Isolation Of Peripheral Blood Mononuclear Cells Research Articles

Human neutrophils can mimic myeloid-derived suppressor cells (PMN-MDSC) and suppress microbead or lectin-induced T cell proliferation through artefactual mechanisms

We report that human conventional CD15+ neutrophils can be isolated in the peripheral blood mononuclear cell (PBMC) layer during Ficoll gradient separation, and that they can impair T cell proliferation in vitro without concomitant neutrophil activation and killing. This effect was observed in a total of 92 patients with organ transplants, lung cancer or anxiety/depression, and in 18 healthy donors. Although such features are typically associated in the literature with the presence of certain myeloid-derived suppressor cell (PMN-MDSC) populations, we found that commercial centrifuge tubes that contained membranes or gels for PBMC isolation led to up to 70% PBMC contamination by CD15+ neutrophils, with subsequent suppressive effects in certain cellular assays. In particular, the suppressive activity of human MDSC should not be evaluated using lectin or microbead stimulation, whereas assays involving soluble or plate-bound antibodies or MLR are unaffected. We conclude that CD15+ neutrophil contamination, and associated effects on suppressor assays, can lead to significant artefacts in studies of human PMN-MDSC.

Immune response to reovirus (REO) in phase I study with chemotherapy in patients with KRAS mutant metastatic colorectal cancer (mCRC).

217 Background: Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is underway in a phase 1 study of mCRC. This study evaluates the nature of immune response by determining the distribution of antigen presenting cells (APCs) and activated T lymphocytes along with the cytokine expression pattern in peripheral circulation. Methods: REO was administered as a 60-minute intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3x106. Serum was collected pre- and post- REO on days 1, pre REO on days 2-5, and days 8, 15, 22, and 29. Peripheral blood mononuclear cells (PBMC) were isolated and stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123. Stained cells were fixed and evaluated by flow cytometry. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Results: Patients mount a robust immune response with dendritic cell maturation at 48 hrs (p < 0.01) followed by activation of cytotoxic T (CD8+) cells at Day 8 (p < 0.01). Cytokine assay indicated upregulation of Interleukin 1 beta (IL-1β; p = 0.004), Granulocyte-macrophage colony-stimulating factor (GM-CSF; p = 0.05), the chemokine Macrophage Inflammatory Proteins (MIP-1β; p = 0.05) at day 15. Furthermore, consistent upregulation of inflammatory cytokine IL-6 was seen from days 3 through 8 (p < 0.05), and decrease in IL-8 at 72 hrs (p = 0.03) was observed. Conclusions: REO induces strong immune response in patients with mCRC. APCs are stimulated within 48 hrs and activated (CD8+ CD70+) T cells within 168 hrs. Cytokine profiling indicates stimulation for maturation of APCs, chemotactic induction for macrophages and activation of T cells as highlighted by release of IL-1β, GM-CSF and MIP-1β respectively. Sustained increased expression of IL-6 (triggering lymphocyte maturation) and downregulation of IL-8 (pro-angiogenic cytokine) is also observed. REO thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells. Clinical trial information: NCT01274624.

A Modified Ficoll-Paque Gradient Method for Isolating Mononuclear Cells from the Peripheral and Umbilical Cord Blood of Humans for Biobanks and Clinical Laboratories.

Although the Ficoll-Paque method is classically used to isolate peripheral blood mononuclear cells (PBMCs), modifications in this method are required for a more rapid and economic output for biobanks and clinical laboratories, particularly in developing countries. In this study, we addressed this issue by modifying the Ficoll-Paque method for the isolation of PBMCs or mononuclear cells from the peripheral and the umbilical cord blood of healthy and diseased (infected, anemic, and chronic obstructive pulmonary disease) adult individuals. In the modified method, we initiated the cell isolation process from the buffy coat layer, which appears in the interface between the plasma and sediments after centrifugation, instead of using the whole blood as described in the classic method. Although the PBMC yield by the modified method was about 12% less than in the classic method, the number of PBMCs isolated by the modified method was more than one million, which is enough for different research/diagnostic purposes, such as multi-omics detection. Assessment of cell viability and purity by hematology analyzer and trypan blue showed no significant difference between the viability and purity of the PBMCs isolated by these two methods in almost all groups, except samples from the infected and cord blood groups, where lower PBMC purity with higher granulocyte contamination were observed. In addition, at delayed processing time points, all parameters for the two methods were decreased in a time-dependent manner, especially at 8, 12, or 24 hours after the sample collection. In summary, the performance of PBMC isolation by the classic and modified methods mainly relies on the PBMC ratio in original samples. The modified method could be preferred for PBMC isolation because of its time and cost savings, especially for the biobanks and clinical laboratories in developing countries.

Short communication: Decreasing the dietary ratio of n-6 to n-3 fatty acids increases the n-3 concentration of peripheral blood mononuclear cells in weaned Holstein heifer calves

Utilization of nutrients to improve overall heifer health is of interest because of the importance of replacement heifers to the dairy industry. The objective of our study was to compare the effect of supplementation of dietary n-3 and n-6 fatty acids (FA) on FA concentrations in peripheral blood mononuclear cells (PBMC) of Holstein calves. Twenty-seven Holstein heifer calves (107 ± 2.6 d of age; 142.6 ± 6.5 kg of body weight) from the university research and teaching herd were randomly assigned to a common TMR supplemented with 1 of 3 treatments: Ca salts of flaxseed FA (Virtus Nutrition, Corcoran, CA) containing 35% 18:3 n-3 (N3), Ca salts of soybean FA (Virtus Nutrition) containing 50% 18:2 n-6 (N6), or a 50:50 mix of N3 and N6. Treatments were supplemented with FA at 4% of dietary dry matter and fed for 30 d. Feed intake was recorded daily, and body weight, wither height, and body condition score were measured weekly throughout the study. On d 28 heifers were vaccinated with a Pasteurella vaccine and the temperature response to the vaccine was recorded. Blood was collected on d 0 and 28 for PBMC isolation. After total lipid extraction and FA methyl ester preparation, FA composition of PBMC was measured. We observed no effect of treatment on body weight gain, body condition score change, or wither height change. Heifers receiving the N3 diet had a lower temperature response to Pasteurella challenge compared with both the mix and N6 diets. Heifers consuming the N3 diet had a greater content of total n-3 FA, α-linolenic acid, and eicosapentaenoic acid in PBMC compared with heifers fed the N6 and mix diets. Heifers receiving the N3 diet also had a lower content of total n-6 FA, linoleic acid, and arachidonic acid in PBMC than heifers fed the N6 and mix diets. In conclusion, our study determined that feeding weaned female Holstein heifers a diet high in n-3 FA increased concentrations of n-3 FA in PBMC.

The effect of 1.5 T cardiac magnetic resonance on human circulating leucocytes.

Investigators have proposed that cardiovascular magnetic resonance (CMR) should have restrictions similar to those of ionizing imaging techniques. We aimed to investigate the acute effect of 1.5 T CMR on leucocyte DNA integrity, cell counts, and function in vitro, and in a large cohort of patients in vivo. In vitro study: peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers, and histone H2AX phosphorylation (γ-H2AX) expression, leucocyte counts, and functional parameters were quantified using flow cytometry under the following conditions: (i) immediately following PBMC isolation, (ii) after standing on the benchside as a temperature and time control, (iii) after a standard CMR scan. In vivo study: blood samples were taken from 64 consecutive consenting patients immediately before and after a standard clinical scan. Samples were analysed for γ-H2AX expression and leucocyte counts. CMR was not associated with a significant change in γ-H2AX expression in vitro or in vivo, although there were significant inter-patient variations. In vitro cell integrity and function did not change with CMR. There was a significant reduction in circulating T cells in vivo following CMR. 1.5 T CMR was not associated with DNA damage in vitro or in vivo. Histone H2AX phosphorylation expression varied markedly between individuals; therefore, small studies using γ-H2AX as a marker of DNA damage should be interpreted with caution. Cardiovascular magnetic resonance was not associated with loss of leucocyte viability or function in vitro. Cardiovascular magnetic resonance was associated with a statistically significant reduction in viable leucocytes in vivo.

PROFILING AND CHARACTERIZING MICRORNAS IN PATIENTS WITH HEREDITARY HEMORRHAGIC TELANGIECTASIA

Hereditary hemorrhagic telangiectasia (HHT) is a rare, autosomal dominant, vascular disorder that affects 1 in 5000 to 8000 individuals worldwide. HHT is characterized by vascular abnormalities, such as telangiectasias and arteriovenous malformations (AVMs). Telangiectasias are dilated skin and mucocutaneous blood vessels that are susceptible to spontaneous hemorrhage. AVMs are direct connections between arteries and veins, without intervening capillaries, and can cause significant right to left shunts. HHT arises from heterozygous mutations in at least 3 genes, ENG, ACVRL1, and SMAD4. Haploinsufficiency of their respective protein products perturb TGF signalling, affecting the angiogenic potential of endothelial cells (ECs) and the migratory capacity of monocytes, contributing to vascular malformations in HHT. MicroRNAs (miRs) are small non-coding RNAs that post-transcriptionally target mRNA transcripts. We aimed to profile and characterize miRs in HHT patient derived early endothelial progenitor cells (eEPCs) and peripheral blood mononuclear cells (PBMCs) to explore their potential role in HHT pathogenesis. EEPCs are early outgrowth ECs cultured from the mononuclear cell layer (buffy coat) of peripheral blood. They have been shown to augment angiogenesis in a paracrine manner by secreting various growth factors and are a widely accepted surrogate for studying EC function 28 HHT patients with confirmed mutations, and 25 controls were recruited. 40 ml of peripheral blood was collected for eEPC culture and PBMC isolation. Total RNA was isolated from PBMCs and eEPCs. PBMC and eEPC expression profiling was conducted with a human miR array analysis. Dysregulated miRs identified from the analyses were then validated with RT-qPCR. Significant differences were determined using a two-tailed t-test. Of the 800 miRs screened, 167 and 121 dysregulated miRs were identified in PBMCs and eEPCs, respectively. Selected PBMC miRs (MiRs-28-5p, -30b-5p, -361-3p and -374a-5p) and eEPC miRs (miRs-132-3p, -221-3p and -424-5p) were validated with RT-qPCR. PBMC miR-361-3p (p=0.025) known to target IGF1, and eEPC miR-132-3p (p=0.047) known to target SMAD2, were found to be significantly decreased compared to controls. Subsequently, IGF1 messenger RNA (mRNA) levels were significantly increased (p=0.005) in PBMCs of HHT patients compared to controls. Our results show dysregulation of select miRs in PBMCs and eEPCs from HHT patients. The finding of increased IGF1 mRNA and its possible relation to decreased miR-361-3p levels is both novel and exciting. This may represent a putative pathogenic mechanism involved in HHT, and may provide a unique miR therapeutic target for the treatment of clinical manifestations. National Institutes of Health (NIH)

Validation of Interferon Induced IFIT Genes in Host Antiviral Defence Against BTV16 Infection : An Initial Report

Whole blood samples were collected from healthy sheep and goat, Peripheral blood mononuclear cells (PBMC) isolation was done from whole blood of both the species and cultured in six well plates using RPMI-1640 media in the presence of 5% CO2 at 37 0C. Bluetongue (virus BTV)-16 infection was given in PBMCs of both the species at 0 h and representative samples were collected at 24, 48 and 72 h. Total RNA was extracted and cDNA was obtained by reverse transcription. All the four differentially expressed genes (IFITs genes) were validated using quantitative real time PCR and the target gene expression was studied in fold change at 24, 48 and 72 h post infection of sheep and goat PBMCs against BTV-16 infection along with mock infected cells as control. Up regulation in target gene expression was found in virus infected PBMCs of both the species at all the time intervals than control PBMCs indicates the antiviral host protective response of those DEGs against BTV-16 infection. NS1 gene expression of BTV16 in sheep and goat PBMCs at different time interval were also determined which showed significant variation in both species.

009 The effect of cardiac magnetic resonance on human circulating leukocytes

Aims Investigators have proposed that cardiovascular magnetic resonance (CMR) should have restrictions similar to those of ionising imaging techniques due to proposed alterations to leukocytes. We aimed to investigate the acute effect of CMR on leukocyte DNA integrity and cell viability in vitro, and in a large cohort of patients in vivo. Methods and results In vitro study: Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers and assessed: 1) immediately following PBMC isolation, 2) after standing on the benchside as a temperature and time control, 3) after a standard CMR scan. Histone H2AX phosphorylation (γ-H2AX), an indicator of DNA damage, and leukocyte counts were quantified using flow cytometry. In vivo study: Blood samples were taken from 64 consecutive consenting patients immediately before and after a standard clinical scan. Samples were analysed for T cell count and γ-H2AX expression. CMR scanning was associated with a significant increase in leukocyte γ-H2AX expression, indicating DNA damage occurs. We also observed a trend towards a significant decrease in absolute leukocyte numbers in vitro following CMR. CMR was not associated with a significant change in γ-H2AX expression in vivo, although there were significant inter-patient variations. There was a significant reduction in circulating T cells following CMR. Conclusion CMR was not associated with DNA damage in vivo. γ-H2AX expression varied markedly between individuals, therefore small studies using γ-H2AX as a marker of DNA damage should be interpreted with caution. CMR was associated with a statistically significant reduction in viable leukocytes, although the clinical relevance of the magnitude is unclear. Further work is warranted to contextualise these findings and delineate their impact.

Impairment of Wnt/β-catenin signaling in blood cells of patients with severe cavitary pulmonary tuberculosis.

Tuberculosis (TB) remains as a leading infectious disease worldwide. Our previous study showed interferon (IFN)-γ and CD3 T cell impairment in patients with severe cavitary pulmonary TB (PTB). However, the cause of the change in immune responses during the progression of TB is still poorly understood. In this study, eight newly diagnosed patients with severe cavitary and mild lesion non-cavity PTB were recruited, and three healthy volunteers were recruited as the control. RNA extracted from blood was tested by whole genome oligo microarrays. A PCR array was used to further test the same samples. Two additional groups of patients were recruited according to the same criteria with healthy control(HC) recruited as well and subjected to peripheral blood mononuclear cell isolation (PBMC)and analysis of TCF-7, β-catenin, cyclin D2, IFN-γ, and tumor necrosis factor (TNF)-α expression in CD14- cells (lymphocytes) and CD14+ cells by quantitative PCR. The changes of expression of β-catenin, CD69+ and IFN-γ by CD3+, CD14- and CD14+ cells in vitro with stimulation of LiCl were tested by flow cytometry. Whole genome oligo microarrays showed a significant decrease in expression of the Wnt signaling pathway in severe PTB patients. Further analysis of the Wnt pathway by PCR array indicated that TCF-7, β-catenin, and cyclin D2 expression was significantly reduced in severe PTB patients compared with mild PTB patients. In the additionally recruited patients, TCF-7, β-catenin, and cyclin D2 were expressed in both CD14+ and CD14- cells, while β-catenin was decreased significantly in CD14- cells compared with CD14+ cells in severe PTB patients, and IFN-γ and TNF-α expression in CD14- cells was also reduced significantly in severe PTB patients. β-catenin can directly trigger T cell activation and IFN-γsecretion in PBMCs stimulated for 24 hours. These findings indicate that Wnt pathway and its key genes, such as β-catenin, were impaired in blood cells of patients with severe PTB. Therefore, Wnt/β-catenin pathway is closely associated with T cell proliferation and TB lesion deterioration.

Efficient and reliable establishment of lymphoblastoid cell lines by Epstein-Barr virus transformation from a limited amount of peripheral blood

Lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV) serve as an unlimited resource of human genomic DNA. The protocol that is widely used to establish LCLs involves peripheral blood mononuclear cell isolation by density gradient centrifugation, however, that method requires as much as 5 ml of peripheral blood. In this study, in order to provide a more simple and efficient method for the generation of LCLs, we developed a new protocol using hemolytic reaction to enrich white blood cells for EBV transformation and found that the hemolytic protocol successfully generated LCLs from a small volume (i.e., 0.1 ml) of peripheral blood. To assess the quality of genomic DNA extracted from LCLs established by the hemolytic protocol (LCL-hemolytic), we performed single nucleotide polymorphism (SNP) microarray genotyping using the GeneChip® 100 K Array Set (Affymetrix, Inc.). The concordances of the SNP genotyping resulting from genomic DNA from LCL-hemolytic (99.92%) were found to be as good as the technical replicate (99.90%), and Kappa statistics results confirmed the reliability. The findings of this study reveal that the hemolytic protocol is a simple and reliable method for the generation of LCLs, even from a small volume of peripheral blood.

Peripheral blood mononuclear cells from neovascular age-related macular degeneration patients produce higher levels of chemokines CCL2 (MCP-1) and CXCL8 (IL-8)

BackgroundInfiltrating immune cells including monocytes/macrophages have been implicated in the pathogenesis of neovascular age-related macular degeneration (nAMD). The aim of this study was to investigate the cytokine and chemokine expression and secretion profile of peripheral blood mononuclear cells (PBMCs) from nAMD patients and the relationship between the cytokine/chemokine expression profile and clinical phenotype of nAMD, including macular fibrosis, macular atrophy or the responsiveness to anti-VEGF therapy.MethodsOne hundred sixty-one nAMD patients and 43 controls were enrolled in this study. nAMD patients were divided into subgroups based on the presence/absence of (1) macular atrophy, (2) macular fibrosis and (3) responsiveness to anti-VEGF therapy; 25–30 ml of peripheral blood were obtained from all participants and 5 ml were used for serum collection, and the remaining were used for PBMC isolation using density gradient centrifugation. Intracellular cytokine expressions by PBMCs following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation were examined using flow cytometry. Cytokine productions in lipopolysaccharides (LPS)-or 1% oxygen -treated PBMC were measured using cytometric bead array (CBA) assay. In addition, cytokine and chemokine levels in the serum were also measured by CBA assay.ResultsPBMCs from nAMD patients secreted higher levels of IL-8, CCL2 and VEGF, especially following LPS and 1% oxygen stimulation, than those from controls. 60~80% of IL-8 producing cells were CD11b+CD3− monocytes. The percentage of CD11b+CD3− IL-8+ was significantly increased in nAMD patients compared to controls. PBMCs from nAMD patients without macular fibrosis produced the highest levels of IL-8 and CCL2, whilst PBMCs from nAMD patients with macular atrophy produced highest levels of VEGF. In addition, PBMCs from patients who partially responded to anti-VEGF produced higher levels of IL-8 compared to the cells from complete responders. Interestingly, serum level of CCL2 was not increased in nAMD patients although there was a trend of increased IL-8 in nAMD patients.ConclusionsPBMCs, in particular monocytes, may contribute to CNV development in nAMD through secreting elevated levels of IL-8, CCL2 and VEGF after they are recruited to the macula. Apart from VEGF, IL-8 and CCL2 may be additional targets for nAMD management.

Isolation of Peripheral Blood Mononuclear Cells Using Vacutainer® Cellular Preparation Tubes (CPTTM).

Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that separates the Ficoll solution from the blood to be drawn. Once blood is drawn into CPTs, they can be centrifuged to separate the PBMC, then shipped (if desired) to a processing lab. The processing lab removes the PBMC from the upper compartment of the tube (above the gel plug), washes the PBMC, and can cryopreserve them using DMSO-containing media, as detailed in this protocol.

Generation of Epstein-Barr Virus Immortalized-B Lymphocytes Cell Line of Orang Asli

Obtaining sustainable source of cells or DNA from a single individual has always been a constraint in biomedical research. Generation of lymphoblastoid cell lines (LCLs) as a surrogate for replacement of isolated peripheral blood mononuclear cells (PBMCs) has substantially accelerated the process of biological investigations. LCLs can be established by transfection of B lymphocytes from peripheral blood with Epstein Barr Virus (EBV), bearing minor genetic and phenotypic alterations. Here we report the generation of LCLs for the indigenous population (Orang Asli) from Peninsular Malaysia. The establishment of cell line is crucial for such isolated populations since the population size is small and sample collections are often challenging. We applied an alternative approach in isolation of PBMCs before proceeding to EBV transfection of Orang Asli samples, of which the BD Vacutainer CPT tubes were used to replace the conventional protocol such as Ficoll-paque or histopaque. We called this approach the “modified method of CPT” (MMC). A total of 59 out of the 64 samples (92%) of Orang Asli were successfully transformed into LCLs by transfecting with EBV supernatant in a single attempt. To verify the success of LCLs transformation and confirm the presence of the human DNA, FCGR3B gene copy number variations of the DNA extracted from the LCLs were typed using Paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR) analysis. The genotype results were compared between the DNA obtained from the LCLs and the original PBMCs from the identical individuals, and results revealed 100% concordance rate. The success of the transformation for LCL for the indigenous populations will eventually allow a more comprehensive and advanced biomedical research to be carried out in the next future without the exhaustion of the biomaterials.

Diagnostic sensitivity and related impact factors of T cell spot test of tuberculosis in acquired immunodeficiency syndrome/tuberculosis patients

Objective To analyze the diagnostic sensitivity and related impact factors of T cell spot test of tuberculosis (T-SPOT.TB) test in acquired immunodeficiency syndrome (AIDS)/tuberculosis (TB) patients. Methods Ninety-two confirmed cases with AIDS/TB coinfection were tested by T-SPOT.TB, and the impact of CD4+ T cells counts on the diagnostic sensitivity was analyzed. Multivariate Logistic analysis was used for the analysis of impact factors of T-SPOT.TB sensitivity. Blood samples of 19 cases with advanced stage AIDS/TB from January 2015 to January 2016 were collected, and peripheral blood mononuclear cell (PBMC) were isolated by Ficoll method, and lymphocytes were isolated by Percoll method. McNemar test was used for the comparison of these two methods. Results Among the 92 patients with AIDS/TB, T-SPOT.TB tests were positive in 51 cases, with positive rate of 55.4%. The sensitivity was 26.3% (10/38) when CD4+ T cell count less than 20/μL, and that was 92.9% (13/14) when CD4+ T cell more than 200/μL. In Logistic analysis, the sensitivity of T-SPOT. TB test in patients with extra-pulmonary tuberculosis was better than that in pulmonary tuberculosis patients (OR=3.042, P=0.038). The sensitivity of T-SPOT.TB was positively correlated with CD4+ T cell count, and the sensitivity increased by 2.889 times when CD4+ T cells increasing 100/μL (OR=3.889, P=0.016). The percentage of lymphocytes in PBMC was also positively correlated with T-SPOT.TB positivity, and the sensitivity increased by 1.393 times when the percentage increasing 30% (OR=2.393, P=0.045). When Percoll was used for lymphocytes isolation, the T-SPOT.TB sensitivity was 52.6% (10/19), and when Ficoll was used for PBMC isolation, the sensitivity was 36.8%(7/19). The difference was not statistically significant (P=0.375). Conclusion The sensitivity of T-SPOT.TB test based on lymphocytes is higher than that based on PBMC. Key words: Acquired immunodeficiency syndrome; Tuberculosis; T-SPOT.TB

Virtual Global Transplant Laboratory Standard Operating Procedures for Blood Collection, PBMC Isolation, and Storage

Research on human immune responses frequently involves the use of peripheral blood mononuclear cells (PBMC) immediately, or at significantly delayed timepoints, after collection. This requires PBMC isolation from whole blood and cryopreservation for some applications. It is important to standardize protocols for blood collection, PBMC isolation, cryopreservation, and thawing that maximize survival and functionality of PBMC at the time of analysis. This resource includes detailed protocols describing blood collection tubes, isolation of PBMC using a density gradient, cryopreservation of PBMC, and thawing of cells as well as preparation for functional assays. For each protocol, we include important considerations, such as timing, storage temperatures, and freezing rate. In addition, we provide alternatives so that researchers can make informed decisions in determining the optimal protocol for their application.

Quality Assurance of Samples and Processes in the Spanish Renal Research Network (REDinREN) Biobank.

Biobanks are useful platforms to build bridges between basic, translational, and clinical research and clinical care. They are repositories of high-quality human biological samples ideal for evaluating their histological characteristics and also their genome, transcriptome, and proteome. The Spanish Renal Research Network Biobank contains more than 76,500 well-preserved frozen samples of a wide variety of kidney diseases, collected from 5450 patients seen by over 70 nephrology services throughout the Spanish territory. To determine and to report the results of the quality control of samples and processes conducted in our biobank, implemented in accordance with the requirements of the ISO 9001:2008 international standard. Two types of quality controls were performed: (1) systematic, that is, measurement of viable peripheral blood mononuclear cells (PBMCs) obtained and purity of nucleic acids and (2) ad-hoc, that is, viability of thawed PBMC, DNA extraction process reproducibility, and the integrity and functionality of nucleic acids, implemented on a routine basis. PBMC isolation by Ficoll yielded reproducible results and its cryopreserved viability was >90%. Acceptable A260/A280 ratios were obtained for the vast majority of the DNA (n = 2328) and RNA (n = 78) samples analyzed. DNA integrity was demonstrated by agarose gels and by β-globulin gene polymerase chain reaction (PCR) amplification of 1327 and 989 bp fragments. DNA of acceptable quality had at least three bands of β-globulin amplified obtained (n = 26/30). RNA integrity number (RIN) determinations obtained RIN numbers ≥7 (n = 87/96). The amplifiability of nucleic acids was confirmed by qPCR and RT-qPCR of β-actin and GAPDH genes. Long storage or delayed processing time did not affect the quality of the samples analyzed. The processes of DNA extraction also yielded reproducible results. These results clearly indicate that our PBMC, DNA, and RNA stored samples meet the required quality standards to be used for biomedical research, ensuring their long-term preservation.

Evaluation of techniques for performing cellular isolation and preservation during microgravity conditions.

Genomic and epigenomic studies require the precise transfer of microliter volumes among different types of tubes in order to purify DNA, RNA, or protein from biological samples and subsequently perform analyses of DNA methylation, RNA expression, and chromatin modifications on a genome-wide scale. Epigenomic and transcriptional analyses of human blood cells, for example, require separation of purified cell types to avoid confounding contributions of altered cellular proportions, and long-term preservation of these cells requires their isolation and transfer into appropriate freezing media. There are currently no protocols for these cellular isolation procedures on the International Space Station (ISS). Currently human blood samples are either frozen as mixed cell populations (within the CPT collection tubes) with poor yield of viable cells required for cell-type isolations, or returned under ambient conditions, which requires timing with Soyuz missions. Here we evaluate the feasibility of translating terrestrial cell purification techniques to the ISS. Our evaluations were performed in microgravity conditions during parabolic atmospheric flight. The pipetting of open liquids in microgravity was evaluated using analog-blood fluids and several types of pipette hardware. The best-performing pipettors were used to evaluate the pipetting steps required for peripheral blood mononuclear cell (PBMC) isolation following terrestrial density-gradient centrifugation. Evaluation of actual blood products was performed for both the overlay of diluted blood, and the transfer of isolated PBMCs. We also validated magnetic purification of cells. We found that positive-displacement pipettors avoided air bubbles, and the tips allowed the strong surface tension of water, glycerol, and blood to maintain a patent meniscus and withstand robust pipetting in microgravity. These procedures will greatly increase the breadth of research that can be performed on board the ISS, and allow improvised experimentation by astronauts on extraterrestrial missions.

Culture and Differentiation of Monocyte Derived Macrophages Using Human Serum: An Optimized Method

Background: Monocyte derived macrophages (MDMs) are appropriate in vitro models to study the function of macrophages in immune related diseases. Not only most of the methods in literature for efficient MDM culture and differentiation are expensive but also they require specific equipment. However, there are some reports indicating that monocyte enrichment is possible through attachment. Purification and differentiation of macrophages occurs in media containing human serum or platelet depleted plasma without extra supplementations. Different variables such as incubation time and serum concentration affect this simple MDM preparation method. Objectives: Here we represent an optimized simple method for MDM preparation from peripheral blood mononuclear cells (PBMC). Materials and Methods: To introduce an optimized method we accomplished the present descriptive study. After PBMC isolation and monocyte enrichment in complete RPMI 1640 growth media, macrophages were cultured and differentiated using human serum. Efficient phagocytosis was evaluated using heat-inactivated Escherichia coli followed by SYBR staining. Geimsa staining of macrophages was accomplished to visualize the typical morphology under light microscopy. Results: The derived macrophages have the typical morphology of differentiated macrophages and are able to phagocyte the heat inactivated SYBR stained E. coli. Conclusions: This optimized method is a simple and cost effective method to prepare MDM with typical morphology representations able to phagocytosis efficiently.

The evaluation of gene expression and enzyme activity of SIRT1 in peripheral blood mononuclear cells isolated from patients with relapsing-remitting multiple sclerosis

Background: Little in known regarding the clinical relevance of SIRT1 in multiple sclerosis (MS). Here, we aimed to evaluate mRNA expression, protein level and enzyme activity of SIRT1 in peripheral blood mononuclear cells (PBMCs) isolated from relapsing –remitting MS patients (RRMS) and healthy controls. Materials and Methods: Twenty patients with RR-MS and twenty two age- and sex-matched healthy subjects were enrolled in this case-control study. Following PBMCs isolation, mRNA expression was evaluated by real time-PCR. SIRT1 activity and SIRT1 protein level were measured using a fluorometric assay and an enzyme-linked immunosorbent assay (ELISA) respectively, in PBMC lysates. Results: There was no statistically significant difference in the mRNA expression of SIRT1 (p=0.56) and its protein levels (p=0.15) between MS patients and healthy subjects. By contrast, SIRT1 enzyme activity were significantly (p=0.008) lower in RRMS patients compared with that in healthy subjects. Conclusion: Our findings demonstrated that enzyme activity of SIRT1 is significantly lower in PBMCs of RRMS patients in comparison with healthy subjects. However, more investigations are essential to clarify the role of SIRT1 in MS pathogenesis. Keywords: enzyme activity, multiple sclerosis, pathogenesis

The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells

BackgroundThe role of CD4+ regulatory T cells (Tregs) and their subsets during HIV infection is controversial. Cryopreserved peripheral blood mononuclear cells (PBMC) are an important source for assessing number and function of Tregs. However, it is unknown if PBMC isolation and cryopreservation affect the expression of CD120b and CD39, markers that identify specific subsets of Tregs. MethodsHIV-uninfected (HIV−) and -infected (HIV+) men were randomly selected from the Multicenter AIDS Cohort Study (MACS). Percentages of CD120b+ and CD39+ Tregs measured by flow cytometry in whole blood and in corresponding fresh and cryopreserved PBMC were compared. ResultsPercentages of CD120b+ Tregs were significantly lower in a) fresh PBMC relative to whole blood, and b) freshly thawed frozen PBMC relative to fresh PBMC when the recovery of viable cryopreserved cells was low. When present, low expression of CD120b in frozen PBMC was reversible by 4h of in vitro culture. In contrast, expression of CD39 on Tregs was not affected by isolation and/or cryopreservation of PBMC, or by relative recovery of cryopreserved PBMC. These findings were unaffected by the HIV status of the donor. ConclusionThe data suggest that percentages of CD120b+ Tregs and CD39+ Tregs can be validly measured in either whole blood or PBMC (fresh and frozen) in HIV− and HIV+ men. However, for measurement of CD120b+ Tregs one type of sample should be used consistently within a given study, and thawed frozen cells may require in vitro culture if recovery of viable cells is low.