High Mean Fluorescence Intensity Values Research Articles

The Challenge of Preoperative Panel Reactive Antibody Positivity in Parathyroid Transplantation.

Identifying suitable recipient criteria and matching recipients with appropriate donors are required to increase survival for parathyroid transplant. This study was undertaken to evaluate transplant survival rates while comparing preoperative panel reactive antibody positivity. The study included 14 hypoparathyroidism patients who presented to our clinic for parathyroid transplant. Preoperative ABO compatibility and negative cross-match tests were prioritized for recipient-donor matching, and panel reactive antibody screening tests were performed. During the 24-month follow-up, we evaluated medication use and serum calcium, phosphorus, and parathormone levels of patients. Preoperative panel reactive antibody positivity was assessed in 3 groups. The HLA class I-positive group (mean fluorescence intensity range, 179-1770) showed decreased medication use and stability in serum calcium levels. The HLA class IIpositive (mean fluorescence intensity range, 85-3959) showed decreased medication use by 25% to 50% and returned to their former prescription doses after 12 months. An opposite pattern was observed in 2 patients with panel reactive antibody positivity for both HLA classes (mean fluorescence intensity range, 462-2289), with 1 patient requiring medication for continuing symptoms and the other patient occasionally taking additional magnesium supplementation, despite decreased medication doses after 12 months. Serum calcium levels remained normal, and parathormone and phosphorus levels were elevated. Improving patient symptoms and having no requirement for intravenous calcium replacement are priorities, and monitoring serum levels is the next important step. Varied panel reactive antibody positivities and survival rates indicate a requirement, and each HLA class could require a proper limitation for the mean fluorescence intensity. Preoperative mean fluorescence intensity cut-off value should be <900. Higher mean fluorescence intensity values in panel reactive antibody screenings could increase risk of short-term graft survival after parathyroid transplant. Further studies should include immunological risk assessments by individualizing the outcome with donor-specific antibodies.

Cutaneous Human Papillomaviruses and the Risk of Keratinocyte Carcinomas.

Cutaneous human papillomavirus (cuHPV) infections may be novel targets for skin cancer prevention and treatment, but critical information regarding the development of virus-positive skin cancers following cuHPV infection has been lacking. In this study, baseline cuHPV infection was measured by serology and viral DNA detection in eyebrow hairs (EBH) and forearm skin swabs (SSW) among 1,008 individuals undergoing routine skin cancer screening exams and followed for incidence of basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cuSCC). Baseline β-HPV detection, particularly in SSW, significantly predicted cuSCC (HR = 4.32; 95% confidence interval, 1.00-18.66), whereas serologic evidence of past β-HPV infection was not associated with cuSCC. Less than 5% of baseline β-HPV types detected in SSW were present in subsequent cuSCC tumors, and cuHPV detected in SSW with higher mean fluorescence intensity values were more likely to be present in cuSCC compared with those with lower levels (P < 0.001). β-HPV-positive cuSCC occurred more often in areas of highly sun-damaged skin than did β-HPV-negative cuSCC. Overall, no clear patterns were observed between baseline β-HPV detection and subsequent development of BCC, or between baseline γ-HPV detection and either cuSCC or BCC. Collectively, these results demonstrate that β-HPV detection in SSW is a significant predictor of cuSCC risk, although evidence suggests only a small subset of cuSCC is etiologically linked to β-HPV infection. SIGNIFICANCE: β-HPV positivity may be a useful biomarker for identifying individuals who could benefit from increased screening or novel cutaneous squamous cell carcinoma prevention strategies.

Expression of ADP receptor P2Y12, thromboxane A2 receptor and C-type lectin-like receptor 2 in cord blood-derived megakaryopoiesis

The ADP receptor P2Y12, the thromboxane A2 receptor (TXA2R) and the C-type lectin-like receptor 2 (CLEC-2) mediate platelet activation by different mechanisms. Only little is known about the expression of the receptors in human megakaryopoiesis. Our study aimed to establish a flow cytometry (FC) method for the measurement of P2Y12, TXA2R, and CLEC-2 on platelets of healthy donors and to monitor receptor expression in ex vivo megakaryopoiesis. We determined mean fluorescence intensity (MFI) values of FITC, PE, or APC labeled antibodies binding to the receptors on platelets of 90 healthy donors. For cord blood-derived megakaryopoiesis (CBMK) differentiation of CD34+ cells was induced by IL-3, SCF, and TPO. At 6 time points between day 0 and day 21 of cell culture the MFI values for CD34, CD41, CD61, P2Y12, TXA2R, and CLEC-2 were measured. Quantitative PCR was used for relative quantification of the corresponding mRNA. Transcription factor (TF) binding sites were predicted by in silico analysis of the genes. Platelets showed expectable high MFI values for the platelet marker CD41 (13,716 median MFI). Lower MFI was found for P2Y12 (2,847 median MFI) and CLEC-2 (1,211 median MFI), whereas, binding of the TXA2R antibody revealed even higher values (21,458 median MFI) than CD41. In CBMK the CD34+ cells were negative for P2Y12, TXA2R, and CLEC-2 at day 0. A maximum of 21-fold and 6-fold increase of P2Y12 and TXA2R MFI values, respectively, was found on day 14 to 17. MFI for CLEC-2 increased by 58-fold within the first week and reached a maximum of 1,572-fold increase within the first two weeks of CBMK. Very similar results were obtained on the RNA level. The differential regulation of receptor expression in CBMK was further supported by significant differences in the numbers and types of TF binding sites. P2Y12 and TXA2R, both upregulated only to a low extent in CBMK, probably, are dispensable for megakaryopoiesis. Furthermore, we speculate that CLEC-2 strongly upregulated in early CMBK is important for megakaryopoiesis.

Panel-Reactive and Donor-Specific Antibodies before Lung Transplantation can Affect Outcomes in Korean Patients Receiving Lung Transplantation.

PurposeData on the distribution and impact of panel reactive antibodies (PRA) and donor specific antibodies (DSA) before lung transplantation in Asia, especially multi-center-based data, are limited. This study evaluated the prevalence of and effects of PRA and DSA levels before lung transplantations on outcomes in Korean patients using nationwide multicenter registry data.Materials and MethodsThis study included 103 patients who received a lung transplant at five tertiary hospitals in South Korea between March 2015 and December 2017. Mortality, primary graft dysfunction (PGD), and bronchiolitis obliterans syndrome (BOS) were evaluated.ResultsSixteen patients had class I and/or class II PRAs exceeding 50%. Ten patients (9.7%) had DSAs with a mean fluorescence intensity (MFI) higher than 1000, six of whom had antibodies with a high MFI (≥2000). DSAs with high MFIs were more frequently observed in patients with high-grade PGD (≥2) than in those with no or low-grade (≤1) PGD. In the 47 patients who survived for longer than 9 months and were evaluated for BOS after the transplant, BOS was not related to DSA or PRA levels. One-year mortality was more strongly related to PRA class I exceeding 50% than that under 50% (0% vs. 16.7%, p=0.007).ConclusionPreoperative DSAs and PRAs are related to worse outcomes after lung transplantation. DSAs and PRAs should be considered when selecting lung transplant recipients, and recipients who have preoperative DSAs with high MFI values and high PRA levels should be monitored closely after lung transplantation.

An Easy and Sensitive Method to Profile the Antibody Specificities of HLA-specific Memory B Cells.

Pretransplant immunological risk assessment is currently based on donor-specific HLA antibodies in serum. Despite being an excellent source for antibodies produced by bone marrow-residing plasma cells, serum analysis does not provide information on the memory B-cell compartment. Although B-cell culture supernatants can be used to detect memory B cell-derived HLA antibodies, low IgG concentrations can preclude detectability of HLA antibodies in luminex single-antigen bead (SAB) assays. Culture supernatants of polyclonally activated B cells from alloantigen exposed (n = 13) or nonexposed (n = 10) individuals were either concentrated 10-fold, or IgG was isolated by using a protein G affinity purification method to increase the IgG concentration. These processed culture supernatants, as well as paired serum samples were tested for the presence of HLA antibodies using luminex SAB analysis. In immunized individuals, 64% were found to have HLA-specific B-cell memory in concentrated supernatants, whereas 82% showed HLA-specific B-cell memory when IgG isolated supernatants were used for HLA antibody detection. IgG-isolated supernatants showed higher mean fluorescence intensity values compared with concentrated supernatants without increased background. In some individuals, HLA-specific B-cell memory was detected in the absence of accompanying serum antibody specificities. We developed a novel, highly sensitive method to assess the HLA-specific memory B-cell compartment using luminex SAB technology. This assay allows direct comparison to the serum compartment and may therefore provide a more complete picture of the humoral alloimmune response in patients with a history of alloantigen exposure.

Association of HLA Types with Non-Specific Binding of Negative Control Beads in Luminex Panel Reactive Antibody (PRA) Screening Assay.

Luminex panel reactive antibody (PRA) screening assays using microbeads are widely used for organ transplantation. Anti-HLA serum reactivity is calculated by correcting for non-specific binding to the negative control (NC) beads. High mean fluorescence intensity (MFI) value of NC beads are observed in some patients and can result in false negative results in the PRA screening assay. We analyzed the clinical characteristics and HLA types of those patients with high MFI values of NC beads. Sixty-six patients with high MFI values of NC beads (> 300) in the PRA LABScreen Mixed assay (One Lambda) tested were included as the high NC group. Age and gender matched controls with low MFI values of NC beads (< 100) (n = 132), tested with PRA, were selected as the low NC group and 207 healthy Koreans were used as normal controls. Association of clinical characteristics and HLA types with the high NC group were analyzed using Chi-square test or Fischer's exact test, as appropriate. The proportion of patients with underlying liver disease was higher in the high NC group compared to the low NC group (18.1% vs. 1.5%, p < 0.001, OR = 14.2). The seropositivity of anti-nuclear antibody and rheumatoid factor, the frequency of use of intravenous immunoglobulin G, anti-thymocyte globulin, and rituximab showed no difference between two groups. The phenotype frequency (PF) of HLA-B46 was higher in the high NC group than in the low NC group (8.0% vs. 3.2%, p = 0.036, OR = 2.8). The PF of HLA-B7 was lower in the high NC group than in the healthy controls (0.0% vs. 6.5%, p = 0.008, OR = 0.1). The PF of HLA-DR1 was lower in the high NC group than in the low NC group (0.8% vs. 6.6%, p = 0.015, OR = 0.1) or healthy controls (0.8% vs. 7.4%, p = 0.003, Pc = 0.042, OR = 0.1). Increased non-specific binding to NC beads was associated with underlying liver disease and HLAB46. HLA-B7 and HLA-DR1 were related to a lower chance of non-specific binding to NC beads. The mechanism of those associations, such as differences in non-specific antibody response according to HLA phenotype or underlying disease, needs to be elucidated in further studies.

OR3 Clinical relevance of preformed “acceptable” donor specific antibodies in kidney transplantation

Preformed HLA cytotoxic antibodies, specific for mismatched HLA molecules of the potential donor, represent an absolute contraindication in kidney transplantation. New techniques, like the Luminex-Single Antigen Beads assay, are very sensitive allowing detection of HLA donor specific antibodies (DSAs) at low mean fluorescence intensity (MFI) values. However, few evidence on the clinical relevance of such low “strength” DSAs have been reported. Graft outcome (follow-up 34.2 ± 19.8 months) of 99 pre-sensitized patients (%FlowPRA class I = 41 ± 33; %FlowPRA class II = 30 ± 36), transplanted between May 2007 and June 2014 on the basis of both CDC-XM and FC-XM negative results, was analyzed. Five patients were excluded from the study because of primary non-function of the graft (3 patients) or death for non-immunologic causes with non-functional graft (2 patients). Forty-seven (47%) patients did not have pre-formed HLA DSAs; the remaining 52 (53%) patients had “acceptable” DSAs (MFI ⩽ 5000) specific for HLA-A/B/C/DR/DQB molecules or anti-DPB/DQA DSAs with high MFI values (>5000). HLA class I DSAs were present in 28 patients; five of these showed DSAs against more than one donor HLA molecules. HLA class II DSAs were present in 19 patients; three of these showed anti-DPB/DQA DSAs with MFI > 5000. The remaining five patients had both HLA class I and class II DSAs; one of these had high level of anti-DP DSA. Analyzing graft outcome of the remaining 94 transplanted patients with functioning graft, we did not evidenced any significant difference between DSA positive patients and DSA negative patients (rejection: 10.4% vs. 6.5%, P = ns; graft loss: 12.5% vs. 10.9%, P = ns). In particular, among the 48 DSA positive patients, three (6.2%) had humoral rejection without graft loss; two (4.2%) had cellular rejection, one of these lost the graft. Six DSA positive patients suffered graft failure that was never due to antibody-mediated rejection related to pre-formed DSAs. The results of this study show that pre-formed DSAs with low mean fluorescence intensity values do not represent a contraindication in kidney transplantation. An accurate evaluation of the “strength” of Luminex-detected DSA allows transplanting patients with clinically “irrelevant” HLA antibodies.

Pretransplant Donor-Specific HLA Class-I and -II Antibodies Are Associated With an Increased Risk for Kidney Graft Failure

Pretransplant risk assessment of graft failure is important for donor selection and choice of immunosuppressive treatment. We examined the relation between kidney graft failure and presence of IgG donor specific HLA antibodies (DSA) or C1q-fixing DSA, detected by single antigen bead array (SAB) in pretransplant sera from 837 transplantations. IgG-DSA were found in 290 (35%) sera, whereas only 30 (4%) sera had C1q-fixing DSA. Patients with both class-I plus -II DSA had a 10 yr graft survival of 30% versus 72% in patients without HLA antibodies (p < 0.001). No significant difference was observed in graft survival between patients with or without C1q-fixing DSA. Direct comparison of both assays showed that high mean fluorescence intensity values on the pan-IgG SAB assay are generally related to C1q-fixation. We conclude that the presence of class-I plus -II IgG DSA as detected by SAB in pretransplant sera of crossmatch negative kidney recipients is indicative for an increased risk for graft failure, whereas the clinical significance of C1q-fixing IgG-DSA could not be assessed due to their low prevalence.

Predictive Value of Donor Specific Antibody Measured by Luminex Single Antigen Assay for Antibody Mediated Rejection after Kidney Transplantation

Background: Luminex panel reactive antibody (PRA) is a method that is well known for its high sensitivity and specificity. By using a single antigen assay, the presence or absence of donor specific antibody (DSA) can be determined and its strength can be quantified in terms of the mean fluorescence intensity (MFI). In this study, we analyzed the correlation between the pre-transplant PRA and DSA measured by the Luminex method and the post-transplant clinical features after kidney transplantation. Methods: A total of 123 pre-transplant sera samples from kidney transplanted patients were tested. Luminex-PRA identification tests were performed using a Luminex fluoroanalyzer and a LifeCodes class I, II ID Kits. Single antigen assay by the Luminex method was used for detecting DSA and its MFI. Results: The positive Luminex-PRA group included more highly-sensitized patients such as women, patients with a previously positive lymphocyte cross match test and patients who were undergoing retransplantation. There was no correlation between the acute rejection rate and positive PRA on the Luminex-PRA. However, pretransplant DSA detected by the single antigen assay was significantly associated with episodes of antibody mediated rejection (P=0.047, OR=10.2), and DSA with higher MFI values (MFI≥3,000) was associated with antibody mediated rejection (P=0.023). Conclusions: Although pre-transplant positive PRA was not correlated with acute rejection episodes, the DSA measured by the Luminex single antigen assay seems to have a predictive value for post-transplant antibody mediated rejection.

Reduced antioxidant capacities in platelets from patients with autoimmune thrombocytopenia purpura (ITP)

Autoimmune thrombocytopenic purpura (ITP) is characterized by an abnormally low platelet count and bleeding risks. The exact triggering event remains elusive. Oxidative stress may play a role in several autoimmune diseases. A direct link between platelets in ITP and oxidative stress has not yet been addressed. The intracellular platelet antioxidant capacity (AOC) in ITP patients in the active phase (n = 24) and remission (n = 12), and 44 healthy controls were analysed with 2′,7′-dichlorodihydrofluorescein diacetate, and in combination with hydrogen peroxide. Enzyme activities (EA) of serum glutathione peroxidase (GPx), glutathione reductase (GRed) and catalase (CAT) were investigated colourimetrically in patients and controls. The AOC of ITP patients in the active phase was drastically reduced, with significantly high mean fluorescence intensity values. Higher GPx activity was observed in both active phase and remission in comparison to healthy controls (p < 0.001), with greater activity observed in active ITP than remission (p = 0.001). However, GRed EA was not elevated indicating that reduced glutathione (GSH) is not comparably recovered as consumed, leading to a decreased bioavailability of GSH and increased oxidative stress. These results suggest that oxidative stress is implicated in active ITP and may play a crucial role in its pathophysiology.

Evaluation of Influenza-Specific Humoral Response by Microbead Array Analysis

Quantitation and determination of antigen specificity of systemic and mucosal immune responses to influenza vaccination is beneficial for future vaccine development. Previous methods to acquire this information were costly, time consuming and sample exhaustive. The benefits of suspension microbead array (MBA) analysis are numerous. The multiplex capabilities of the system conserve time, money and sample, while generating statistically powerful data. To demonstrate the use of the assay by comparing the humoral influenza-specific responses of two cohorts from two countries that differed in circulating influenza strains and rates of influenza vaccination. Influenza hemagglutinin (HA) from different strains were coated on microbeads and incubated with serum samples to capture immunoglobulin (Ig) A(1) and IgG(1) host antibodies. Statistically significant differences in IgA(1) and IgG(1) exist between the serum samples from Winnipeg (Manitoba) donors and those from Kenyan (Africa) donors. Data were compared using Mann-Whitney nonparametric tests. The Winnipeg donors had higher mean fluorescence intensity values, with significant P values for anti-HA IgA(1) to A/Wyoming/3/2003 (P=0.044), A/Vietnam/1203/2004 (P=0.0179), A/New Caledonia/20/99 (P<0.0001) and B/Tokyo/53/99 (P=0.0002). No differences were seen between the groups in their response to B/Jilin/20/2003. The Winnipeg donors had higher mean fluorescence intensity values, with significant P values for anti-HA IgG(1) to A/Wyoming/3/2003 (P=0.0135), B/Tokyo/53/99 (P=0.006) and B/Jilin20/2003 (P=0.026). Influenza-specific IgA(1) and IgG(1) antibodies were successfully detected using MBA technology. A significant difference in antibody response was observed between Winnipeg and Kenyan donor serums. MBA analysis is a relatively quick and cost-effective method for serum antibody analysis. The potential to simultaneously assay small sample volumes for a multitude of antigens makes this method invaluable for future vaccine response monitoring.