenotypic Profiling of Peripheral Blood Immune Cells in Patients with Hematologic Malignancies after Sars-Cov-2 Infection

Introduction:The spread of SARS-CoV-2 virus continues to pose a major public health threat. Patients with cancer are thought to be at increased risk from SARS-COV-2 infection due to the immunodeficiency that results from the underlying neoplasm and treatment. The immune response to this infection has been the subject of great interest, with an extreme variation in clinical severity between infected individuals. Variation in the immune cell response (B, T, and NK lymphocytes, monocytes, and myeloid derived suppressor cells (MDSCs), among others) and their function have been hypothesized to be responsible for this range of presentation.Methods:Two patients with a history of hematologic malignancies were matched with three non-cancer patients with similar baseline clinical characteristics and severity of COVID related illness. The critical group (CG) was defined as those requiring mechanical ventilation (MV) due to COVID related respiratory failure and the non-critical group (NCG) were hospitalized but did not require MV. All samples studied were obtained from peripheral blood and processed within 4-hours of collection. Peripheral blood mononuclear cell (PBMC) were isolated using ficoll density gradient separation. Flowcytometric analysis using CytekTM Aurora was done on fresh PBMC samples. Thirty antibody-based flow markers were used to identify 54 distinct immune cell populations. IRB approval was obtained.Results:Critical Group (CG):The CG included case 1, a 47 year-old (y.o.) female (F) with a history (hx) of acute myeloid leukemia and had an matched related donor allogeneic hematopoietic stem cell transplant (alloHSCT) 10-years prior remaining in remission, with hematologic recovery, and off immunosuppressants treated with remdesivir and coritcosteroids for COIVD directed therapy; and case 2, a 55 y.o. F with a hx of HIV treated with corticosteroids for COIVD directed therapy (see Figure 1a).Non-Critical Group (NCG):The NCG included case 3, a 73 y.o male (M) with hx of relapsed/refractory Philadelphia chromosome negative Acute Lymphoblastic Leukemia with loss of CD19 and CD22 expression following treatment with blinatumumab and inotuzumab, and most recently treated with decitabine/venetoclax; case 4, a 66 y.o. M with hx of cardiomyopathy; and case 6, a 54 y.o. M with hx of obesity. None of the NCG cases were treated with COVID directed therapy.See Table 1 for further clinical information.Immunophenotypic expression:Flow cytometry gating strategy done as outlined in Fig 1a. Case 1 had a high proportion of B-cells, CD8+ T-cells, and cells with exhaustion markers (CD8+CD94+ T-cells, CD4+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1-CD94+ NK T-cells, Lag3+Cd11b- non-TB leukocytes) and MDSC immunophenotypes compared with matched case 2. Case 3 also had a high proportion of exhaustion markers (Lag3+CD39 low B-cells, CD8+PD1+ T-cells, CD8+CD94+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1+CD94+ NK T-cells, PD1-CD94+ NK T-cells, Lag3+CD11b+, Lag3+CD11b- non-TB leukocytes) and high expression of immunosuppressive Treg and all MDSC; although high expression of granulocytic MDSC. Case 2 had a significant number of exhaustion and immunosuppressive cells as well. Cases 4 and 5 had a higher predominance of all T-cell subtypes and also had variable expression of exhaustion and immunosuppressive immunophenotypes (See Fib 1b).Conclusion:In our study of one critical and one non-critical patient with a history of hematologic malignancy matched with three non-cancer patients we demonstrate the high predominance of exhaustion markers (Lag3,PD1,CD94) and immunosuppressive cell types (Treg, granulocytic and monocytic MDSC). These findings are consistent with the fact that both CG and NCG, as hospitalized patients, represent the most severely ill COVID patient cohort. Of notable interest to the cancer population, cases 1 and 3 had a significant number of exhaustion and immunosuppressive immunophenotypes, suggestive of baseline exhaustion following alloHSCT even years after engraftment in case 1 and attenuated functional immunity in a patient undergoing active treatment in case 3. Interestingly, case 3 had lower expression of all MDSC, a known treatment effect of decitabine. Paired cytokine measurement and its effect on immunophenotype is underway. Additionally, we plan to present an atlas of the peripheral immune cell response on fifteen additional non-cancer COVID patients.DisclosuresNo relevant conflicts of interest to declare.

Increased Expression of B Lymphocyte Induced Maturation Protein 1 (BLIMP1) in Patients with Common Variable Immunodeficiency (CVID).

Common variable immunodeficiency (CVID) is a primary immune deficiency disorder characterized by a failure in B cell differentiation, impaired immunoglobulin production,and defect in response to vaccines. As a result of defective B cell maturation and differentiation in CVID, the affected patients commonly present with reduced numbers of memory B cell and antibody-secreting plasma cells. B-cell lymphoma 6 protein (BCL6) and B lymphocyte induced maturation protein 1 (BLIMP1) molecules are two important transcription factors that have key roles in the maturation of B cells to plasma cells. Hence, in the current survey, we aimed to evaluate the mRNA and protein expression levels of BCL6 and BLIMP1 in B lymphocytes isolated from peripheral blood in CVID patients. We collected blood samples from 12 CVID patients and 12 healthy controls. We isolated peripheral blood mononuclear cells (PBMCs) using Ficoll density gradient separation. Then, CD19+ B cells were purified using MACS. The protein expression and transcriptional level of BCL6 and BLIMP1 were respectively measured using flow cytometry and real-time PCR. Our results showed that the BLIMP1 mRNA expression, as well as BLIMP1 protein expression, were significantly higher in CVID patients compared to control subjects (p=0.009 and p=0.007, respectively). However, we found no significant difference in mRNA and protein expression of BCL6 between patients and healthy controls. According to our findings, increased mRNA and protein expression levels of BLIMP1 could be involved in defective maturation of B cells in patients with CVID and elucidate mechanistic insights into the pathogenesis of this disorder.

MONOCYTE-DERIVED DENDRITIC CELLS ISOLATION AND PHENOTYPIC CHARACTERIZATION

Dendritic cell (DC) is the most powerful inducers and regulators of immune responses, responsible for interaction within the immune system. The ability of DC to induce or regulate/suppress immune responses led to attention in their immunotherapeutic use in various disease types. The aim of the presented study was to generate in vitro CD14+ enriched peripheral blood monocytes (PBMCs) following the culture with human monocyte-derived dendritic cell serum-free differentiation media over ten days with phenotypical analysis and morphological identification for functional studies. In vitro, peripheral blood of human donors samples were collected for the purification of blood CD14+ monocytes that represent the most common origin for DC precursors based on ficoll density gradient separation and human monocyte-derived dendritic cell enriched differentiation medium. After PBMCs isolation, the cell viability, cell yield were determined, the monocyte-derived dendritic cell surface marker expression was detected by flow cytometric analysis after staining with specific fluorescence-conjugated monoclonal antibodies. in vitro culture and differentiation of human monocytes into DCs and their subsequent maturation into mature DC constitute a critical first step for different downstream analysis ranging from an understanding of both human DC biology and function to their use in clinical diagnostics and therapeutic design for different disease.

EGF, GDNF, and IGF-1 influence the proliferation and stemness of ovine spermatogonial stem cells in vitro.

The objective of the present study was to purify sheep spermatogonial stem cells (SSCs) from testicular isolate using combined enrichment methods and to study the effect of growth factors on SSC stemness during culture. The testicular cells from prepubertal male sheep were isolated, and SSCs were purified using Ficoll gradients (10 and 12%) followed by differential plating (laminin with BSA). SSCs were cultured with StemPro®-34SFM, additives, and FBS for 7days. The various doses (ng/ml) of growth factors, EGF at 10, 15, and 20, GDNF at 40, 70, and 100 and IGF-1 at 50, 100, and 150 were tested for the proliferation and stemness of SSCs in vitro. The stemness in cultured cells was assessed using SSC markers PLZF, ITGA6, and GFRα1. Ficoll density gradient separation significantly (p < 0.05) increased the percentage of SSCs in 12% fraction (35.1 ± 3.8 vs 11.2 ± 3.7). Subsequently, purification using laminin with BSA plating further enriched SSCs to 61.7 ± 4.7%. GDNF at 40ng/ml, EGF at 15 and 20ng/ml and IGF1 at 100 and 150ng/ml significantly (p < 0.05) improved proliferation and stemness of SSCs up to 7days in culture. GDNF at 40ng/ml outperformed other growth factors tested and could maintain the ovine SSCs proliferation and stemness for 36days. The combined enrichment method employing density gradient centrifugation and laminin with BSA plating improves the purification efficiency of ovine SSCs. GDNF at 40ng/ml is essential for optimal proliferation and sustenance of stemness of ovine SSCs in vitro.

Saturated fatty acids, obesity, and the nucleotide oligomerization domain–like receptor protein 3 (NLRP3) inflammasome in asthmatic patients

Both obesity and high dietary fat intake activate the nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome. We aimed to examine NLRP3 inflammasome activity in the airways of obese asthmatic patients after macronutrient overload and in immune cells challenged by inflammasome triggers. Study 1 was a cross-sectional observational study of nonobese (n=51) and obese (n=76) asthmatic adults. Study 2 was a randomized, crossover, acute feeding study in 23 asthmatic adults (n=12 nonobese and n=11 obese subjects). Subjects consumed 3 isocaloric meals on 3 separate occasions (ie, saturated fatty acid, n-6 polyunsaturated fatty acid, and carbohydrate) and were assessed at 0 and 4hours. For Studies 1 and 2, airway inflammation was measured based on sputum differential cell counts, IL-1β protein levels (ELISA), and sputum cell gene expression (Nanostring nCounter). In Study 3 peripheral blood neutrophils and monocytes were isolated by using Ficoll density gradient and magnetic bead separation and incubated with or without palmitic acid, LPS, or TNF-α for 24hours, and IL-1β release was measured (ELISA). In Study 1 NLRP3 and nucleotide oligomerization domain 1 (NOD1) gene expression was upregulated, and sputum IL-1β protein levels were greater in obese versus nonobese asthmatic patients. In Study 2 the saturated fatty acid meal led to increases in sputum neutrophil percentages and sputum cell gene expression of Toll-like receptor 4 (TLR4) and NLRP3 at 4hours in nonobese asthmatic patients. In Study 3 neutrophils and monocytes released IL-1β when challenged with a combination of palmitic acid and LPS or TNF-α. The NLRP3 inflammasome is a potential therapeutic target in asthmatic patients. Behavioral interventions that reduce fatty acid exposure, such as weight loss and dietary saturated fat restriction, warrant further exploration.

Combined dextran and ficoll separation yields pure populations of chicken peripheral blood mononuclear cells - short communicationia

The ficoll density gradient method, the most widely used method for isolating avian peripheral blood mononuclear cells (PBMCs), results in significant contamination with nucleated thrombocytes. This thrombocyte contamination can affect both immunophenotyping and functional assays of T cells. We used full blood samples from ten vaccinated male layer hybrid chickens for isolating PBMCs, and have developed a low cost, combined dextran and ficoll density gradient separation method which, in comparison to the standard ficoll density gradient separation, markedly reduces thrombocyte contamination of the isolated PBMCs. An ex vivo T cell proliferation assay showed an overall reduced background proliferation in unstimulated and stimulated samples after combined dextran and ficoll separation of PBMCs, resulting in a statistically significant difference upon in vitro NDV challenge.

Process automation in manufacturing of mesenchymal stromal cells.

Process automation in manufacturing of mesenchymal stromal cells.

Evaluation of Isolation Methods for Circulating Tumor Cells (CTCs)

Background: Detection of CTCs is a poor prognostic factor for many cancer types; however, their very low frequency represents an obstacle for their detection. The objective of the current study was to compare the performance of commonly used methods for CTCs isolation. Methods: The evaluated methods using spiking experiments of MCF7, SKBR3 and MDA MB-231 breast cancer cell lines were (i) ficoll density gradient separation (DGS), (ii) red blood cell lysis (Erythrolysis) isolation, (iii) positive immunomagnetic selection (EpCAM Dynal beads), (iv) two different negative immunomagnetic separation systems (Dynal vs Miltenyi CD45 beads) as well as (v) the Cell Search platform and (vi) the ISET system. Results: The recovery rates of Erythrolysis and DGS were 39% and 24%, respectively. Magnetic isolations are ranked from the worse to the best recovery rate as follows:, Myltenyi-anti-CD45 microbeads (24%); Dynal-anti-EpCAM beads (75%); Dynabeads-anti-CD45 (97%). CTCs isolation from blood samples using the CellSearch and ISET systems revealed that the recovery rate for Cell Search and ISET was 52% and 95%, respectively. Conclusions: Dynal-anti-CD45 beads have the best recovery rate compared to other magnetic methods. Furthermore the recovery rate of ISET was higher compared to Cell Search, especially for the more aggressive MDA-MB 231 cell line.

Red Blood Cell Depletion for Pediatric Major ABO Mismatch BMT: Evaluation of the Risk of Hemolysis and Comparison of Two Techniques

Introduction: Major ABO mismatch occurs when the recipient has preformed isoagglutinins against the donor. Since unmanipulated bone marrow transplant (BMT) grafts contain donor red blood cells (RBCs), major ABO mismatch BMT infusions can cause hemolytic reactions that lead to renal dysfunction. To prevent this complication, RBC depletion of the graft can be performed by various techniques. Strong evidence-based recommendations regarding RBC depletion are lacking. In particular, it is unclear what residual volume of incompatible RBCs in the graft is safe, especially for pediatric patients.Methods: We conducted a retrospective cohort study of pediatric patients who had major ABO mismatch BMT at a single center. No patients had pre-BMT plasmapheresis to reduce isoagglutinin titers. RBC depletion was performed on the BMT grafts with either hydroxyethyl starch (HES) sedimentation or Ficoll density gradient separation. All patients received similar supportive care around the BM infusion that included hyperhydration and premedication with acetaminophen, diphenhydramine, and hydrocortisone. Serum creatinine the day after the BM infusion was compared to creatinine the day of the BM infusion. Since 2006 all patients had urine screened for blood post-BMT.Results: Sixty-three patients were identified who received a major ABO mismatch BMT between 9/2004 and 6/2015 (39 had RBC depletion with HES, 24 with Ficoll). Compared to Ficoll RBC depletion, patients who received BM grafts that had RBC depletion with HES received significantly more incompatible RBCs (Table 1). Hemoglobinuria was significantly more common in HES patients, but evidence of post-BM infusion renal impairment was not (Table 1). All patients had donor engraftment with a similar time to neutrophil engraftment for both groups (Table 1). Considering only the HES patients, 8/8 (100%) patients with >25% rise in creatinine had hemoglobinuria compared to 8/21(38%) patients with ≤25% rise in creatinine (p=0.003). Also among just the HES patients, the median amount of incompatible RBCs infused was not significantly different between patients with (0.74 ml/kg) and without (0.62 ml/kg) hemoglobinuria (p=0.42), or between patients with (0.56 ml/kg) and without (0.62 ml/kg) a >25% rise in creatinine (p=0.86).Table 1: Comparison of patients who had RBC depletion with HES vs. Ficoll Table 1HES n=39Ficoll n=24p-valueMedian volume of RBCs/patient weight0.62 ml/kg0.04 ml/kg<0.0001Number with urine positive for blood*16/29 (55%)1/20 (5%)0.0003Number with >25% rise in creatinine8 (21%)5 (21%)1.0Number with >50% rise in creatinine4 (10%)0 (0%)0.29Median day of neutrophil engraftment20210.11*considering only patients transplanted after 2006.Conclusion: Our study is the first to analyze pediatric major ABO mismatch BMT infusion hemolysis after two different RBC depletion techniques: HES and Ficoll. Our results suggest that RBC depletion with Ficoll achieves less residual RBCs in the BM graft and likely less resulting hemoglobinuria. However, after both techniques hemolysis-induced renal impairment is rare. The volume of residual incompatible RBCs in the infused BM graft does not appear to strongly determine if clinical hemolysis occurs. The volume that is safe likely depends on other variables that influence the risk of clinical hemolysis. DisclosuresNo relevant conflicts of interest to declare.

P024 : POTENTIAL FOR A FALSE POSITIVE B CELL FLOW CYTOMETRY CROSSMATCH WITH THE USE OF FLUORESCEIN ISOTHYOCYANATE-GOAT-ANTI-HUMAN IMMUNOGLOBULIN-G ANTIBODY

Background In flow cytometry crossmatches (FCXM), sera from AB blood group male donors with none or minimal anti-HLA IgG antibodies are used as the negative control serum (NCS). As NCS can give higher median channel fluorescence (MCF) on certain donor cells, it is advised to use cells treated with phosphate buffer saline (PBS) as an additional negative control for comparison. Aim We examined whether using PBS or NCS as the negative control and subsequent staining with FITC-goat-anti-human IgG polyclonal antibody (g α IgG pAb; Jackson Immunoresearch Laboratory; Cat# 109-096-098) or FITC-mouse-anti-human IgG monoclonal antibody (m α IgG mAb; BD biosciences; Cat# 555786; Clone: G18-145) impacts FCXM results. Methodology Peripheral blood ( N = 5) and serum ( N = 5) were collected from human subjects. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient separation. In 96 well V-bottom plate, 300,000 PBMC were incubated with 30 μl of serum for 20 min at 22 °C, washed and stained with 10 μl each of anti-CD3 PerCP and of anti-CD19 PE, and with either 10 μl (1:100 diluted) of FITC-gαIgG pAb or 60 μl of FITC-mαIgG mAb for 30 min at 4 °C. The cells were washed and flow cytometry was performed using BD FACS Canto II instrument and the data was analyzed using the DiVa software. A channel shift of >40 from the NCS for T cell and a shift of >50 for B cells is considered positive here. Results PBMCs treated with PBS gave positive B cell channel shift when stained with g α IgG pAb, but not when stained with m α IgG mAb ( Fig. 1 ). Unlike m α IgG mAb, the g α IgG pAb seems to bind non-specifically to B cells in the absence of human serum. Conclusion This observation cautions that in a clinical FCXM testing, the non use of NCS can lead to a false positive FCXM result. It is also anticipated that a technical error of not adding patient’s serum to the donor cells can lead to positive B cell crossmatch reporting. Using m α IgG mAb or any other α IgG antibody that does not give non-specific binding to donor cells is recommended for FCXM testing.

P023 : TAGGING CELLS IN FLOW CYTOMETRY CROSSMATCH: PLATE METHOD PROVIDES HIGHER B CELL PERCENTAGE AS COMPARED TO TUBE METHOD

In flow cytometry crossmatches (FCXM), B cell percentages of certain renal transplant patients (although not on Rituximab treatment) do not make it to laboratory set cut-off of 500 B cell events or 1000 B cell events. This is uncommon in case of donor cells. Loss of cells during washing steps is expected when cells are tagged in tubes. To determine if plate method of tagging peripheral blood mononuclear cells (PBMC) will provide higher B cell percentages as compared to tube method. Peripheral blood was collected from 23 human subjects. PBMC were isolated by Ficoll density gradient separation. In 96 well V-bottom polystyrene tissue culture (TC) treated plate (Cat# 3894, Corning Inc., Corning, NY, USA), untreated plate (Cat# 651161, Greiner Bio-One, Frickenhausen, Germany), and in 5 ml polystyrene round-bottom tubes (Cat# 352054, BD, 1 Becton Drive, Franklin Lakes, NJ, USA), 300,000 PBMC were incubated with 30 μl of negative control serum (NCS) for 20 min at room temperature, washed twice, and stained with 10 μl each of anti-CD3 PerCP, anti-CD19 PE, and FITC-goat-anti-human IgG polyclonal antibody (1:100 diluted; g α IgG pAb; Jackson Immunoresearch Laboratory; Cat# 109-096-098) for 30 min at 4 °C. The cells were washed and flow cytometry was performed using BD FACSCanto II instrument and the data was analyzed using the DiVa software. CD19+ B cell percentages were significantly higher when the cells were tagged in TC treated plates as compared to those in tubes (9 ± 4% vs. 4 ± 2%, P = 0.0001). Expectedly, the higher B cell percentage found with cells tagged in TC treated plate were associated with a lower T cell percentages: 72 ± 13% when tagged in TC treated plates and 77 ± 12% when tagged in tubes ( P = 0.002). In addition, no difference in B cell percentages (10 ± 4 vs. 10 ± 4) or in T cell percentages (80 ± 4 vs. 80 ± 3) was identified in FCXM performed in TC treated or in untreated plates. When FCXMs were performed in tubes and in plates in parallel, channel shifts for anti-T or B cell specific IgG antibodies in human serum were identical. This observation cautions that cells tagged or stained in plates have different percentage of cells as compared to that in tubes. In FCXM, plate method of tagging helps acquiring more B cell events as compared to tube method of tagging cells.

Characteristic DNA methylation profiles in peripheral blood monocytes are associated with inflammatory phenotypes of asthma

Epigenetic changes including DNA methylation caused by environmental exposures may contribute to the heterogeneous inflammatory response in asthma. Here we investigate alterations in DNA methylation of purified blood monocytes that are associated with inflammatory phenotypes of asthma. Peripheral blood was collected from adults with eosinophilic asthma (EA; n = 21), paucigranulocytic asthma (PGA; n = 22), neutrophilic asthma (NA; n = 9), and healthy controls (n = 10). Blood monocytes were isolated using ficoll density gradient and immuno-magnetic cell separation. Bisulfite converted genomic DNA was hybridized to Illumina Infinium Methylation27 arrays and analyzed for differential methylation using R/Bioconductor packages; networks of gene interactions were identified using the STRING database. Compared with healthy controls, differentially methylated CpG loci were identified in EA (n = 413), PGA (n = 495), and NA (n = 89). We found that 223, 237, and 72 loci were significantly hypermethylated in EA, PGA, and NA, respectively. Nine genes were common to all three phenotypes and showed increased methylation in asthma. Three pathway networks were identified in EA, involved in purine metabolism, calcium signaling, and ECM-receptor interaction. In PGA, two networks were identified, involved in neuroactive ligand-receptor interaction and ubiquitin mediated proteolysis. In NA, one network was identified involving sFRP1 as a key node, over representing the Wnt signaling pathway. We have identified characteristic alterations in DNA methylation that are associated with inflammatory phenotypes of asthma and may contribute to the disease mechanisms. This network-based characterization may help in the development of epigenetic biomarkers and therapeutic targets for asthma.