外周全血中与老化相关的DNA甲基化标记的来源

机体老化与癌症、神经退行性疾病等许多复杂疾病相关。目前,研究者已在外周全血中识别了大量的与老化相关的DNA甲基化标记,这些标记可能反映外周血白细胞在机体老化过程中发生的变化,也可能反映外周血中与年龄相关的细胞构成比例的变化。文章利用3组正常个体外周全血DNA甲基化谱,采用Spearman秩相关分析识别了与老化相关的CpG甲基化位点(age-related DNA methylation CpG sites, arCpGs)并评价了其可重复性;利用去卷积算法估计了各外周血样本中髓性和淋巴性细胞的比例并分析了其与年龄的相关性;比较了在外周全血、CD4+T细胞和CD14+单核细胞中识别的arCpGs的一致性。结果显示,在独立外周全血数据中识别的arCpGs具有显著的可重复性(超几何检验,P=1.65×10^-11)。外周血髓性和淋巴性细胞的比例分别与年龄显著正、负相关(Spearman秩相关检验,P<0.05,r≤0.22),它们间DNA甲基化水平差异较大的CpG位点倾向于在外周全血中被识别为arCpGs。在CD4+T细胞中识别的arCpGs与在外周全血中识别的arCpGs显著交叠(超几何检验,P=6.14×10^-12),且99.1%的交叠位点在CD4+T细胞及外周全血中的DNA甲基化水平与年龄的正、负相关性一致。尽管在CD14+单核细胞中识别的arCpGs与在外周全血中识别的arCpGs并不显著交叠,但是在交叠的51个arCpGs中,有90.1%的位点在CD14+单核细胞、外周全血以及CD4+T细胞中的DNA甲基化水平与年龄的正、负相关性一致,提示它们可能主要反映细胞间共同的改变。在外周全血中识别的arCpGs主要反映某些白细胞共同或特异的DNA甲基化改变,但是也有一部分反映外周血细胞比例构成的变化。     

Deconvolution of Blood Microarray Data Identifies Cellular Activation Patterns in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease with a complex spectrum of cellular and molecular characteristics including several dramatic changes in the populations of peripheral leukocytes. These changes include general leukopenia, activation of B and T cells, and maturation of granulocytes. The manifestation of SLE in peripheral blood is central to the disease but is incompletely understood. A technique for rigorously characterizing changes in mixed populations of cells, microarray expression deconvolution, has been applied to several areas of biology but not to SLE or to blood. Here we demonstrate that microarray expression deconvolution accurately quantifies the constituents of real blood samples and mixtures of immune-derived cell lines. We characterize a broad spectrum of peripheral leukocyte cell types and states in SLE to uncover novel patterns including: specific activation of NK and T helper lymphocytes, relationships of these patterns to each other, and correlations to clinical variables and measures. The expansion and activation of monocytes, NK cells, and T helper cells in SLE at least partly underlie this disease''s prominent interferon signature. These and other patterns of leukocyte dynamics uncovered here correlate with disease severity and treatment, suggest potential new treatments, and extend our understanding of lupus pathology as a complex autoimmune disease involving many arms of the immune system.     

Quantitative reconstruction of leukocyte subsets using DNA methylation

Background

Cell lineage-specific DNA methylation patterns distinguish normal human leukocyte subsets and can be used to detect and quantify these subsets in peripheral blood. We have developed an approach that uses DNA methylation to simultaneously quantify multiple leukocyte subsets, enabling investigation of immune modulations in virtually any blood sample including archived samples previously precluded from such analysis. Here we assess the performance characteristics and validity of this approach.

Results

Using Illumina Infinium HumanMethylation27 and VeraCode GoldenGate Methylation Assay microarrays, we measure DNA methylation in leukocyte subsets purified from human whole blood and identify cell lineage-specific DNA methylation signatures that distinguish human T cells, B cells, NK cells, monocytes, eosinophils, basophils and neutrophils. We employ a bioinformatics-based approach to quantify these cell types in complex mixtures, including whole blood, using DNA methylation at as few as 20 CpG loci. A reconstruction experiment confirms that the approach could accurately measure the composition of mixtures of human blood leukocyte subsets. Applying the DNA methylation-based approach to quantify the cellular components of human whole blood, we verify its accuracy by direct comparison to gold standard immune quantification methods that utilize physical, optical and proteomic characteristics of the cells. We also demonstrate that the approach is not affected by storage of blood samples, even under conditions prohibiting the use of gold standard methods.

Conclusions

Cell mixture distributions within peripheral blood can be assessed accurately and reliably using DNA methylation. Thus, precise immune cell differential estimates can be reconstructed using only DNA rather than whole cells.     

Neuropeptide Y is expressed by rat mononuclear blood leukocytes and strongly down-regulated during inflammation

Neuropeptide Y (NPY), a classical sympathetic comediator, regulates immunological functions including T cell activation and migration of blood leukocytes. A NPY-mediated neuroimmune cross-talk is well conceivable in sympathetically innervated tissues. In denervated, e.g., transplanted organs, however, leukocyte function is not fundamentally disturbed. Thus, we hypothesized that NPY is expressed by blood leukocytes themselves and regulated during inflammation. NPY mRNA and peptide expression were analyzed in mononuclear leukocytes isolated from the blood vessels of healthy rat kidneys, as well as from the blood vessels of isogeneic and allogeneic renal grafts transplanted in the Dark Agouti to Lewis or in the Fischer 344 to Lewis rat strain combination. Depending on the donor strain, acute allograft rejection is either fatal or reversible but both experimental models are characterized by massive accumulation of intravascular leukocytes. Leukocytes, predominantly monocytes, isolated from the blood vessels of untreated kidneys and isografts expressed high amounts of NPY mRNA and peptide, similar to expression levels in sympathetic ganglia. During acute allograft rejection, leukocytic NPY expression drastically dropped to approximately 1% of control levels in both rat strain combinations. In conclusion, NPY is an abundantly produced and tightly regulated cytokine of mononuclear blood leukocytes.     

Cell type–specific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.     

Role of ICAM-1 in chronic hepatic allograft rejection in the rat

The pathogenesis of hepatic allograft rejection remains unclear. We aimed to clarify the early role of intercellular adhesion molecule-1 (ICAM-1)-mediated cell recruitment in chronic hepatic rejection. Liver transplantation was performed from Lewis to Lewis rats (isograft controls) and from Lewis to Brown Norway rats (allograft rejection group). The allografted rats were treated with either ICAM-1 antisense oligonucleotides (10 mg. kg(-1). day(-1) x 6 days ip) or a control preparation (either ICAM-1 missense oligonucleotide or normal saline). Hepatic leukocyte recruitment in vivo was studied on day 6 by using intravital microscopy. Liver histology, biochemistry, and survival rates were also examined. Leukocyte adhesion in terminal hepatic venules was significantly increased in the rejection group compared with isograft controls. Antisense ICAM-1 in the allografted group effectively reduced leukocyte adhesion. Histology and liver chemistry were less deranged in the antisense-treated groups compared with control-treated allografted rats. In the allograft groups, survival was significantly prolonged in the antisense-treated rats (42.3 +/- 1.2 days) compared with the controls (25.2 +/- 2.7 days). These results showed that early leukocyte recruitment in the hepatic microvasculature of rejecting allografts is ICAM-1 dependent and suggest that impacting on early cell recruitment can significantly ameliorate chronic rejection.     

Clonotype analysis of human alloreactive T cells: a novel approach to studying peripheral tolerance in a transplant recipient

The recognition of allo-MHC and associated peptides on the surface of graft-derived APC by host T cells (direct pathway allorecognition) plays an important role in acute rejection after organ transplantation. However, the status of the direct pathway T cells in stable long term transplants remains unclear. To detect alloreactive T cell clones in PBL and the allograft during the transplant tolerance, we utilized RT-PCR instead of functional assays, which tend to underestimate their in vivo frequencies. We established alloreactive CD4+ and CD8+ T cell clones from peripheral blood sampled during the stable tolerance phase of a patient whose graft maintained good function for 9 years, 7 without immunosuppression. We analyzed the sequence of TCR Vbeta and Valpha genes and made clonotype-specific probes that allowed us to detect each clone in peripheral blood or biopsy specimens obtained during a 1-year period before and after the rapid onset of chronic rejection. We found an unexpectedly high level of donor HLA-specific T cell clonotype mRNA in peripheral blood during the late tolerance phase. Strong signals for two CD4+ clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reduction in direct pathway T cell clonotype mRNA in peripheral blood and the graft. Our data are inconsistent with the hypothesis that direct pathway T cells are involved only in early acute rejection events and suggest the possibility that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.     

Blocking the monocyte chemoattractant protein-1/CCR2 chemokine pathway induces permanent survival of islet allografts through a programmed death-1 ligand-1-dependent mechanism

Islet allografts are subject to rapid rejection through host cellular immune responses involving mononuclear cell recruitment and tissue injury. Interruption of leukocyte recruitment through chemokine receptor targeting is of therapeutic benefit in various experimental models, but little is known about the contribution of chemokine pathways to islet allograft rejection. We found that murine islets produce monocyte chemoattractant protein-1 (MCP-1; CCL2) in vitro and that islet allograft rejection was associated with intragraft expression of MCP-1 and its receptor, CCR2. We therefore investigated whether MCP-1 and CCR2 are required for the rejection of fully MHC-disparate islet allografts. Wild-type mice treated with blocking anti-MCP-1 mAb plus a brief, subtherapeutic course of rapamycin had long-term islet allograft survival, in contrast to the effect of treatment with either mAb or rapamycin alone. CCR2(-/-) mice treated with rapamycin also maintained islet allografts long-term. Both MCP/CCR2- and rapamycin-sensitive signals were required for maximal proliferation of alloreactive T cells, suggesting that MCP-1/CCR2 induce rejection by promoting alloreactive T cell clonal expansion and homing and migration. Prolonged islet allograft survival achieved by blockade of the MCP-1/CCR2 pathway plus rapamycin therapy was accompanied by a mononuclear cell infiltrate expressing the inhibitory receptor, programmed death-1 (PD-1), and its ligand (PD-L1, B7-H1), and prolongation of islet allograft survival was abrogated by anti-PD-L1 mAb therapy. These data show that the blockade of MCP-1 binding to CCR2 in conjunction with subtherapeutic immunosuppression can have profound effects on islet allograft survival and implicate the expression of the PD-1/PD-L1 pathway in the regulation of physiologic responses in vivo.     

Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection

Acute allograft rejection is a major complication postlung transplantation and is the main risk factor for the development of bronchiolitis obliterans syndrome. Acute rejection is characterized by intragraft infiltration of activated mononuclear cells. The ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11) are potent chemoattractants for mononuclear cells and act through their shared receptor, CXCR3. Elevated levels of these chemokines in bronchoalveolar lavage fluid have been associated with human acute lung allograft rejection. This led to the hypothesis that the expression of these chemokines during an allogeneic response promotes the recruitment of mononuclear cells, leading to acute lung allograft rejection. We performed studies in a rat orthotopic lung transplantation model of acute rejection, and demonstrated increased expression of CXCL9 and CXCL10 paralleling the recruitment of mononuclear cells and cells expressing CXCR3 to the allograft. However, CXCL9 levels were 15-fold greater than CXCL10 during maximal rejection. Inhibition of CXCL9 decreased intragraft recruitment of mononuclear cells and cellular expression of CXCR3, resulting in lower acute lung allograft rejection scores. Furthermore, the combination of low dose cyclosporin A with anti-CXCL9 therapy had more profound effects on intragraft leukocyte infiltration and in reducing acute allograft rejection scores. This supports the notion that CXCL9 interaction with cells expressing CXCR3 has an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of acute lung allograft rejection.     

A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX(3)CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-alpha-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX(3)CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX(3)CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX(3)CR1 Ab. Treatment with the anti-CX(3)CR1 Ab significantly prolonged allograft survival from 7 +/- 1 to 49 +/- 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.     

The Presence of HLA-E-Restricted,CMV-Specific CD8+ T Cells in the Blood of Lung Transplant Recipients Correlates with Chronic Allograft Rejection

The human cytomegalovirus (CMV) immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA)-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC). UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8⁺ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS). Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation.     

HLA-DR4 molecules in neuroendocrine epithelial cells associate to a heterogeneous repertoire of cytoplasmic and surface self peptides

Expression of MHC class II genes by epithelial cells is induced in inflammatory conditions such as autoimmunity and organ transplantation. Class II ligands generated by the epithelial cell processing mechanisms are unknown, although some unique epitopes have been described in epithelial cells that B cells could not generate. Epithelial cells are the targets of autoreactive T cell responses in autoimmune diseases and of transplant rejection processes, which may involve recognition of cell type-specific epitopes. In the present report, we have compared the DR4-associated repertoire and the intracellular distribution of class II, invariant chain (Ii), and DM molecules between a human DR4-, Ii-, and DM-transfected rat neuroendocrine epithelial cell line and a homozygous DR4 (DRB1*0401) lymphoblastoid B cell line, by mass spectrometry sequencing techniques, and immunoelectron microscopy. The epithelial cells chosen for transfection, RINm5F, are rat insular cells widely used for human studies of autoimmune diabetes. The results revealed a remarkably heterogeneous pool of self protein-derived peptides from the cell surface and various intracellular compartments, including the cytosol and secretory vesicles in epithelial cells, compared with a very restricted homogeneous repertoire in lymphoblastoid B cell lines, where few epitopes from surface molecules were predominant. The generation of distinct DR4-associated peptide repertoires in these two cell types could be due to the effect of several factors including differences in subcellular location of Ii and DM molecules, differential DO expression, and cell type-specific mechanisms of class II ligand generation. This is specially relevant to processes involving epithelial T cell interactions such as organ-specific autoimmunity and transplant rejection.     

Dynamic expression of Qa-2 during acute graft rejection

Human leukocyte antigen (HLA)-G exhibits immunotolerogenicity and is related to allograft acceptance. Qa-2 is the murine homolog of HLA-G; it has structure and functions similar to those of HLA-G. We investigated the dynamic expression of Qa-2 in skin allografts by immunohistochemistry and on peripheral blood lymphocyte subsets by flow cytometry during the entire process of acute graft rejection (AGR) with a murine skin transplantation model to determine its relationship with the pathological changes of allografts and the influence of immunosuppressive therapy. In grafts without immunosuppressive treatment, Qa-2 did not exhibit obvious changes in syngeneic and allogeneic recipients. In contrast, with immunosuppressant-treated grafts, positive expression of Qa-2 was observed. It remained at high levels in the immunosuppressant-treated syngeneic group; however, it became weakly positive and even negative in infiltrating inflammatory cells as AGR advanced, but it remained strongly positive in other skin tissues throughout the AGR process. Qa-2 expression on CD4(+) and CD8(+) peripheral blood lymphocyte subsets remained stable at a normal level in the non-immunosuppressant-treated syngeneic group. Immunosuppressive treatment can also significantly upregulate Qa-2. In the allogeneic groups, decreased expression was observed when AGR was at histological grades 1 to 2 (well before gross rejection was observed). Qa-2 was upregulated again after the graft was rejected completely. The results suggest that the increase in Qa-2 may be attributed to the use of immunosuppressive treatments. Moreover, Qa-2 expression decreased significantly with AGR progression, suggesting that it may be a potential marker for predicting AGR, especially in the presence of immunosuppressive agents.     

The role of the CC chemokine, RANTES, in acute lung allograft rejection

Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.     

DNA methylation arrays as surrogate measures of cell mixture distribution

ABSTRACT: BACKGROUND: There has been a long-standing need in biomedical research for a method that quantifies the normally mixed composition of leukocytes beyond what is possible by simple histological or flow cytometric assessments. The latter is restricted by the labile nature of protein epitopes, requirements for cell processing, and timely cell analysis. In a diverse array of diseases and following numerous immune-toxic exposures, leukocyte composition will critically inform the underlying immuno-biology to most chronic medical conditions. Emerging research demonstrates that DNA methylation is responsible for cellular differentiation, and when measured in whole peripheral blood, serves to distinguish cancer cases from controls. RESULTS: Here we present a method, similar to regression calibration, for inferring changes in the distribution of white blood cells between different subpopulations (e.g. cases and controls) using DNA methylation signatures, in combination with a previously obtained external validation set consisting of signatures from purified leukocyte samples. We validate the fundamental idea in a cell mixture reconstruction experiment, then demonstrate our method on DNA methylation data sets from several studies, including data from a Head and Neck Squamous Cell Carcinoma (HNSCC) study and an ovarian cancer study. Our method produces results consistent with prior biological findings, thereby validating the approach. CONCLUSIONS: Our method, in combination with an appropriate external validation set, promises new opportunities for large-scale immunological studies of both disease states and noxious exposures.     

MicroRNAs hsa-miR-99b,hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

Background

Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients.

Methods

miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets.

Results

Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology.

Conclusion

This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.     

Role of the L3T4+ T cell in allograft rejection

Pancreatic islet and fetal pancreas allotransplantation has been used to examine the role of the L3T4+ T cell in allograft rejection. Tissues were grafted into recipient animals depleted of peripheral L3T4+ T cells by in vivo administration of GK1.5 (anti-L3T4) monoclonal antibody to ask the question: is there a requirement for the L3T4+ T cell in graft rejection? Data show that the requirement for the L3T4+ T cell depends on either the type of tissue transplanted or type of the antigenic disparity between donor and recipient. Data also indicate that islet allograft acceptance achieved after GK1.5 treatment of the recipient is not due to tolerance induction. We therefore conclude that the cellular requirements for allograft rejection are determined by the type of tissue transplanted and the genetic disparity between donor and recipient.     

Leukocyte isolation by sedimentation: the effect of rouleau-promoting agents on leukocyte differential count.

The rouleau-promoting agents dextran and polyvinylpyrrolidone (PVP) were used to accelerate erythrocyte sedimentation in order to harvest the leukocyte rich plasma (LRP). The objective of the work was to determine if agent concentration or blood: agent ratio had any effect on the leukocyte differential count and if so at what agent concentration and agent:blood ratio did the LRP leukocyte differential count most closely match the whole blood leukocyte differential count. With both sedimentation agents the data clearly indicate that both parameters effect LRP differential counts and that low concentrations of sedimentation agents are most important in obtaining LRP differential counts which most closely match the whole blood differential counts.