Concordance of three point of care testing devices with clinical chemistry laboratory standard assays and patient-reported outcomes of blood sampling methods.

BACKGROUND: Point of care testing (POCT) devices have been developed to facilitate immediate results with the potential to aid screening for new disease and enable patients to self-monitor their disease. Non-communicable diseases (NCDs) are the major cause of mortality globally and are increasing in prevalence as the population ages. Allied health care professionals (AHPs) are skilled in undertaking risk assessment and delivering preventative advice, providing opportunities to access large proportions of the population who may not visit their doctor, within non-traditional community settings. There is evidence of high levels of support from public, patients and health professionals for engaging AHPs in risk-targeted early case detection of certain NCDs. Thus, POCT devices offer a potential alternative to traditional venous blood collection, as novel care pathways for increasing early case detection and access to preventative care. The objectives of this study were to: (i) determine the concordance of the specific POCT devices with laboratory-based standard assays employed within clinical biochemistry laboratories. (ii) compare the sampling experience of both methods via patient-reported experiences. METHODS: A prospective, two-centre study was undertaken involving 158 participants who provided informed consent. Venous blood was collected for traditional assays of HbA1c, creatinine/ estimated Glomerular-Filtration-Rate (eGFR) and vitamin-D. Capillary blood was collected by finger prick test and also assayed for the same biochemical indices (Nova StatSensor (creatinine/eGFR); Siemens DCA-Vantage (HbA1C); CityAssays (vitamin-D)). All users were provided with device training. Participants reported any discomfort experienced by each simultaneously applied method (randomised in order) via a 100 mm Visual-Analogue-Scale. RESULTS: Results for each POCT device and the laboratory standard were analysed by Bland-Altman plots to determine assay concordance. POCT devices demonstrated good concordance with laboratory testing, with at least 95% of all samples being within two standard deviations, for each of the devices tested. The majority of participants reported less discomfort with POCT than venepuncture, with the average reported discomfort being 17/100 mm less for POCT compared to venous blood sample collection on the visual analogue scale. CONCLUSIONS: The POCT devices demonstrated acceptable concordance with laboratory-based assays, and patients reported lower levels of discomfort compared to traditional means of blood collection. This study demonstrates the potential of using these devices as acceptable methods for opportunistic testing of "at-risk" individuals within non-traditional community care settings.

Serum-free light chains adjusted for renal function are a potential biomarker for post-transplant lymphoproliferative disorders.

Post-transplant lymphoproliferative disease (PTLD) is a serious complication of solid organ transplantation. As early diagnosis remains challenging, we investigated the utility of serum-free light chain (FLC) and heavy chain/light chain pairs (HLC) as diagnostic biomarkers. Pre-treatment serum FLC and HLC levels were measured in 20 patients at their first diagnosis of B cell PTLD and in 14/20 patients during follow-up. Results were compared to serum FLC/HLC levels of 90 matched PTLD-free transplanted controls. Renal dysfunction was common in both cohorts, and combined FLC levels were often elevated above the conventional upper limit of normal (45.7 mg/L). Combined FLC levels were higher in patients with PTLD than in transplant controls (p = 0.013), and levels above the conventional ULN were associated with PTLD (OR 3.2, p = 0.05). Following adjustment to cystatin C as a marker of renal function an even stronger association was found for a (dimensionless) threshold value of 37.8 (OR 8.9, p < 0.001). In addition, monoclonal proliferation (abnormal FLC ratio, using an established renal range cutoff) was more common in PTLD than in controls (3/20 vs. 2/90, p = 0.04). Following therapy, at the time of protocolised restaging, patients experiencing subsequent sustained complete remission displayed lower FLC levels than those not experiencing such remission (p = 0.053). No relationship with HLC results was seen. Elevated polyclonal FLC levels (especially when adjusted for renal function) and monoclonal proliferation are a potential biomarker for PTLD diagnosis and disease surveillance. However, prospective validation is necessary before FLC measurement should be incorporated in follow-up of transplant recipients and PTLD management.

TGFß modulates cell-to-cell communication in early epithelial-to-mesenchymal transition.

AIMS/HYPOTHESIS: A key pathology in diabetic nephropathy is tubulointerstitial fibrosis. The condition is characterised by increased deposition of the extracellular matrix, fibrotic scar formation and declining renal function, with the prosclerotic cytokine TGF-ß1 mediating many of these catastrophic changes. Here we investigated whether TGF-ß1-induced epithelial-to-mesenchymal transition (EMT) plays a role in alterations in cell adhesion, cell coupling and cell communication in the human renal proximal tubule. METHODS: Whole-cell and cell compartment abundance of E-cadherin, N-cadherin, snail, vimentin, ß-catenin and connexin-43 was determined in human kidney cell line (HK)2 and human proximal tubule cells with or without TGF-ß1, using western blotting and immunocytochemistry, followed by quantification by densitometry. The contribution of connexin-43 in proximal tubule cell communication was quantified using small interfering RNA knockdown, while dye-transfer was used to assess gap junctional intercellular communication (GJIC). Functional tethering was assessed by single-cell force spectroscopy with or without TGF-ß1, or by immunoneutralisation of cadherin ligation. RESULTS: High glucose (25 mmol/l) increased the secretion of TGF-ß1 from HK2 cells. Analysis confirmed early TGF-ß1-induced morphological and phenotypical changes of EMT, with altered levels of adhesion and adherens junction proteins. These changes correlated with impaired cell adhesion and decreased tethering between coupled cells. Impaired E-cadherin-mediated adhesion reduced connexin-43 production and GJIC, these effects being mimicked by neutralisation of E-cadherin ligation. Upregulation of N-cadherin failed to restore adhesion or connexin-43-mediated GJIC. CONCLUSIONS/INTERPRETATION: We provide compelling evidence that TGF-ß1-induced EMT instigates a loss of E-cadherin, cell adhesion and ultimately of connexin-mediated cell communication in the proximal tubule under diabetic conditions; these changes occur ahead of overt signs of renal damage.

Cytomegalovirus seropositivity is independently associated with cardiovascular disease in non-dialysis dependent chronic kidney disease.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of early death in patients with chronic kidney disease (CKD). Previous work has described an association between Cytomegalovirus (CMV) seropositivity and CVD amongst patients with dialysis dependent end stage renal disease. Whether CMV seropositivity is associated with CVD in non-dialysis dependent CKD has not been established. AIM: Investigate whether past CMV infection is associated with prevalent CVD in patients with non-dialysis dependent CKD. DESIGN: A retrospective observational study using the Renal Impairment in Secondary Care cohort, a study evaluating bio-clinical determinants of outcomes in patients with progressive CKD. METHODS: We assayed cryopreserved serum samples collected at inception for anti-CMV IgG antibodies from 764 patients with stages 2 to 5 CKD (pre-dialysis) and investigated its relationship with prevalent CVD. RESULTS: Median estimated glomerular filtration was 24 ml/min/1.73 m2 (IQR 19-32). Sixty-eight percent of patients were CMV seropositive. CMV seropositivity was associated with older age, non-Caucasian ethnicity, diabetes and higher social deprivation index score. On univariable analysis, CMV seropositivity correlated with higher systolic blood pressure (P = 0.044), prevalent CVD (P < 0.001), ischaemic heart disease (P < 0.001) and cerebrovascular disease (P = 0.022). On multivariable analysis, CMV seropositive patients nearly twice as likely to have CVD compared to seronegative patients [Odds Ratio (OR) = 1.998, CI 1.231-3.242, P = 0.005]. CONCLUSIONS: In patients with non-dialysis CKD, CMV seropositivity is independently associated with a higher prevalence of CVD.

Donor HLA-C genotype has a profound impact on the clinical outcome following liver transplantation.

Late allograft dysfunction is a significant problem following liver transplantation and its pathogenesis is uncertain. HLA-C is the major inhibitory ligand for killer immunoglobulin-like receptors (KIRs) that regulate the cytotoxic activity of natural killer (NK) cells. HLA-C alleles can be allocated into two groups, termed HLA-C1 and HLA-C2, based on their KIR specificity. HLA-C2 interactions are more inhibiting to NK cell activation. We studied the clinical importance of HLA-C genotype in a large liver transplant cohort and found that possession of at least one HLA-C2 allele by the donor allograft was associated with less histological evidence of chronic rejection and graft cirrhosis, a 16.2% reduction in graft loss (p = 0.003) (hazard ratio: 2.7, 95% CI 1.4-5.3) and a 13.6% improvement in patient survival (p = 0.01) (hazard ratio: 1.9, 95% CI 1.1-3.3) at 10 years. Transplantation of an HLA-C2 homozygous allograft led to a particularly striking 26.5% reduction in graft loss (p < 0.001) (hazard ratio: 7.2, 95% CI 2.2-23.0) at 10 years when compared to HLA-C1 homozygous allografts. Donor HLA-C genotype is therefore a major determinant of clinical outcome after liver transplantation and reveals the importance of NK cells in chronic rejection and graft cirrhosis. Modulation of HLA-C and KIR interactions represents an important novel approach to promote long-term graft and patient survival.

Focal segmental necrotizing glomerulonephritis in rheumatoid arthritis.

We report ten patients with rheumatoid arthritis (RA) who developed a focal segmental necrotizing glomerulonephritis (FSNGN) and extracapillary proliferation typical of vasculitic glomerulonephritis. Five patients also had extrarenal vasculitis. Renal presentation was with renal impairment (n = 9) (median creatinine 726 mumol/l, range 230-1592 mumol/l), microscopic haematuria (n = 8) and proteinuria (n = 10). Nine patients were seropositive for rheumatoid factor and nine had bone erosions. Serum from four of five patients tested by indirect immunofluorescence was positive for antineutrophil cytoplasmic antibody (ANCA) with perinuclear staining. Only three patients had penicillamine or gold therapy. Treatment was with prednisolone and cyclophosphamide (six patients, two of whom were also plasma-exchanged), prednisolone and azathioprine (two patients) and prednisolone alone (two patients). There was a marked improvement in renal function in eight patients. Two patients with dialysis-dependent renal failure recovered renal function, although in one patient this was transient and she required further dialysis 4 months later. Two other patients progressed to dialysis at 3 months and 1 year respectively. Four patients died, one remains dialysis-dependent, and four continue to have good renal function at 5 year follow-up (median creatinine 148.5 mumol/l, range 120-193 mumol/l). One patient was lost to follow-up at 5 years. FSNGN should be considered in all patients with RA and renal impairment, proteinuria and/or microscopic haematuria. This diagnosis appears to be more likely in patients with clinical extrarenal vasculitis, bone erosions or who are seropositive. In these cases, an urgent renal biopsy is indicated.

The relationship between high-sensitivity CRP and polyclonal free light chains as markers of inflammation in chronic disease.

INTRODUCTION: Serum concentrations of polyclonal free light chains (FLC) represent the activity of the adaptive immune system. This study assessed the relationship between polyclonal FLC and the established marker of innate immunity, C-reactive protein (CRP), in chronic and acute disease. METHODS: We utilized four cross-sectional chronic disease patient cohorts: chronic kidney disease (CKD), diabetes, vasculitis and kidney transplantation; and a longitudinal intensive care case series to assess the kinetics of production in acute disease. RESULTS: There was a weak association between polyclonal FLC and high-sensitivity CRP (hs-CRP) in the study cohorts. A longitudinal assessment in acute disease showed a gradual increase in FLC concentrations over time, often when CRP levels were falling, demonstrating clear differences in the response kinetics of CRP and FLC in this setting. CONCLUSION: Polyclonal FLC and hs-CRP provide independent information as to inflammatory status. Prospective studies are now required to assess the utility of hs-CRP and polyclonal FLC in combination for risk stratification in disease populations.

The relationship between albuminuria, MCP-1/CCL2, and interstitial macrophages in chronic kidney disease.

Glomerular-derived proteins may activate tubular cells to express the macrophage-directed chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Macrophages at interstitial sites have a central role in directing renal scarring. We have prospectively assessed the relationship between albuminuria, urinary MCP-1/CCL2, interstitial macrophage infiltration, in situ damage, and clinical outcomes in a large group of patients with chronic kidney disease. We studied 215 patients and quantified albumin-creatinine ratio (ACR), urinary MCP-1/CCL2, interstitial macrophage numbers, and in situ damage. ACR correlated with urinary MCP-1/CCL2 (correlation 0.499; P<0.001), interstitial macrophage numbers (correlation 0.481; P<0.001), and index of chronic damage (correlation 0.363; P<0.001). Macrophage numbers closely correlated with in situ damage (correlation 0.755; P<0.001). By multivariate analysis ACR, urinary MCP-1/CCL2, and interstitial macrophage numbers were interdependent. By Kaplan-Meier survival analysis albuminuria, urinary MCP-1/CCL2, interstitial macrophages, and chronic damage predict the outcome. ACR, macrophage numbers, chronic damage, and creatinine independently predicted renal survival. The association of ACR with other variables was strongest in patients with less advanced disease states. There is a close association between albuminuria, urinary MCP-1/CCL2, and interstitial macrophage infiltration with in situ damage and clinical outcomes. These findings support the hypothesis that albuminuria triggers tubular MCP-1/CCL2 expression with subsequent macrophage infiltration. These processes may represent the dominant pathway for the progression of renal injury before the establishment of advanced renal scarring.

Activation of endothelial cells in thrombosis and vasculitis.

The endothelium participates actively in homeostatic mechanisms such as the regulation of vascular tone and maintenance of a nonthrombotic environment, as well as directing biological responses such as leukocyte trafficking to inflammatory sites. Disruption of these processes leads to disease. In the antiphospholipid antibody syndrome autoantibodies provoke the endothelium to develop a prothrombotic surface. In systemic vasculitides associated with presence of antineutrophil cytoplasm antibodies, it is likely that the autoantibodies incite premature neutrophil activation, disrupted neutrophil-endothelium interactions and endothelial damage. This review considers how normal endothelial functions may be subverted in disease and how active endothelial responses may contribute to disease.

Assessment of disease activity in systemic vasculitis.

The systemic vasculitides are a group of inflammatory disorders characterised by relapses and remission. Before the introduction of immunosuppressive drugs, mortality was unacceptably high. Immunosuppressive therapy has had a therapeutic impact, but at the cost of increased risk of infection and other adverse effects. Differentiating infection from active disease can be difficult, and the inappropriate prescription of immunosuppressive drugs can be fatal. Hence disease indices which can aid physicians to identify the active phase of disease and enable early treatment, will be valuable in the management of this group of disorders.

Neutrophil priming and apoptosis in anti-neutrophil cytoplasmic autoantibody-associated vasculitis.

BACKGROUND: Interactions between anti-neutrophil cytoplasmic autoantibody (ANCA) and primed neutrophils (PMNs) may be central to the pathogenesis of primary small vessel vasculitis. PMNs from patients are primed, expressing proteinase 3 (PR3) on the cell surface, which permits interaction with ANCA. In vitro ANCA activates primed PMN to degranulate and generate a respiratory burst. Resultant reactive oxygen species are important in triggering apoptosis, but the fate of PMN in ANCA-associated vasculitis is unknown. Failure to remove apoptotic PMN in a nonphlogistic manner may sustain the inflammatory response. METHODS: PMNs from patients or controls were isolated, and the basal production of superoxide was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome C. ANCA antigen expression on apoptotic PMN was assessed at 0, 12, and 18 hours by flow cytometry using dual staining with FITC-conjugated annexin V and PE-conjugated anti-murine IgG against monoclonal ANCA. Apoptosis was also assessed by morphology. In further studies, apoptotic PMNs were opsonized with monoclonal anti-myeloperoxidase (MPO) or anti-proteinase-3 (PR3) or irrelevant isotype-matched IgG (N IgG) and phagocytosis by macrophages was measured using interaction assays. Cytokines interleukin-8 (IL-8) and interleukin-1 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Proteinase-3 expression (active 63.04 +/- 5.6% of total number of cells, remission 51.47 +/- 7.9% of total number of cells, control 17.7 +/- 4.7% of total number of cells, P < 0.05) and basal superoxide production (active 6.9 +/- 0.8 nmol/L x 10(6) cells, remission 5.15 +/- 0.4 nmol/L/10(6) cells, control 3.63 +/- 0.3 nmol/L/10(6) cells, P < 0.001) were significantly greater with freshly isolated PMN from patients than controls. PR3 expression and superoxide generation were positively correlated. PMN from patients with active disease became apoptotic at a greater rate than those of controls (at 18 hours, patients 72.3 +/- 3.9% apoptosis, controls 53.2 +/- 2.7% apoptosis, P < 0.05). PR3 and MPO expression were significantly greater on PMN isolated from patients at 12 and 18 hours. Opsonization of apoptotic PMN with ANCA significantly enhanced recognition and phagocytosis by scavenger macrophages (anti-MPO 88.95 +/- 6.27, anti-PR3 93.98 +/- 4.90, N IgG 44.89 +/- 3.44, P < 0.01) with increased secretion of IL-1 (anti-PR3 34.73 +/- 6.8 pg/mL, anti-MPO 42.01 +/- 12.3 pg/mL, N IgG 8.04 +/- 6.3 pg/mL, P < 0.05) and IL-8 (anti-PR3 8.97 +/- 0.93 ng/mL, anti-MPO 8.45 +/- 1.46 ng/mL, N IgG 0.96 +/- 0.15 ng/mL, P < 0.01). CONCLUSION: In vivo circulating PMNs are primed as assessed by PR3 expression and basal superoxide production, thereby enhancing their inflammatory potential. These PMNs undergo apoptosis more readily, at which times they express PR3 and MPO on their surface. These antigens may then provide targets for ANCA. Opsonization of apoptotic PMN will enhance clearance by macrophages but will also trigger the release of pro-inflammatory cytokines that may contribute to chronic inflammation.

T lymphocyte adhesion mechanisms within inflamed human kidney: studies with a Stamper-Woodruff assay.

Renal inflammatory conditions are characterized by mononuclear cell recruitment to sites of inflammation. We have developed a modified Stamper-Woodruff assay system to analyze mechanisms of functional T cell adhesion to cryostat sections of renal biopsy material from patients with vasculitic glomerulonephritis (GN) and acute allograft rejection. Peripheral blood T cells adhered to intraglomerular, periglomerular, and tubulointerstitial regions of the cortex. Blocking monoclonal antibodies against tissue expressed ICAM-1, VCAM-1, and the CS-1 domain of fibronectin (CS-1Fn) differentially attenuated T cell adhesion. Glomerular adhesion in vasculitic GN and tubulointerstitial adhesion in acute rejection were particularly sensitive to both anti-ICAM-1 and anti-VCAM-1 antibodies, indicating a prominent role for ICAM-1 and VCAM-1 at glomerular sites in vasculitis and at tubulointerstitial sites in rejection. Furthermore, using KL/4 cells (LFA-1 expressing) and Jurkat cells (VLA-4 expressing), we demonstrated specific LFA-1/ICAM-1- and VLA-4/VCAM-1-mediated interactions within glomerular and tubulointerstitial compartments. Jurkat cells also adhered to VCAM-1-free sites, and binding was inhibitable by anti-CS-1Fn antibody, thereby demonstrating a role for VLA-4/fibronectin interactions especially at intraglomerular sites in acute rejection where VCAM-1 is notably absent. We therefore propose a prominent functional role for ICAM-1, VCAM-1, and CS-1 domain fibronectin in T cell recruitment to the inflamed kidney.

Interleukin-8: A pathogenetic role in antineutrophil cytoplasmic autoantibody-associated glomerulonephritis.

BACKGROUND: In neutrophil trafficking, the role of interleukin-8 (IL-8) is location dependent. Tissue IL-8 directs transmigration, whereas intravascular IL-8 frustrates this process. The bystander damage of glomerular endothelium by antineutrophil cytoplasmic autoantibody (ANCA)-activated neutrophils is believed to be an early event in the pathogenesis of ANCA-associated glomerulonephritis. We have studied the role of IL-8 in this process. METHODS: Intraglomerular expression of IL-8 in patients with ANCA-associated glomerulonephritis was studied by in situ hybridization and immunohistochemistry and location of neutrophils by serial section immunohistochemistry. In vitro, we analyzed ANCA-stimulated neutrophil IL-8 production by enzyme-linked immunosorbent assay, and the IL-8 attributable effect of ANCA-stimulated neutrophil supernatant by chemotactic and transendothelial assays. RESULTS: There was intraglomerular expression of IL-8 at segmental, crescentic, and parietal epithelial sites. IL-8 protein expression colocalized to intraglomerular neutrophils; many localized within glomerular capillary loops, suggesting failed trafficking to tissue IL-8. ANCAs differentially stimulated time- and dose-dependent neutrophil IL-8 production, and ANCA-stimulated neutrophil supernatant demonstrated potent IL-8-dependent chemotactic activity and inhibited transendothelial migration of normal human neutrophils toward an IL-8 gradient. CONCLUSION: Despite heavy tissue expression of IL-8 in ANCA-associated GN, the production of IL-8 by ANCA-stimulated neutrophils within the intravascular compartment may frustrate neutrophil transmigration, encourage intravascular stasis, and contribute to bystander damage of glomerular endothelial cells.

In situ analysis of C-C chemokine mRNA in human glomerulonephritis.

BACKGROUND: Glomerular and tubulointerstitial accumulations of macrophages and T cells are a prominent feature of immune inflammatory glomerulonephritis. The C-C family of chemokines are major mononuclear-cell chemoattractants and may be central to the recruitment of these cells. METHODS: Using in situ hybridization (ISH) we analyzed the expression of mRNA for the C-C chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha and beta (MIP-1alpha, MIP-1beta) and RANTES in renal biopsy material from twenty patients with glomerulonephritis. RESULTS: In overt inflammatory glomerulonephritides, chemokine transcripts were differentially expressed by glomerular and tubulointerstitial leukocyte infiltrates, glomerular parietal and proximal tubular epithelial cells and endothelial cells. There was little expression in minimal change nephropathy and normal tissue. Expression of individual chemokines correlated with intrarenal T cell and macrophage infiltrates. Combined immunohistochemistry and ISH demonstrated that 56.9% of cells expressing MCP-1 mRNA were CD68+ve (monocytes/macrophages) and 53% of infiltrating CD68 +ve cells were MCP-1 mRNA positive. CONCLUSIONS: These studies indicate that the in situ production of C-C chemokines by resident and infiltrating cells may play a crucial role in regulating macrophage and T-cell recruitment in glomerulonephritis.