Mean Fluorescence Intensity Values Research Articles

Molecular characterization of a cathepsin L1 highly expressed in phagocytes of pacific oyster Crassostrea gigas

Cathepsin L1 (CTSL1) is a lysosomal cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of immune response against pathogen infection based on the proteolytic activity. In the present study, a CTSL1 homologue (designated as CgCTSL1) was identified from Crassostrea gigas. It contained a typically single Pept_C1 domain with three conserved catalytically essential residues (Gln25, His135 and Asn178). The mRNA of CgCTSL1 was ubiquitously expressed in oyster tissues with the highest expression level in important immune tissues such as gill and hemocytes. CgCTSL1 proteins were mainly detected in gill and hepatopancreas by immunohistochemistry. Recombinant CgCTSL1 (rCgCTSL1) exhibited proteolytic activity to cleave the substrate Ac-FR-amino-4-trifluoromethyl coumarin (AFC) in a dose-dependent manner, and the inhibitor could reduce its proteolytic activity. After the interference of CgCTSL1 mRNA, the proteolytic activity of oyster hemocytes was significantly down-regulated with the released AFC fluorescence value decreasing from 375.84 to 179.21 (p < 0.05). Flow cytometry analysis revealed that the expression of CgCTSL1 protein was higher in phagocytes with the mean fluorescence intensity (MFI) value of 21,187 (4.13-fold, p < 0.01) compared to the MFI value of 5,130 in non-phagocytic hemocytes. The further confocal analysis demonstrated that the actively phagocytic hemocytes with green bead signals were co-localized with stronger CgCTSL1 positive signals. The mRNA expression levels of CgCTSL1 in phagocyte-like sub-populations of granulocytes and semi-granulocytes were 298.12-fold (p < 0.01) and 2.75-fold (p < 0.01) of that in agranulocytes, respectively. Western blotting analysis of the hemocyte proteins revealed that CgCTSL1 was relatively abundant in granulocytes and semi-granulocytes compared to that in agranulocytes. These results collectively suggested that CgCTSL1, a CTSL1 homologue highly expressed in phagocyte-like hemocytes, was possibly involved in cellular immune response dependent on its conserved proteolytic activity, which might provide clues for the divergence between phagocytes and non-phagocytic hemocytes as well as the identification of promising molecular markers for phagocytes in oyster C. gigas.

Repeated Freezing and Thawing Does Not Affect HLA Antibody Titer

Introduction For deceased donor organ transplantations, we are not currently using virtual crossmatches (XMs) and preliminary negative T cell XMs are mandatory for kidney and pancreas allocation in Korea. Korean Network for Organ Sharing (KONOS) distributes transplant candidates’ sera to 21 OPO laboratories and KONOS preliminary XMs are performed in these laboratories using frozen stored sera. We are performing more than 40% of nationwide KONOS XMs in the Korea Organ Donation Agency (KODA) laboratory, and we had difficulties in keeping the frozen sera from more than 20,000 transplant candidates in small aliquots to avoid HLA antibody deterioration. We investigated the extent of change of antibody titer after repeated freezing and thawing. Materials and Methods Two different sera (serum A and B), which were tested positive for both class I and class II HLA antibodies were selected for the investigation. Sodium azide (0.1% in final concentration) was added to the sera as preservative, and freezing-thawing cycles were repeated for 20 times. A total of 7 serum samples (1, 3, 6, 9, 12, 15, and 20 times frozen-thawed; labeled #1, #3, #6, #9, #12, #15, and #20) were used for HLA antibody screening test (One Lambda, USA), and 3 samples (1, 9, and 20 times frozen-thawed) were used for HLA antibody single antigen assay (One Lambda, USA) and T cell flow cytometry XM (FCXM) test. To avoid prozone phenomenon, 1:1 (neat), 1:5, and 1:20 diluted sera were tested by screening assay and 1:20 sera for both serum A and B were used for the investigation. HLA antibody strength in mean fluorescence intensity (MFI) values and FCXM result in MFI ratios were compared. Test results of #20 sample compared to #1 sample were calculated as percentage values and variabilities of the test results among different samples of repeated freezing-thawing were calculated as coefficient of variation (CV) values. Results and Discussion The results are summarized in Table 1. There was little change in MFI values after 20 times of repeated freezing-thawing cycles: percentage of #20 vs # 1 sample as baseline (100%) was in the range of 99-110% for screening assay and 90-136% for single antigen assay. For serum A, change of MFI values of 5 class I and 5 class II antibodies of the #1, #9 and #20 samples are shown (Fig. 1 and Fig. 2). Variability of the test results among different samples of repeated freezing-thawing was low, and most of the CV values (7 out of 8) were <10% (2.2-6.9%). T cell FCXM results (3 XMs) also showed little change in MFI ratios (103-107%) and low CV values (4.2-6.7%). Unlike the notion that antibodies are deteriorated after repeated freezing-thawing, the strength of HLA antibodies was little affected after 20 times of freezing-thawing cycles.Conclusion HLA antibody strength is little affected by up to 20 times of repeated freezing and thawing. For preliminary HLA XM tests for deceased donor organ transplantation, serum samples from transplant candidates can be stored frozen without aliquoting.

Regulatory T cells as a biomarker for response to adalimumab in rheumatoid arthritis

Regulatory T cells as a biomarker for response to adalimumab in rheumatoid arthritis

Longitudinal change in the serology of antibodies to Chlamydia trachomatis pgp3 in children residing in a trachoma area

A serologic test for antibodies to chlamydial antigen pgp3 may be a useful tool for trachoma surveillance. However, little is known about the stability of antibody status over time, or factors associated with seroreversion/conversion. A cohort of 2,111 children ages 1–9 years in Tanzania were followed for one year in the absence of mass azithromycin. At baseline and follow-up, they were evaluated for trachoma, chlamydial infection, and antibodies to chlamydial antigen pgp3. At baseline, 31% of children were seropositive for pgp3 antibodies and 6.4% seroreverted to negative over one year. Of those seronegative, 9.8% seroconverted over the year. The seroreverters had lower baseline mean fluorescence intensity (MFI-BG) values compared to the seropositives who remained positive (Odds Ratio = 0.04 for every unit increase in log10MFI-BG, 95% CI = 0.02–0.09), and were more likely to live in communities with trachoma <5% (p < 0.008). While seroconversion was expected, seroreversion was unexpected. The low seroprevalence rate reported from low endemic areas may be due to seroreversion as well as lack of exposure.

Quantitative assessment of informative immunophenotypic markers increases the diagnostic value of immunophenotyping in mature CD5-positive B-cell neoplasms.

The data on the clinical utility of the quantitative assessment of immunophenotypes in distinguishing mature CD5-positive B-cell neoplasms is limited. The study aim was to assess the diagnostic value of the quantitative assessment of a panel of 18 markers and to identify the most informative ones. The immunophenotype of the neoplastic population was determined in diagnostic specimens from 188 patients. BD FACSCanto II flow cytometer and FACSDiva software were used to analyze the positivity/negativity and mean fluorescence intensity (MFI) of the surface expression of 18 markers. Advanced data mining methods were used to define the key differential diagnostic features of CLL/SLL (chronic lymphocytic leukemia/small lymphocytic lymphoma), MCL (mantle cell lymphoma), and CD5+ MZL (marginal zone lymphoma). The most informative markers for the distinction of CLL/SLL, MCL, CD5+ MZL, including atypical cases, were the MFI values of CD79b, CD20, CD23, CD43, CD38, CD11c, FMC7, CD200, kappa light chain, and their combinations. CD23 and CD200 were the most discriminant between CLL/SLL and MCL and CD23 plus CD79b between CLL/SLL and CD5+ MZL. The quantitative analysis of the most informative markers failed to accurately distinguish MCL and CD5+ MZL. The study highlights the data mining methods for the analysis and selection of the most informative immunophenotypic markers and for the design of a predictive model (diagnostic classifier), minimizing the subjectivity of expert-based assessment. Our data confirmed that the quantification of the expression of informative markers increases the diagnostic value of immunophenotyping in mature CD5+ B-cell neoplasms. © 2017 International Clinical Cytometry Society.

Clinical Utility of Complement Dependent Assays in Kidney Transplantation.

Formation of antibodies against polymorphic HLA molecules on donor endothelium is central to the pathogenesis of antibody-mediated rejection, the dominant cause of long-term kidney allograft loss. Although introduction of the single-antigen bead assay has greatly facilitated the immune risk assessment of transplant recipients, it is recognized that not all IgG HLA antibodies detected using this method are equally relevant. In recent years, novel assays (C4d, C1q, C3d) have been developed to interrogate the complement-activating potential of anti-HLA antibodies in vitro, with the hypothesis that complement-fixing antibodies are more immediately injurious to the graft compared with noncomplement-binding antibodies. Although initial studies demonstrated the potential of these assays to risk-stratify antibodies beyond the conventional limited metric of mean fluorescence intensity values, new data from recent analyses challenge some of these early findings. In this review, we examine the technical aspects of these assays and key studies that evaluated the discriminant capacity of these tests to predict numerous outcomes in kidney transplantation. We discuss conflicting data and emerging controversies in the context of recent experimental evidence which offer new insights into the major factors that influence complement activation. Finally, we provide our perspective on the current role and utility of complement diagnostic assays as 1 variable in the multifactorial risk assessment and management of kidney transplant recipients.

35 Interspecies Somatic Cell Nuclear Transfer Embryos that Form Nucleoli do not Always Activate Mitochondrial Functional Differentiation at the Time of Embryonic Genome Activation

Embryonic genome activation (EGA) is a complex process that needs a good orchestration of all biochemical processes at the time of maternal-to-embryonic transition. Mitochondria are strictly dependent on the nucleus for their correct activity as ~1500 mitochondrial genes have nuclear localisation. The finding of transcriptional activation and accumulation of mRNAs related to mitochondrial biogenesis (Mtango et al. 2008 Reprod. Fertil. Dev. 20, 846-859) around the time of EGA confirmed the role of nucleus in this process. Studying mitochondria behaviour in interspecies somatic cell nuclear transfer (iSCNT) embryos (Lagutina et al. 2010 Reproduction 140, 273-285), we have found that at the time of EGA, mitochondria activation could be demonstrated by JC-1 accumulation. We suggested that comparison of the mean green fluorescence intensity (FI) that corresponds to the fluorescence of the monomeric form of the dye and correlates to relative mitochondrial mass (Mancini et al. 1997) in iSCNT and control nuclear transfer (NT) could serve as a test to assess EGA in iSCNT embryos. The aim of this study was to estimate nuclear-cytoplasmic interaction in iSCNT embryos that formed nucleoli at the time of EGA (Lagutina et al. 2011 Reproduction 141, 453-465) such as embryos derived from bovine oocytes and bovine (control), buffalo or ovine donor nuclei, and from porcine oocytes and porcine (control), horse, or rabbit nuclei. Embryos 72 h after activation were stained with 2 μM JC-1 in SOF-HEPES with 10% FCS at 37°C for 1 h. Images were collected using a fluorescein isothiocyanate (FITC) filter and analysed with Adobe Photoshop Elements 2 (Adobe Systems, San Jose, CA, USA). The data are presented as mean FI of the embryo. To demonstrate the effect of EGA inhibition on mitochondria, bovine and porcine NT embryos were cultured in medium supplemented with 25 μg/mL α-amanitin (AA) from 48 to 72 h after activation. The analyses of mean FI of the embryos showed that ovine and buffalo nuclei were able to support mitochondrial mass accumulation in iSCNT embryos with bovine oocytes equal to control bovine NT embryos (35 ± 11.2; 41.9 ± 14.8; 36.2 ± 7.6, respectively) that was significantly higher than in bovine embryos treated with AA (15.4 ± 4.9; P &lt; 0.05). In the iSCNT embryos composed of porcine oocytes and equine or rabbit nuclei, mean FI values (20 ± 13.4; 18.3 ± 5.5, respectively) were comparable with those in porcine NT embryos treated with AA (16.2 ± 6.2), and were significantly lower than in porcine control (91 ± 47.7; P &lt; 0.05) NT embryos, demonstrating the inability of equine and rabbit nuclei to properly govern the porcine mitochondria mass growth at the time of EGA. In conclusion, nucleolus formation and activation of nuclear encoded mitochondrial genes at the time of EGA cannot serve, per se, as a marker of correct embryonic genome activation in iSCNT embryos, because, in our conditions, no iSCNT embryos developed to blastocyst (Lagutina et al. 2010 Reproduction 140, 273-285). This knowledge about behaviour of different embryo compartments at the time of EGA could extend our understanding of the whole process. This work was funded by Translink (EU FP7 no. 603049) and Xenoislet projects (EU FP7 no. 601827).

CD26 expression is down-regulated on CD8+ T cells in patients with Hashimoto's thyroiditis

The immune mechanism underlying Hashimoto's thyroiditis (HT) remains unclear. CD26, also known as dipeptidyl peptidase 4 (DPP-4), is a multifunctional molecule involved in the pathophysiology of autoimmune diseases. This study aimed to investigate the role of CD26 in the pathogenesis of HT. Peripheral blood was drawn from 20 healthy controls and 31 HT patients (19 mild HT patients and 12 severe HT patients). Plasma sCD26 concentrations were measured by ELISA, and sCD26 enzymatic activity was assessed using a luciferase-based assay. The expression levels of membrane-bound CD26 were analyzed by flow cytometry. Plasma sCD26 concentrations were lower in HT patients than in healthy controls, although the difference in sCD26 concentrations between the two groups did not reach statistical significance (P=0.07). The percentages of CD8+ T cells and Tc1 cells with CD26 expression were decreased in HT patients compared with those in healthy controls, and the mean fluorescence intensity (MFI) values of CD26 on CD8+ T cells and Tc17 cells in HT patients were significantly lower than in healthy controls (P<0.05). In HT patients, the expression of CD26 on CD8+ T cells and Tc subsets was decreased in the hypothyroidism group compared with that in the euthyroid group (P<0.05). These results suggest that the sCD26 concentrations and membrane-bound CD26 levels on CD8+ T cells are aberrant in HT and that the reduced CD26 expression may be involved in the progression of HT.

Cancer-related mRNA expression analysis using a novel flow cytometry-based assay.

Cancer-related gene expression data mostly originate from unfractionated bulk samples, leading to "expression averaging" of heterogeneous populations. Multicolor flow cytometry (FCM) may distinguish heterogeneous populations based on the phenotypic characterization of single-cells, but is not applicable for RNA targets. Here, we evaluated the PrimeFlow™ RNA assay, a novel FCM-based assay designed to measure gene expressions, in two cancer entities with high and low RNA target levels. Neuroblastoma (NB) cell lines were studied for MYCN gene expression by PrimeFlow™ and compared with the gold standard, RT-qPCR. Dilution series of NB cells (0.10-11%) were prepared to evaluate performance in small cell populations. Diagnostic material of de novo acute myeloid leukemia (AML) patients was used to measure Wilms' tumor 1 (WT1) expression in bulk leukemic cells and rare subsets, e.g. leukemic stem cells (LSCs). FCM analysis was performed on a FACSCanto II (BD Biosciences) using Infinicyt™ (Cytognos® ) for data analysis. mRNA expression was reported by normalized mean fluorescence intensity (MFI) values and staining indices. MYCN mRNA quantified by PrimeFlow™ significantly correlated with RT-qPCR and remained detectable in small (0.1%) populations. Using PrimeFlowTM , WT1 levels were shown to be significantly higher in AML patient samples with WT1 overexpression, previously defined by RT-qPCR. Moreover, WT1 overexpression was distinguishable between heterogeneous cell populations and remained measurable in rare LSCs. PrimeFlow™ is a sensitive technique to investigate mRNA expressions, with high concordance to RT-qPCR. High (MYCN) and subtle (WT1) overexpressed mRNA targets can be quantified in heterogeneous and rare subpopulations e.g. LSCs. © 2017 International Clinical Cytometry Society.

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.

Clinical risk stratification of paediatric renal transplant recipients using C1q and C3d fixing of de novo donor-specific antibodies

IntroductionWe have previously shown that children who developed de novo donor-specific human leukocyte antigen (HLA) antibodies (DSA) had greater decline in allograft function. We hypothesised that patients with complement-activating DSA would have poorer renal allograft outcomes.MethodsA total of 75 children developed DSA in the original study. The first positive DSA sample was subsequently tested for C1q and C3d fixing. The primary event was defined as 50% reduction from baseline estimated glomerular filtration rate and was analysed using the Kaplan–Meier estimator.ResultsOf 65 patients tested, 32 (49%) and 23 (35%) tested positive for C1q and C3d fixing, respectively. Of the 32 C1q-positive (c1q+) patients, 13 (41%) did not show concomitant C3d fixing. The mean fluorescence intensity values of the original immunoglobulin G DSA correlated poorly with complement-fixing positivity (C1q: adjusted R2 0.072; C3d: adjusted R2 0.11; p < 0.05). C1q+ antibodies were associated with acute tubulitis [0.75 ± 0.18 (C1q+) vs. 0.25 ± 0.08 (C1q−) episodes per patient (mean ± standard error of the mean; p < 0.05] but not with worse long-term renal allograft dysfunction (median time to primary event 5.9 (C1q+) vs. 6.4 (C1q−) years; hazard ratio (HR) 0.74; 95% confidence ratio (CI) 0.30–1.81; p = 0.58]. C3d-positive (C3d+) antibodies were associated with positive C4d histological staining [47% (C3d+) vs. 20% (C3d−); p = 0.04] and with significantly worse long-term allograft dysfunction [median time to primary event: 5.6 (C3d+) vs. 6.5 (C3d−) years; HR 0.38; 95% CI 0.15–0.97; p = 0.04].ConclusionAssessment of C3d fixing as part of prospective HLA monitoring can potentially aid stratification of patients at the highest risk of long-term renal allograft dysfunction.

A bead-assisted flow cytometry method for the semi-quantitative analysis of Extracellular Vesicles

Most experimental approaches commonly employed for the characterization and quantitation of EVs are time consuming, require of specialized instrumentation and often are rather inaccurate. To circumvent the caveats imposed by EV small size, we used general and specific membrane markers in bead assisted flow cytometry, to provide a semi-quantitative measure of EV content in a given sample. EVs were isolated from in vitro cultured cells-conditioned medium and biological fluids by size exclusion chromatography and coupled to latex beads to allow their detection by standard flow cytometers. Our analyses demonstrate a linear correlation between EV concentration and Mean Fluorescence Intensity values in samples cleared of protein contaminants. Comparison with one of the most widespread method such as NTA, suggests a similar linear range and reliable accuracy to detect saturation. However, although detection of the different biomarkers is feasible when tested on ultracentrifugation-enriched samples, protein contamination impairs quantitation of this type of samples by bead-based flow cytometry. Thus, we provide evidence that bead-assisted flow cytometry method is an accurate and reliable method for the semiquantitative bulk analysis of EVs, which could be easily implemented in most laboratories.

P153 Utility of the virtual crossmatch for living donor evaluations

Aim Our institution performs 750–900 flow cytometric crossmatches (FCXM) annually in support of living-donor renal transplantation. The FCXM is costly, time consuming, and has interpretational conundrums (i.e., FCXM + donor specific antibody (DSA) negative or FCXM−/DSA+). By combining high resolution HLA typing with solid-phase antibody (SAB) testing, recipients should be transplantable based solely on a virtual crossmatch (vXM). Given the increased utilization of the vXM, our aim was to assess its utility in living donor evaluations. Methods We reviewed 100 living donor FCXMs and retrospectively performed a vXM for each pair. vXM was performed only on cases with available donor typing and SAB specificity testing. Additionally, ΔMESF values of the FCXM, FlowPRA results, and mean fluorescence intensity (MFI) values of DSA were analyzed. Results Initially, there was a 75% correlation between FCXM and vXM: 60 cases were negative for both FCXM and vXM; 15 cases were positive for both. However, 20 cases were FCXM+ (mostly B-cell) but vXM−, which tended to occur in non-alloimmunized recipients. 3/20 had autoimmune disease and 8/20 were transplanted without development of DSA. Although deemed compatible, the remaining 12 were not transplanted. These 20 cases were considered False Positive FCXMs. 5 cases were FCXM−/vXM+. Of these, 4 had Class I DSAs and 1 had both Class I and II DSAs; however, all 5 had low MFI values. (Table 1). Thus, reclassifying the false positive FCXMs, the final correlation was 95%. Download : Download high-res image (146KB) Download : Download full-size image Conclusions Though the initial FCXM/vXM correlation rate was only 75%, there are key characteristics such as differences in FCXM, ΔMESF, PRA values, and DSA MFI values that can help interpret discrepant result. Notably, there were 4 times more false positive FCXMs then false positive vXMs. Reclassifying the False Positive FCXM cases increased the correlation to 95%. These data support the utility of the vXM as a replacement for a physical FCXM for most living donor evaluations.

P159 Comparison of single antigen bead and platelet crossmatch assays

Aim Satisfactory increments may be achieved in refractory patients with platelets that are antigen negative for corresponding HLA antibody. However, no HLA antibody test or cutoff is universally used. Our small study sought to identify an appropriate single antigen bead assay (SAB) and Mean Fluorescent Intensity (MFI) cutoff to select HLA mismatched platelets in lieu of platelet crossmatch (PLT XM). Methods HLA sensitized patient sera (n = 17; CPRA 24–100%, median 93%) were tested by One Lambda™ Class I IgG-SAB and C1q-SAB and Immucor™ C3d-SAB, and PLT XM against 8 Class I typed platelets. Cumulative MFI for donor specific antibodies (DSA) in 36 patient-donor pairs were calculated. The vendor’s recommended cutoff for C3d-SAB, 500 MFI for C1q-SAB and 3000, 5000 and 10,000 MFI for IgG-SAB were used to assign an expected result to compare with the observed PLT XM result. MFI values from IgG-SAB negative, PLT XM positive (presumed false negative) pairs were replaced with MFI values from 1:10 diluted serum to correct for complement interference (Mod IgG-SAB). Results were stratified by MFI and sensitivity and specificity were calculated. Results No SAB was 100% concordant with PLT XM. Two positive PLT XMs were likely due to ABO incompatibility. Two negative PLT XMs occurred in the presence of moderately strong B44 DSA. C1q-SAB compares favorably with PLT XM. C3d-SAB has the highest specificity, but lowest sensitivity. IgG- and Mod IgG-SAB (3000 MFI) have excellent sensitivity but poor specificity. When Mod IgG-SAB cutoff is raised to 5000 MFI or 10,000 MFI, specificity increases but sensitivity decreases. Mod IgG-SAB (5000 MFI) sensitivity is comparable to C1q-SAB and specificity is improved when B44 DSA Conclusions C1q-SAB is reported to correlate with acceptable platelet increments and correlates well with PLT XM in our study. Mod IgG-SAB (5000 MFI) that ignores anti-B44 DSA

P269 Prevalence of HLA antibodies in kidney transplanted patients

Aim The prevalence and the strength of HLA antibodies were analyzed in kidney transplanted patients in Siriraj Hospital Bangkok, Thailand. Methods From 2008–2016, post transplanted serum samples from 185 patients were tested for HLA antibodies by Luminex single antigen beads(One Lambda). class I antibodies were found in 82 patients and class II antibodies were found in 98 patients. The donors were typed for HLA-A, -B, DR and DQ and DSA were determined. The strength of antibodies were determined by mean fluorescence intensity (MFI) values of the beads. Results The frequencies of HLA antibodies were similar for HLA-A(69.5%), HLA-B(78%), HLA-DR(66.3%), HLA-DQ(65.3%), but lower for HLA-C(17%), HLA-DP(13.3%). The frequencies for DSA was found higher for HLA-DQ(44.9%) than HLA-A (21.9%), HLA-B(12.2%) and HLA-DR(14.3%). The median MFI values of antibodies were similar for HLA-A(5,143), HLA-B(5,866), HLA-DR(5,279), whereas the values were lower for HLA-C(4,446) and were higher for HLA-DQ(8,842) and HLA-DP(8,920). In addition, the frequencies of patients with MFI >5000 were found higher for HLA-DQ(76.6%) than HLA-DP(69.2%), HLA-A(68.4%), HLA-B(65.6%), HLA-DR(55.4%) and HLA-C(35.7%) Conclusions The incidence of anti-HLA-C and DP were lower compared to anti-HLA-A, B, DR and DQ, however the strength of anti-HLA-DP was high and similar to anti-HLA-DQ.

P155 Performing a virtual crossmatch: A semi-automated approach

Aim The aim of this method is to obtain clear, fast, and accurate virtual crossmatch results which enhance communication with our clinical programs. Methods Virtual crossmatching is essential for heart and lung transplantation due to the need for short ischemia times and is increasingly used in kidney transplantation to facilitate organ allocation. There are currently no reliable tools for performing a virtual crossmatch. Our previous method was to manually assess the patient’s antibody profile and compare to the HLA typing of the potential donor. For patients with extensive history, this process was untimely and cumbersome. Our initial improvement was to export all Luminex Single Antigen (LSA) Bead results into a Microsoft Excel file, which was then manually sorted to generate a report with patient and donor HLA typings as well as current sample and historical peak mean fluorescent intensity values (MFIs) for donor specific antibodies (DSA). Most recently, we developed a semi-automated approach by creating a Microsoft Excel Workbook with a specifically written macro that sorts the exported data automatically and calculates cumulative MFI values. Results Once the HLA typing of the potential donor is entered into the workbook and the LSA beads are chosen, the MFI values will be automatically pulled into the current and historical tables. These values are then calculated automatically to obtain a current and historical cumulative MFI value. The sample dates of the peak historical MFI are also included (Fig. 1). The new approach has been validated and implemented into clinical practice. Conclusions We found the semi-automated workbook to be a reliable tool which reduces clerical errors, saves time, provides clear data interpretation, and can be easily shared with our transplant programs. In addition, the workbook includes an optional graph of DSA MFIs over time. This additional functionality provides a straightforward antibody assessment in relation to the potential donor. Download : Download high-res image (356KB) Download : Download full-size image

Quantitative Evaluation of the Impact of Ethylenediaminetetraacetic Acid Pretreatment on Single-Antigen Bead Assay.

BackgroundEthylenediaminetetraacetic acid (EDTA) pretreatment has been shown to overcome complement interference in the single-antigen bead (SAB) assay. However, a quantitative evaluation of its impact on the assay for preemptive application to diverse clinical samples is still lacking.MethodsSerum samples from 95 renal transplant candidates were tested with and without EDTA-pretreatment in parallel. Changes in mean fluorescence intensity (MFI) values were analyzed to determine the impact of EDTA-pretreatment and the characteristics of complement interference.ResultsMFI values from EDTA-treated and untreated sera showed good correlations (r = 0.99) and were linear after excluding outliers (slopes, 1; intercepts, −63.7 and −24.2 for class I and II, respectively). Using an assay cutoff of 2000 MFI, positive/negative assignments were concordant for 99% of the 9215 class I beads and 9025 class II beads tested. As defined by an MFI increment above 4000 after EDTA pretreatment, complement interference affected 172 class I beads in 12 samples (12.6%) and 60 class II beads in 7 samples (7.4%), and the findings were supported in 83% and 86% of these samples by dilution studies. In a case study, EDTA pretreatment prevented falsely low MFI values and facilitated the interpretation of titration curves. Finally, EDTA pretreatment reduced the coefficient of variance (CV) by 2.1% and 2.4% for class I and II beads respectively (P < 0.0001).ConclusionsIt is safe to preemptively treat all clinical samples with EDTA before SAB assay to prevent false negative results or falsely low MFI values. EDTA pretreatment has the added benefit of improved assay precision.

A model of acute antibody mediated rejection in mouse renal transplantation

Objective To establish a model of acute antibody mediated rejection (AMR) in mouse kidney transplantation. Methods 6-8 weeks old male C3H (H2k) and BALB/c (H2d) mice were used as skin or kidney graft donors and recipients, respectively. All animals were provided by Beijing vital river experimental animal technical limited company. Recipient animals were divided into 2 groups. AMR group, recipient animals were presensitized by transplanting skin grafts before one week of kidney transplantation (n=10). Cell mediated rejection (CMR) group, recipient animals received kidney grafts without being presensitized by skin transplantation (n=10). Normal balb/c mice acted as control (n=3). Animal survival time was recorded and renal allograft function of creatinine (Cr) and blood urea nitrogen (BUN) was monitored. On the fifth day after kidney transplantation, recipient animals were sacrificed, analysis of renal graft histology based on Banff 2013 for rejection diagnosis, immunohistochemistry for mouse IgG, complement fragment C3d deposition detection intragraft, and flow cytometry for circulating donor specific antibody (DSA)-IgG, IgM detection were performed. Results The mean survival time of AMR group was (4.4±1.1) d and longer than 60 days for CMR goup. Graft function of serum Cr [ (415.6±25.6) μmol/L] and BUN [ (2 394.0±94.0) pmol/L] were remarkably increased on postoperative 2 d in AMR group. CMR control group showed normal serum levels of Cr [ (77.8±7.2) μmol/L] and BUN [ (1 343.0±233.9) pmol/L] on 60 days after kidney transplantation. On 5 d after kidney transplantation, the macroscopic morphology of renal grafts in AMR group was significantly swelling while grafts in CMR group were normal. The circulating IgG DSA level in AMR group was remarkably higher than CMR group with mean fluorescence intensity (MFI) values 223.0±29.0 vs. 134.5±39.6. The serum IgM DSA level did not change significantly with MFI values 9.2±0.1 vs. 10.0±0.7. Grafts in AMR group demonstrated dilation of peritubular capillaries (PTC), peritubular capillaritis, glomerulitis, glomerular/tubular necrosis and interstitial inflammation, hemorrhage, and edema. Extensively deposition of IgG and C3d intragraft were also observed in AMR group, while mild changes presented in CMR group which showed no significant difference from the normal control BALB/c mice. Conclusion We successfully established a model of acute AMR in C3H→BALB/c mouse kidney transplantation by presensitizing recipient mice using donor-strain derived skin transplantation at 1 week prior to kidney transplantation, which showed significantly different from non-presensitized recipient mice. Key words: Kidney transplantation; Antibody mediated rejection; Model, animal; Mice

Replicon Particle Expressing the E2 Glycoprotein of Bovine Viral Diarrhea Virus Immunization and Evaluation of Antibody Response.

The objective of this study was to develop a new antigen delivery system using an alphavirus replicon particle (RP) to induce humoral antibody responses against bovine viral diarrhea virus (BVDV) recombinant antigen produced from envelope glycoprotein E2. An alphavirus RP expressing the E2 glycoprotein of BVDV was used for immunization of pigs. A fluorescent microsphere immunoassay (FMIA) has been applied to detect BVDV E2 antigen-specific antibody isotype in pig immunized with alphavirus RP. Full-length BVDV E2 (aa 1-375) was cleaved into several pieces, eight E2 DNA fragments, including full-length DNA, were cloned into expression vector pHUE, and the recombinant proteins expressed in BL-21 (DE3) Escherichia coli. After successful conjugation of purified proteins with microsphere beads, a multiplex FMIA platform was constructed, and BVDV E2 alphavirus-based RP-immunized animal serum samples were tested in the presence of bead-bound antigen targets. The results were represented as mean fluorescence intensity (MFI); the MFI values were converted to sample value/positive value (S/P) ratios. BVDV E2 (aa 1-183) showed the highest MFI values of eight recombinant E2 fragments when the specific activity of each fragment was tested. In immunized animals, data for BVDV E2-specific IgA, IgG, and IgM in serum and only IgG and IgA in oral fluids were recorded. The MFI values for the positive serum sample showed a 100-fold increase compared with the negative serum sample. Antibody isotype to BVDV E2 antigens showed that IgG > IgM > IgA in serum, whereas IgG > IgA > IgM in oral fluids. The data presented in this study suggested that boosting with the same doses of alphavirus RP in 3-week intervals may potentially enhance antibody response. The experimental results demonstrate that alphavirus RP-expressing BVDV E2 antigen induces antibody response in pig.

Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART)

To analyze the concentration of growth factors secreted by autologous cumulus cell clusters during in vitro human embryo co-culture and investigate the impact of culture supplementation with autologous cumulus cell clusters on embryo development and pregnancy rate as compared to standard culture procedure. Randomized prospective comparative study. Private IVF clinic. 90 IVF patient couples ranging at the age between 35 and 40 (mean age of 36.5) agreed to participate in this study. In this IVF group, 64 first trials, 22 s trials and 4 third trials were included. 48 cases have been terminated at day 3 of culture and 42 of them have been continued up to day 5. Of the 48 cases assigned for day-3 culture, 208 MII-oocytes were randomly assigned for co-culture and 197 MII-oocytes were assigned for standard culture. The selection was done by alternating the oocyte immediately after stripping to co-culture or control, starting always with co-culture. All samples were analyzed with Milliplex factor-specific magnetic-bead panels with phycoerythrin fluorescent factor-specific antibodies using immunofluorescence cytometry technology by Lab Supplies Inc. laboratories in Athens, Greece. The MFI (Mean Fluorescence Intensity) values were calibrated and the readings were translated in pg/ml and calculated against the background values of culture medium alone. The statistical analysis of all data were processed with the Two-tailed T-test form. 36 IVF cases terminated at day 3 were randomly chosen. Supernatants from 17 cases were analyzed for seven different growth factors, namely, epidermal growth factor (EGF), fibroblast growth factor 1 and 2 (FGF-1 and FGF-2), vascular endothelial growth factor A, C and D (VEGF-A, VEGF-C and VEGF-D) and Leptin. Supernatants from 19 cases were analyzed for Insulin-like Growth Factor I and II (IGF-I and IGF-II). VEGF-A and VEGF-C were found at statistically significant levels (p < 0.005) in culture supernatant samples where the autologous cumulus clusters were present but were not detectable in supernatants of embryos cultured alone. IGF-I was detected at considerable levels in supernatant samples where the autologous cumulus clusters were present. IGF-I was not detectable in supernatant samples of embryos cultured alone. IGF-II was detected in significant amounts (p < 0.05) in samples of all groups. The highest concentration level of IGF-II was detected in supernatant samples of embryos in standard culture. The investigated factors, among other substances, may be causally connected to the beneficial effect observed on embryo development. Our findings suggest that co-culture with autologous cumulus cell clusters improves the outcome of embryo culture in IVF programs.