Luminex-based Assay Research Articles

The good, the bad, and the ugly of luminex donor-specific crossmatch.

The presence of donor-specific antibodies directed against human leukocyte antigen significantly influences renal transplant because of antibody-mediated rejection. We performed the screening of pre-renal transplant patients for preformed anti-HLA antibodies using anti-human globulin augmented-complement-dependent lymphocytotoxicity crossmatch (AHG-CDCXm), luminex donor-specific crossmatch (LumXm) and HLA antibody screening. Seven hundred and fifty-four patients were assessed for LumXm. HLA antibody screening was possible in 325 out of 754 cases. All the three investigations viz. CDCXm, HLA antibody screening and LumXm was performed in 325 patients. All CDCXm positive patients (10/325, 3.08%) were also positive with LumXm and HLA antibody screen whereas 14 cases (4.31%) with CDCXm negative were positive with luminex-based assays. LumXm and HLA antibody screening were both positive in 24 (7.38%) cases, LumXm and HLA antibody screening were both negative in 275 (84.63%) cases and LumXm negative and HLA antibody screening was positive in 22 (6.76%) cases. However, there were four cases (1.23%) which were positive in LumXm in spite of being negative in HLA antibody testing. Single Antigen Bead (SAB) assay was performed in all patients positive for HLA antibody test. We suggest that LumXm is a useful and sensitive technique for the detection of anti-HLA antibodies in pre-transplant renal patients. However, other measures such as luminex antibody screen, SAB assay, history of the donor, and the class of antibodies involved should be taken into consideration for pre-transplant work up of renal patients.

Human Immunoglobulin G Cannot Inhibit Fibrinogen Binding by the Genetically Diverse A Domain of Staphylococcus aureus Fibronectin-Binding Protein A.

The fibronectin-binding protein A (FnBPA) is a cell surface-associated protein of Staphylococcus aureus which mediates adherence to the host extracellular matrix and is important for bacterial virulence. Previously, substantial sequence diversity was found among strains in the fibrinogen-binding A domain of this protein, and 7 different isotypes were described. The effect of this sequence diversity on the human antibody response, in terms of both antibody production and antibody function, remains unclear. In this study, we identify five different FnBPA A domain isotypes based on the sequence results of 22 clinical S.aureus isolates, obtained from the same number of patients suffering from bacteremia. Using a bead-based Luminex technique, we measure the patients' total immunoglobulin G (IgG) against the 7 FnBPA isotypes at the onset and during the time course of bacteremia (median of 10 serum samples per patient over a median of 35days). A significant increase in IgG against the FnBPA A domain, including the isotype carried by the infecting strain, is observed in only three out of 22 patients (14%) after the onset of bacteremia. Using a Luminex-based FnBPA-fibrinogen-binding assay, we find that preincubation of recombinant FnBPA isotypes with IgG from diverse patients does not interfere with binding to fibrinogen. This observation is confirmed using an alternative Luminex-based assay and enzyme-linked immunosorbent assay (ELISA). IMPORTANCE Despite the many in vitro and murine in vivo studies involving FnBPA, the actual presence of this virulence factor during human infection is less well established. Furthermore, it is currently unknown to what extent sequence variation in such a virulence factor affects the human antibody response and the ability of antibodies to interfere with FnBPA function. This study sheds new light on these issues. First, the uniform presence of a patient's IgG against FnBPA indicates the presence and importance of this virulence factor during S.aureus pathogenesis. Second, the absence of an increase in antibody production in most patients following bacteremia indicates the complexity of S.aureus-host interactions, possibly involving immune evasion or lack of expression of FnBPA during invasive infection. Finally, we provide new insights into the inability of human antibodies to interfere with FnBPA-fibrinogen binding. These observations should be taken into account during the development of novel vaccination approaches.

Donor-specific HLA-DQ antibodies may contribute to poor graft outcome after heart transplantation.

BACKGROUNDHLA-DQ donor-specific antibodies (DSA) are implicated in allograft dysfunction after renal and lung transplantation. Limited data exists on the impact of HLA-DQ antibodies on heart transplant patients.OBJECTIVETo investigate the impact of DSA formation on allograft function and outcomes in heart transplant patients.DESIGNRetrospective cohort study.SETTINGCollating post-transplantation patient data from computerized database in a tertiary hospital in Riyadh, Saudi Arabia from January 2006 to October 2014.PATIENTS AND METHODSWe excluded recipients with positive preoperative complement-dependent-cytotoxicity crossmatch grafts and those with preformed DSA. Anti-HLA antibodies were identified using Luminex-based assay in sera collected before transplantation with a routine endomyocardial biopsy the first year and then annually.MAIN OUTCOME MEASURESPrimary outcome measures were all-cause mortality, development of antibody mediated rejection, treated acute cellular rejection (ACR) and cardiac allograft vasculopathy (CAV).SAMPLE SIZE127 patients.RESULTSDSA formation occurred in 43/127 (34%), with 33/43 (77%) targeting HLA-DQ antigens alone (n=7) or in combination with -DR, -A or B antibodies (n=26). Most (76%) were male and the mean (SD) age was 36 (14) years. Ten patients developed -A, -B or -DR antibodies without -DQ antibodies also present. Treated ACR (P=.011), reduced left ventricular ejection fraction (P<.001), CAV development (P=.003), and all-cause mortality (P=.01) were all significantly more prevalent in the DSA-positive cohort.CONCLUSIONHLA-DQ donor-specific antibodies were the most common type detected and may play a significant role in poor outcomes post-cardiac transplantation. This emphasizes the importance of HLA-DQ matching and monitoring for DSA formation in order to minimize post-transplantation immunological risk.LIMITATIONSRetrospective design comes with inherent biases, results from single institute, with a particularly young cohort.

Effects of preexisting donor-specific HLA antibodies for graft failure in un-manipulated haploidentical hematopoietic stem cell transplantation

目的探讨单倍体相合造血干细胞移植中HLA供者特异性抗体（DSA）对干细胞植入的影响以及处理方法。方法采用免疫磁珠液相芯片技术，对2016年6月至2017年5月拟行单倍体相合造血干细胞移植患者进行HLA抗体及DSA的检测，对已完成移植患者进行DSA与植入失败相关性分析，检测移植前后DSA水平，探索针对DSA的处理方法。结果共检测了92例拟行单倍体相合造血干细胞移植患者的HLA抗体，其中16例（17.4%）存在HLA抗体，6例（6.5%）DSA阳性。在常规清髓性预处理单倍体相合移植中，26例DSA阴性患者中有24例成功植入，仅有2例发生植入失败，而采用常规预处理的4例DSA阳性患者中仅有1例成功植入，其余3例发生植入失败，二组患者植入率差异有统计学意义[92.3%（24/26）对25.0%（1/4），χ2=8.433，P=0.004]。多因素分析显示，DSA是影响供者干细胞植入的唯一因素[OR=12.0（95% CI 1.39~103.5），P=0.024]。6例DSA阳性的患者中，4例次在移植时采取了针对DSA的措施，均获得供者干细胞顺利植入，其中3例HLA-Ⅰ类DSA阳性患者（首次移植2例、二次移植1例）在输入供者干细胞之前输入供者血小板，另1例HLA-Ⅰ、HLA-Ⅱ类DSA并存患者在二次移植时更换供者并给予全身放疗、利妥昔单抗及供者血小板输注。结论DSA是导致单倍体相合造血干细胞植入失败的关键因素，移植前应进行常规检查，DSA阳性患者应选用DSA阴性供者；无合适供者时，应采取适当措施降低DSA水平以促进干细胞植入。

Analytical performance of a commercial multiplex Luminex-based cytokine panel in the rat

IntroductionMultiplex immunoassays are an important tool in biomarker research during preclinical drug development. However, information regarding analytical performance of commercial multiplex assays for animal species is often limited. To be able to correctly interpret study results, a fit-for-purpose validation approach is recommended. The objective of our study was to provide a realistic example of what level of validation can be expected from this type of assay, using a rat cytokine panel. MethodsThe analytical performance of a commercial Luminex-based multiplex assay comprising IFN-γ, IL-6, IL-10, IL-12p70, IP-10 and TNF-α was evaluated in Sprague-Dawley rat plasma and serum. Calibration curve, working range, precision, accuracy, selectivity, parallelism, dilutional linearity, prozone effect and sample stability were assessed. ResultsAnalytical performance in plasma and serum was comparable. Precision and accuracy results for all analytes were acceptable with coefficient of variation (CV) and relative error (RE) often below 15%, except for serum IL-6. Selectivity results varied per analyte with several cytokines showing CV>30% and no single minimum required dilution (MRD) could be identified. In addition, some striking differences between recombinant and endogenous protein results were observed. A pronounced prozone effect was detected for IP-10. Analytes in samples stored at −70°C were stable (RE<30%) from 1 up to 6months depending on the analyte. DiscussionThe results illustrate the challenges encountered during validation of commercial animal Luminex-based multiplex assays, revealing analytical limitations such as matrix and prozone effects. The Milliplex rat cytokine panel under investigation was deemed suitable for relative quantification of exploratory type biomarkers.

Nasal IL-25 predicts the response to oral corticosteroids in chronic rhinosinusitis with nasal polyps

Nasal IL-25 predicts the response to oral corticosteroids in chronic rhinosinusitis with nasal polyps

Clinical utility of C3d binding donor-specific anti-human leukocyte antigen antibody detection by single antigen beads after kidney transplantation-a retrospective study.

Development of donor-specific antibodies (DSA) after renal transplantation is known to be associated with worse graft survival, yet determining which specificities in which recipients are the most deleterious remains under investigation. This study evaluated the relationship of the complement binding capacity of post-transplant de novo anti-human leukocyte antigen (HLA) antibodies with subsequent clinical outcome. Stored sera from 265 recipients previously identified as having de novo DSA were retested for DSA and their C3d binding capacity using Luminex-based solid-phase assays. Most recipients had anti-HLA class II-reactive DSA (class I = 12.5%, class II = 68.7%, class I and class II = 18.9%). The recipients that had C3d binding DSA (67.5%) had a significantly higher incidence of antibody-mediated rejection and any rejection. They also had significantly lower kidney survival, with the lowest survival in those that had both anti-HLA class I and class II C3d binding DSA. Concurrent biopsy comparison revealed a 96.2% positive predictive value and 47.4% negative predictive value for C4d peritubular capillary (Ptc) deposition. Anti-HLA class I and class II C3d binding DSA carried a twofold and 1.5-fold increased risk of kidney loss, respectively, in multivariate analysis.

Development of an Extended-Specificity Multiplex Immunoassay for Detection of Streptococcus pneumoniae Serotype-Specific Antigen in Urine by Use of Human Monoclonal Antibodies.

Current pneumococcal vaccines cover the 10 to 23 most common serotypes of the 92 presently described. However, with the increased usage of pneumococcal-serotype-based vaccines, the risk of serotype replacement and an increase in disease caused by nonvaccine serotypes remains. Serotype surveillance of pneumococcal infections relies heavily on culture techniques, which are known to be insensitive, particularly in cases of noninvasive disease. Pneumococcal-serotype-specific urine assays offer an alternative method of serotyping for both invasive and noninvasive disease. However, the assays described previously cover mainly conjugate vaccine serotypes, give little information about circulating nonvaccine serotypes, and are currently available only in one or two specialist laboratories. Our laboratory has developed a Luminex-based extended-range antigen capture assay to detect pneumococcal-serotype-specific antigens in urine samples. The assay targets 24 distinct serotypes/serogroups plus the cell wall polysaccharide (CWP) and some cross-reactive serotypes. We report that the assay is capable of detecting all the targeted serotypes and the CWP at 0.1 ng/ml, while some serotypes are detected at concentrations as low as 0.3 pg/ml. The analytical serotype specificity was determined to be 98.4% using a panel of polysaccharide-negative urine specimens spiked with nonpneumococcal bacterial antigens. We also report clinical sensitivities of 96.2% and specificities of 89.9% established using a panel of urine specimens from patients diagnosed with community-acquired pneumonia or pneumococcal disease. This assay can be extended for testing other clinical samples and has the potential to greatly improve serotype-specific surveillance in the many cases of pneumococcal disease in which a culture is never obtained.

Antigenicity of the 2015-2016 seasonal H1N1 human influenza virus HA and NA proteins.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015–2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015–2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2–4 fold lower for the 2015–2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Comparative Assessment of Anti-HLA Antibodies Using Two Commercially Available Luminex-Based Assays.

Allospecific anti-HLA antibodies (Abs) are associated with rejection of solid organ grafts. The 2 main kits to detect anti-HLA Ab in patient serum are commercialized by Immucor and One Lambda/ThermoFisher. We sought to compare the performance of both platforms. Background-adjusted mean fluorescence intensity (MFI) values were used from both platforms to compare sera collected from 125 pretransplant and posttransplant heart and lung transplant recipients. Most HLA class I (94.5%) and HLA class II (89%) Abs with moderate to high MFI titer (≥4000) were detected by both assays. A modest correlation was observed between MFI values obtained from the 2 assays for both class I (r = 0.3, r2 = 0.09, P < 0.0001) and class II Ab (r = 0.707, r2 = 0.5, P < 0.0001). Both assays detected anti-class I and II Ab that the other did not; however, no specific HLA allele was detected preferentially by either of the 2 assays. For a limited number of discrepant sera, dilution resulted in comparable reactivity profiles between the 2 platforms. Immucor and One Lambda/ThermoFisher assays have a similar, albeit nonidentical, ability to detect anti-HLA Ab. Although the correlation between the assays was present, significant variances exist, some of which can be explained by a dilution-sensitive "prozone" effect.

The caspase-8/RIPK3 signaling axis in antigen presenting cells controls the inflammatory arthritic response

BackgroundCaspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, but maintains functions beyond cell death that involve suppression of receptor-interacting serine-threonine kinases (RIPKs). A genome-wide association study meta-analysis revealed an SNP associated with risk of rheumatoid arthritis (RA) development within the locus containing the gene encoding for caspase-8. Innate immune cells, like macrophages and dendritic cells, are gaining momentum as facilitators of autoimmune disease pathogenesis, and, in particular, RA. Therefore, we examined the involvement of caspase-8 within these antigen-presenting cell populations in the pathogenesis of an arthritis model that resembles the RA effector phase.MethodsCreLysMCasp8flox/flox and CreCD11cCasp8flox/flox mice were bred via a cross between Casp8flox/flox and CreLysM or CreCD11c mice. RIPK3–/–CreLysMCasp8flox/flox and RIPK3–/–CreCD11cCasp8flox/flox mice were generated to assess RIPK3 contribution. Mice were subjected to K/BxN serum-transfer-induced arthritis. Luminex-based assays were used to measure cytokines/chemokines. Histological analyses were utilized to examine joint damage. Mixed bone marrow chimeras were generated to assess synovial cell survival. Flow cytometric analysis was employed to characterize cellular distribution. For arthritis, differences between the groups were assessed using two-way analysis of variance (ANOVA) for repeated measurements. All other data were compared by the Mann-Whitney test.ResultsWe show that intact caspase-8 signaling maintains opposing roles in lysozyme-M- and CD11c-expressing cells in the joint; namely, caspase-8 is crucial in CD11c-expressing cells to delay arthritis induction, while caspase-8 in lysozyme M-expressing cells hinders arthritis resolution. Caspase-8 is also implicated in the maintenance of synovial tissue-resident macrophages that can limit arthritis. Global loss of RIPK3 in both caspase-8 deletion constructs causes the response to arthritis to revert back to control levels via a mechanism potentially independent of cell death. Mixed bone marrow chimeric mice demonstrate that caspase-8 deficiency does not confer preferential expansion of synovial macrophage and dendritic cell populations, nor do caspase-8-deficient synovial populations succumb to RIPK3-mediated necroptotic death.ConclusionsThese data demonstrate that caspase-8 functions in synovial antigen-presenting cells to regulate the response to inflammatory stimuli by controlling RIPK3 action, and this delicate balance maintains homeostasis within the joint.

P212 EDTA fully overcomes the prozone effect in luminex-based HLA antibody assays regardless of technical modifications

Aim Our laboratory has recently reported that serum treatment with EDTA is sufficient to overcome the prozone effect. However, it has been reported that in rare cases, specimens are partially resistant to EDTA treatment, and serum dilutions are still necessary. Although we have not encountered such case in our laboratory, we sought to determine if differences in the technical aspects of the assay may contribute to EDTA’s effectiveness. Methods We screened samples sent to our laboratory for HLA antibody testing for suspected prozone effect, based on MFI values of >15,000. Selected samples were tested in three protocols. Protocol #1: to determine the presence of prozone effect, serum samples were frozen, thawed, centrifuged (14,000 rpm for 10 min), and tested at neat, 1:4, 1:16, 1:64, and 1:264 by single antigen (SA) bead assays (One Lambda). Protocol #2: to test the clinically-validated method used by our lab, samples were frozen, thawed, centrifuged, treated with EDTA (4 mM), and tested at neat only; incubation and washing was performed in a 1 μM filter plate on a vacuum plate-washing system (Biotek ELx50). Protocol #3: to test the effect of EDTA in fresh samples with the manual washing method, fresh serum samples were centrifuged, treated with EDTA, and then tested at neat only; incubation and washing was performed in a traditional round-bottom plate. Data were acquired on a Luminex-200 and analyzed with HLAFusion software (One Lambda). Results We identified prozone effects in 18 patients’ samples: 8 patients’ class I antibodies, 8 patients’ class II antibodies, and 2 patients’ class I and II antibodies. Following EDTA treatment, fresh and frozen samples showed similar MFI values for the corresponding assignments, including 138 class I and 103 class II specificities. In addition, all determined specificities showed higher MFI values in EDTA-treated samples than the peak MFI value in sample with dilution only, regardless of which technical protocol was used. Conclusions Samples with prozone effect resistant to EDTA treatment were not detected in our study, independent of technical variability for sample processing. EDTA treatment fully overcomes prozone effect in our testing system. Factors causing low efficacy of EDTA in rare cases remain unknown.

Development and validation of a Luminex assay for detection of a predictive biomarker for PROSTVAC-VF therapy.

Cancer therapies can provide substantially improved survival in some patients while other seemingly similar patients receive little or no benefit. Strategies to identify patients likely to respond well to a given therapy could significantly improve health care outcomes by maximizing clinical benefits while reducing toxicities and adverse effects. Using a glycan microarray assay, we recently reported that pretreatment serum levels of IgM specific to blood group A trisaccharide (BG-Atri) correlate positively with overall survival of cancer patients on PROSTVAC-VF therapy. The results suggested anti-BG-Atri IgM measured prior to treatment could serve as a biomarker for identifying patients likely to benefit from PROSTVAC-VF. For continued development and clinical application of serum IgM specific to BG-Atri as a predictive biomarker, a clinical assay was needed. In this study, we developed and validated a Luminex-based clinical assay for measuring serum IgM specific to BG-Atri. IgM levels were measured with the Luminex assay and compared to levels measured using the microarray for 126 healthy individuals and 77 prostate cancer patients. This assay provided reproducible and consistent results with low %CVs, and tolerance ranges were established for the assay. IgM levels measured using the Luminex assay were found to be highly correlated to the microarray results with R values of 0.93–0.95. This assay is a Laboratory Developed Test (LDT) and is suitable for evaluating thousands of serum samples in CLIA certified laboratories that have validated the assay. In addition, the study demonstrates that discoveries made using neoglycoprotein-based microarrays can be readily migrated to a clinical assay.

Abstract 1849: The role of leptin signaling in the promotion of obesity-associated tumorigenesis and cancer stem cell characteristics in a transgenic mouse model of basal-like breast cancer

Abstract Background: Epidemiological studies have linked obesity to a greater risk of breast cancer-specific mortality. This association may be mediated in part by enhanced cancer stem cell (CSC) enrichment, as CSCs are thought to be the primary drivers of tumor initiation, growth, and metastasis. We and others have have demonstrated that obesity promotes CSC enrichment in pre-clinical models of basal-like breast cancer, but only when leptin signaling is present. These findings, coupled with evidence that elevated serum leptin and breast tumor leptin receptor expression are linked to a worse breast cancer prognosis, suggest that leptin-induced CSC enrichment may be a key contributor to obesity-associated breast cancer progression. Methods: MMTV-Wnt-1 transgenic mice, which develop spontaneous basal-like mammary tumors, were fed a control diet (10% kcal from fat) or a diet-induced obesity regimen (DIO, 60% kcal from fat). Mice were euthanized when tumors reached 1.5 cm in diameter. RNA sequencing was performed on 4 tumors/diet group. Serum hormones and cytokines were measured via luminex-based assay. ALDH activity, a putative CSC marker, was quantified by an ALDH Activity Assay Kit (abcam). Quantitative RT-PCR was used to determine expression of CSC-related genes, leptin, and the leptin receptor. Two mammary tumor cell lines isolated from the MMTV-Wnt-1 model (M-Wnt and E-Wnt cells) and human MDA-MB-231 were exposed in vitro to pooled serum from DIO or control mice (2% concentration), and cell viability, migration, invasion and expression of CSC related genes were measured. Results: Tumor incidence and growth rate were greater in DIO versus control mice. RNA sequencing revealed an upregulation in a CSC gene signature in tumors from DIO versus control mice. Significant upregulation in several of these genes, including aldh1a, oct4, twist1, twist2, and akt3, was confirmed by qRT-PCR (P &amp;lt; 0.05). ALDH activity in the DIO (relative to control) tumors was also significantly increased (P &amp;lt; 0.05). Serum levels and mammary tissue expression of leptin and tumor expression of the leptin receptor were significantly elevated in DIO versus control mice (P &amp;lt; 0.05). Exposure to serum from DIO mice significantly increased (relative to control) M-Wnt, E-Wnt, and MDA-MB-231 cell viability, migration, invasion, and expression of CSC-related genes in vitro (P &amp;lt; 0.05). The role of leptin signaling in these effects and in vitro measures of CSC enrichment will be explored through additional in experiments. Conclusions: Obesity promotes MMTV-Wnt-1 mammary tumor incidence and growth rate, possibly via CSC enrichment. Preliminary findings suggest increased leptin signaling may underlie obesity-associated CSC enrichment and may contribute to the breast cancer burden in obese women. Citation Format: Subreen A. Khatib, Laura W. Bowers, Emily L. Rossi, Shannon B. McDonell, Claire G. Lineberger, David A. Cavazos, Linda A. deGraffenried, Stephen D. Hursting. The role of leptin signaling in the promotion of obesity-associated tumorigenesis and cancer stem cell characteristics in a transgenic mouse model of basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1849. doi:10.1158/1538-7445.AM2017-1849

Multiplex detection in tonsillar tissue of all known human polyomaviruses

BackgroundIn the past few years, eleven new human viruses have joined the two previously known members JCPyV and BKPyV of the Polyomaviridae family, by virtue of molecular methods. Serology data suggest that infections with human polyomaviruses (HPyVs) occur since childhood and the viruses are widespread in the general population. However, the viral persistence sites and transmission routes are by and large unknown. Our previous studies demonstrated that the four new HPyVs – KIPyV, WUPyV, MCPyV and TSPyV – were present in the tonsils, and suggested lymphoid tissue as a persistent site of these emerging human viruses.We developed a Luminex-based multiplex assay for simultaneous detection of all 13 HPyVs known, and explored their occurrence in tonsillar tissues of children and adults mostly with tonsillitis or tonsillar hypertrophy.MethodsWe set up and validated a new Luminex-based multiplex assay by using primer pairs and probes targeting the respective HPyV viral protein 1 (VP1) genes. With this assay we tested 78 tonsillar tissues for DNAs of 13 HPyVs.ResultsThe multiplex assay allowed for simultaneous detection of 13 HPyVs with high analytical sensitivity and specificity, with detection limits of 100–102 copies per microliter, and identified correctly all 13 target sequences with no cross reactions. HPyV DNA altogether was found in 14 (17.9%) of 78 tonsils. The most prevalent HPyVs were HPyV6 (7.7%), TSPyV (3.8%) and WUPyV (3.8%). Mixed infection of two HPyVs occurred in one sample.ConclusionsThe Luminex-based HPyV multiplex assay appears highly suitable for clinical diagnostic purposes and large-scale epidemiological studies. Additional evidence was acquired that the lymphoid system plays a role in HPyV infection and persistence. Thereby, shedding from this site during reactivation might take part in transmission of the newly found HPyVs.

Development of a BNP1-32 Immunoassay That Does Not Cross-React with proBNP.

Plasma B-type natriuretic peptide (BNP) concentration reflects cardiac dysfunction and assists in determining the diagnosis and prognosis of heart failure (HF). Current BNP assays overestimate circulating bioactive BNP1-32 concentrations as they also detect less active BNP metabolites and proBNP. A specific BNP1-32 assay with negligible cross-reactivity to proBNP and/or BNP metabolites may be advantageous. We developed a Luminex-based specific BNP1-32 immunoassay and compared results obtained from 3 other BNP assays (a Luminex-based total-BNP assay, our BNP RIA, and the commercially available Abbott Architect BNP assay) in plasma from 42 patients with HF and 22 healthy controls. The BNP1-32 assay showed 57% cross-reactivity with BNP2-32, but ≤0.1% cross-reactivity to BNP3-32, other BNP metabolites, and proBNP; its detection limit was 0.35 ng/L; and intra- and interassay CVs were <15%. BNP immunoreactivity increased with HF severity (median concentrations being 0.3, 0.8, 26.2, and 17.3 ng/L in healthy controls and 40.7, 139, 465, and 1778 ng/L in HF patients for the BNP1-32, total-BNP, BNP RIA, and Abbott BNP assays respectively). The fold increase between HF cases with the New York Heart Association (NYHA) class IV was significantly greater with the BNP1-32 assay than the Abbott BNP (P = 0.026) and the BNP RIA (P < 0.0001) but not the total-BNP assay. We have developed the first assay that measures BNP1-32 in plasma without interference by proBNP. Analysis of larger patient cohorts is now required to compare the performance of this assay with current less specific assays for the diagnosis or prognosis of HF.

The Impact of Donor-Specific Anti-Human Leukocyte Antigen (HLA) Antibody Rebound on the Risk of Antibody Mediated Rejection in Sensitized Kidney Transplant Recipients.

BACKGROUND Donor-specific anti-HLA antibody (DSA) detected on Luminex-based single antigen assay (LSA) has become the subject of desensitization based upon the results of previous studies. We retrospectively investigated the impact of preoperative DSA on the incidence of antibody mediated rejection (AMR) in patients desensitized using a protocol based on rituximab and rabbit antithymocyte globulin (rATG). MATERIAL AND METHODS Nine patients (Group 1, 9/327, 2.8%) were complement dependent cytotoxicity crossmatch (CDC-XM) positive and underwent desensitization with rituximab (375 mg/m²), intravenous immunoglobulin (IVIG; 400 mg/kg), plasmapheresis, and rATG. Twenty-two patients (Group 2, 22/327, 6.7%) were CDC-XM negative but DSA positive on LSA and had received desensitization with rituximab and rATG, while 55 patients (Group 3, 55/327, 16.8%) were CDC-XM and DSA negative with a calculated panel reactive antibody (cPRA) ≥50%. Another 241 patients (Group 4, 241/327, 73.7%) were CDC-XM and DSA negative with a cPRA <50%. RESULTS Recipients with DSA (Group 2) experienced more AMR than other groups (p<0.01). More de novo DSAs also developed in Group 2 (p<0.001). The mean fluorescence intensity (MFI) of DSA of patients with AMR tended to rebound (p=0.01). CONCLUSIONS Patients who were CDC-XM negative but DSA positive status were at a higher risk of developing AMR even though they had received desensitization with rATG and rituximab. A more intense desensitization protocol is needed for these recipients. Patients with MFI rebound of DSA should be carefully monitored for the risk of AMR.

Human platelet antigen antibody induction in uncomplicated pregnancy is associated with HLA sensitization.

Alloimmunization against human platelet antigens (HPAs) during pregnancy is rare but can lead to severe bleeding disorders, such as fetal and neonatal alloimmune thrombocytopenia. In a cohort of 241 uncomplicated pregnancies, we investigated the immunogenicity of HPA mismatches and correlated HLA sensitization with HPA antibody formation. HPA antibodies were measured with a Luminex-based multiplex assay. HPA mismatches were observed in 109 of 241 pregnancies (45%), but child-specific HPA antibodies were only found in two of 109 cases (2%), indicating a low immunogenicity. Only nine of 241 women (4%) had detectable HPA antibodies. HLA sensitization was identified as a strong and independent predictor for HPA antibody formation (hazard ratio, 10.2; 95% confidence interval, 1.8-193; p = 0.006), whereas the number of pregnancies was not. Our observational data indicated a low immunogenicity of HPA and suggest that a broader immune response-inferred by HLA sensitization-is probably associated with HPA antibody induction.

High-risk human papillomavirus seroprevalence in men and women of six different ethnicities in Amsterdam, the Netherlands: The HELIUS study

BackgroundEthnic variations in the (sero)prevalence of Human Papillomavirus (HPV) and HPV related diseases have been observed previously. We explored if high-risk HPV (hrHPV) seropositivity indeed differs among 6 ethnic groups in Amsterdam the Netherlands and assessed if hrHPV seroprevalence is higher among women than men within each ethnic group, both after adjustment for confounders. MethodsFrom the multi-ethnic HEalthy Life In an Urban Setting (HELIUS) study in Amsterdam (the Netherlands) we randomly selected 4637 men and women aged 18–44 years with a Dutch, South-Asian Surinamese, African Surinamese, Ghanaian, Moroccan, or Turkish ethnicity. Blood samples were tested for HPV-16,−18,−31,−33,−45,−52, and −58 antibodies using a validated Luminex-based multiplex serology assay. We assessed the association of both ethnicity and gender with hrHPV seropositivity using logistic regression models with generalised estimating equations. ResultsThe hrHPV seroprevalence in Dutch, South-Asian Surinamese, African Surinamese, Ghanaian, Moroccan, and Turkish participants was 18%, 12%, 23%, 19%, 17%, and 15% in men, and 30%, 22%, 34%, 31%, 14%, and 15% in women, respectively. HrHPV seroprevalence of non-Dutch men did not differ significantly from Dutch men. HrHPV seroprevalence was significantly higher among African Surinamese women, and significantly lower among Moroccan and Turkish women when compared to Dutch women. These differences were not significant anymore after adjustment for demographic, health, and sexual behavioural differences between ethnicities. HrHPV seroprevalence varied by age, age of sexual debut, and lifetime sexual partners among women but not among men. Seroprevalence of hrHPV was higher among women than among men, except in the Turkish group. ConclusionAmong women hrHPV seroprevalence differed by ethnicity, yet among men no pronounced differences were observed across ethnicities.

Minimizing the risk of allo-sensitization to optimize the benefit of allogeneic cardiac-derived stem/progenitor cells

Allogeneic human cardiac-derived stem/progenitor cells (hCPC) are currently under clinical investigation for cardiac repair. While cellular immune response against allogeneic hCPC could be part of their beneficial-paracrine effects, their humoral immune response remains largely unexplored. Donor-specific HLA antibodies (DSA-HLA-I/DSA-HLA-II), primary elements of antibody-mediated allograft injury, might present an unidentified risk to allogeneic hCPC therapy. Here we established that the binding strength of anti-HLA monoclonal antibodies delineates hCPC proneness to antibody-mediated injury. In vitro modeling of clinical setting demonstrated that specific DSA-HLA-I of high/intermediate binding strength are harmful for hCPC whereas DSA-HLA-II are benign. Furthermore, the Luminex-based solid-phase assays are suitable to predict the DSA-HLA risk to therapeutic hCPC. Our data indicate that screening patient sera for the presence of HLA antibodies is important to provide an immune-educated choice of allogeneic therapeutic cells, minimize the risk of precipitous elimination and promote the allogeneic reparative effects.