Median Fluorescence Intensity Values Research Articles

Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland

The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms – Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland.The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) – applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings.The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria (P-score), in line with the results of the EuroFlow quality assessment rounds performed by the EuroFlow expert laboratories(Kalina et al., 2015). Central analysis of data allowed identification of deviations from the standardized procedures and technical issues (e.g. failure to perform correct instrument setup and improper compensation).In summary, here we show that inter-laboratory cross-platform standardization of 8-color flow cytometric measurements in clinical laboratories is feasible and allows for fully comparable MedFI results across BD FACSCanto II and BC Navios instruments. However, adherence to standardized protocols is crucial. Thus, training of the laboratory personnel in the EuroFlow standardized procedures is highly recommended to prevent errors in instrument setup and sample preparation.

Effect of Single Sensitization Event on Human Leukocyte Antigen Alloimmunization in Kidney Transplant Candidates: A Single-Center Experience.

Human leukocyte antigen alloimmunization is caused by exposure to HLA antigens through transfusion, pregnancy, or transplant. Our study objective was to present the rate of positivity of anti-HLA antibody considering the effects of a single sensitization event in kidney transplant candidates at our center. Our study reviewed 606 kidney transplant candidates. Patient sera were analyzed using Luminex xMAP technology. Panel reactive antibody positivity rates and antibody strengths in patients were analyzed according to a single sensitization event. Our findings showed 246 patients (40.6%) with a panel reactive antibody > 0, of which 97 (39.4%) were sensitized from a single event, 119 (48.4%) were sensitized by multiple events, and 30 (12.2%) had no known sensitizing event. Considering patients sensitized by a single event with a panel reactive antibody > 0, we found that 25.8% had received transplant only, 49.5% had previous pregnancy only, and 24.7% had received transfusion only. The strength of antibodies was significantly higher in patients with previous transplant procedures than in those with transfusion for HLA-A (P < .01), HLA-B (P < .05), HLA-C (P < .05), HLA-DR (P < .001), HLA-DQ (P < .05), and HLA-DP (P < .05). Similarly, we observed significantly higher median fluorescence intensity values for HLA-A, -DR, -DQ, and -DP loci in patients with a previous transplant procedure versus pregnancy. The strength of antibodies against HLA-DR was significantly higher in patients with a previous pregnancy compared with those with transfusion (P < .01). This study documents the profile of HLA alloimmunization in kidney transplant candidates. In particular, transplant procedures appear to have a greater immunologic impact, followed by pregnancy and transfusion. Our data confirm and are in accordance with those of several studies in which the sensitization events were associated with higher prevalence of anti-HLA antibodies.

Reason and Resolution of High Negative Control Beads in Solid-Phase Immunoassay.

The Luminex technology is the most sensitive diagnostic method for HLA antibody detection and identification. However, the interpretation of immunoassays is commonly affected by the artifact, and non-specific background. Sera from some patients show high negative control bead (NC) value, which makes assessing and interpretation of HLA antibodies difficult. In this study, we evaluated the effect of Adsorb Out reagent, dithiothreitol (DTT), and Ethylenediaminetetraacetic acid (EDTA) on the NC median fluorescence intensity value by comparing treated versus untreated patient sera. In addition, we wanted to identify whether kidney disease and administered medication influenced high NC median fluorescence intensity values by comparing patient versus control results. HLA antibody screening was performed on 3500 serum samples. Sera were analyzed using the standard protocol for Luminex antibody screening. Sera with high NC values were preincubated with Adsorb Out, DTT, and EDTA. Screening of these sera was then performed. We found that 4% of samples showed high NC values. Adsorb Out, DTT, and EDTA decreased the NC values at 723.5 (299.25-1443) versus 85 (34-218; P < .001), at 723.5 (299.25-1443) versus 184 (106-597; P < .001), and at 723.5 (299.25-1443) versus 455 (131-1177; P = .004). These succeeded in bringing back NC values to normal range in 69.2%, 43%, and 30% of treated sera, respectively. In addition, the differences of corticoids, immunosuppressive, and heparin drugs between patients and controls were statistically significant (P < .001, < .001, and = .043). However, presence of kidney disease was not significant between these groups. All pretreatments had an important effect in decreasing negative control values, with Adsorb Out having highest efficiency. Serum-specific components could contribute to high negative control bead median fluorescence intensity values. Further studies are needed to determine the adequate pretreatment of patient sera.

It’s about time: The development and validation of a rapid optimized single antigen bead (ROB) assay protocol for LABScreen

The LABScreen single antigen bead assay (SAB) is a method widely used for the identification and monitoring of human leukocyte antigen (HLA) antibodies in patients pre-and post-transplant. While accurate testing of patient samples is key for optimal patient care, time can also be important, especially during deceased donor workups or post-transplant assessments. Here we describe the development and validation of the Rapid Optimized SAB (ROB) protocol, a modified version of the One Lambda LABScreen SAB (OLSAB) procedure, which reduces assay time from 85 to 25min (>70% reduction) without impacting assay quality or sensitivity. Optimization steps included shortened centrifugation cycles and reduced serum and secondary antibody incubation times in combination with increased secondary antibody concentration. Linear regression analysis of baseline median fluorescence intensity (MFI) values showed excellent correlation between the ROB and OLSAB protocols (r2>0.98) for both class I and class II antibodies in 58 sera tested in two HLA laboratories. Importantly, the ROB protocol demonstrated a trend towards improved inter-laboratory MFI concordance when compared to the OLSAB procedure (r2=0.9816 vs 0.9451), especially for HLA antibody specificities in the 500–2000 MFI range (r2=0.7824 vs 0.6313). Implementation of the ROB protocol will expedite HLA antibody testing and may improve reproducibility of the SAB assay.

Evaluation of Fluorescence Microsphere Immunoassay for Antibody Detection to Rift Valley Fever Nucleocapsid Protein and Glycoproteins

Rift Valley Fever Virus (RVFV) is a zoonotic disease that infects ruminants including cattle, sheep, goats, camels and buffalo. There is a need to create a safe, rapid, and accurate assay that can simultaneously detect antibodies directed against multiple RVFV antigens. We describe the improvement and validation of a fluorescence microsphere immunoassay (FMIA) for the detection of IgG antibodies against the RVFV glycoproteins and the immunodominant nucleocapsid (N) protein. The purpose of this assay is to simultaneously screen and confirm sera from animals for positive or negative disease status. Well‐characterized vaccinated and experimentally infected sheep and calf sera were used for the evaluation of the assay. Recombinant viral proteins were produced then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. An unrelated viral antigen coupled control bead set was added to the reaction in order to account for non‐specific binding of antibodies to the antigen of the antigen‐bead complex. Median Fluorescence Intensity (MFI) results were converted to Sample/Positive ratios in order to standardize test results. Our results revealed highest MFI values for the detection of IgG antibodies against the RVFV N protein, indicating that this RVFV antigen could serve as a good candidate for a screening assay. The glycoprotein Gn target has been shown to differentiate vaccinated from non‐vaccinated animals in a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The FMIA demonstrated a high correlation (R2) of 0.91 when compared side by side with plaque reduction neutralization tests. The addition of further RVFV targets such as the non‐structural proteins NSs and NSm is currently under investigation. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay will provide the basis for the development of a multiplex sero‐diagnostic assay system that can simultaneously screen for several ruminant diseases including transboundary animal diseases.Support or Funding InformationThis material is based upon work supported by the Kansas State NBAF Transition Funds, Kansas State University College of Veterinary Medicine, University of Pretoria Department of Veterinary Tropical Diseases, the USDA Agricultural Research project number 5430‐32000‐005‐00D and the U.S. Department of Homeland Security under Grant Award Number DHS‐2010‐ST‐061‐AG0001. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security.

Proteome analysis of circulating exosomes in health and breast cancer

Microvesicles were isolated from blood plasma and total blood of healthy females and breast cancer patients by filtration and ultracentrifugation. According to flow cytometry, different subpopulations of exosomes were represented in blood of healthy donors and cancer patients at different levels with median fluorescence intensity (MFI) values in both groups arranged in the following order: CD24/СD9 > СD9/СD81 > CD9/CD63 = CD24/CD63. Concentration of exosomes in blood plasma of healthy females estimated by nanoparticle tracking analysis (NTA) did not exceed (3.71 ± 1.15) × 107 particles/mL of blood and did not differ from that in plasma of breast cancer patients, which averaged (3.99 ± 1.03) × 107 particles/mL of blood. Concentration of total exosomes in blood (including exosomes from plasma and blood cell surface-bound exosomes) did not depend on the presence/absence of a tumor; the values were (7.66 ± 0.7) × 107 particles/mL of healthy blood and (9.4 ± 1.24) × 107 particles/mL of blood from cancer patients. Comparative analysis of exosomes using 2-D electrophoresis with subsequent analysis of 2-D proteomic maps revealed proteins missing in blood or differentially expressed in healthy females and breast cancer women. The data presented provide the possibility for identification of exosomal proteomic markers and isolation of tumor-specific exosomes, which contributes to the development of breast cancer diagnostics.

Surface CD5 Protein Risk Stratifies Chronic Lymphocytic Leukemia

Introduction: Chronic Lymphocytic Leukemia (CLL) is a malignancy characterized by B-lymphocytes with aberrant expression of CD5. In normal T-lymphocytes, CD5 is an important regulator of T-cell receptor signaling. In CLL, CD5 acts as a repressor of B-cell receptor (BCR) signaling, whereby phosphorylated CD5 anchors SHP-1 at the cell membrane, resulting in inhibition of BCR-mediated second messenger signaling. The significance of CD5 on outcomes of CLL patients has not been evaluated. Because BCR signaling is an oncogenic stimulus in CLL, we hypothesized that higher levels of CD5 may be associated with superior clinical outcomes.Methods: Median fluorescence intensity (MFI) of CD5 on CLL cells was determined by flow cytometry. CLL samples and clinical data were obtained from patients enrolled in IRB-approved protocols at the Duke University and Durham VA Medical Centers. Molecular prognostic markers were measured as described previously. Descriptive and time to event (Cox proportional hazard and Kaplan-Meier) statistical analyses were performed in the statistical environment, R.Results: Between March 2011 and April 2015, 961 CLL samples were evaluated from 352 unique CLL patients. One to 10 samples were evaluated per CLL patient. The mean CD5 MFI was calculated for each unique patient, with a median of 206.59 (range 0.50 - 768.31) and median standard deviation of 33.02 (range 0.09 - 315.46) for CLL patients with multiple samples collected. 150 patients received therapy (44%), and there was no significant difference in CD5 MFI mean or standard deviation between patients who received and did not receive therapy. CD5 MFI values did not significantly differ among CLL demographic or prognostic groups. Cox proportional hazard ratio analyses showed that higher CD5 MFI is associated with longer time to therapy (TTT, p = 0.037), but not overall survival. Based on the distribution of CD5 MFI, we divided the CLL cohort into thirds, with low CD5 defined as MFI < 137, high CD5 defined as MFI > 283.5, and medium CD5 with MFI between these values. Kaplan-Meier analyses demonstrated a significant difference in TTT between these three groups (p = 0.004), with higher CD5 MFI associated with longer TTT. Moreover, CD5 MFI added to established molecular prognostic markers to risk stratify CLL patients (p < 0.0001 for CD38 and IGHV, p = 0.0003 for FISH, and p = 0.004 for ZAP70). Importantly, for those with good prognostic markers such as IGHV mutation or low CD38 expression, lower CD5 identified patients with shorter TTT (p = 0.01 for IGHV mutated; p = 0.0009 for CD38 negative). However, TTT was not significantly different in unmutated IGHV CLL patients or CD38 positive CLL patients with varying degrees of CD5 expression.Conclusions: Surface CD5 expression varies among CLL patients. For most individual patients, there appears to be a low level of variability in CD5 expression, regardless of CLL-directed therapy. Higher CD5 expression is associated with superior clinical outcomes in CLL, consistent with prior in vitro determination of CD5 as a negative regulator of BCR signaling. The relevance of CD5 in risk-stratifying IGHV mutated and CD38 negative CLL patients is of particular relevance, since these subgroups of CLL are particularly dependent on BCR-signaling as an oncogenic process. These findings are important both for prognostication and for development of therapies for this group of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

P008 Case scenario, performance differences of the two commercially available solid phase single antigen reagents

Aim Luminex single antigen technology for antibody identification has enabled laboratories to provide highly accurate virtual crossmatch results prior to transplantation. Both manufacturers, One Lambda (OL) and Immucor (IC) single antigen reagent panels differ in the HLA alleles composite and density. We report here two cases that single antigen panel for each behave significantly different. Method Serum of Patient (RH) and (AM) were tested with OL and IC single antigen panels. Flow cytometry and CDC crossmatchs of both patients were performed with their corresponding donors. Results Serum of (RH) and (AM) tested with OL single antigen panel that showed the following DSA’s, anti DQB1*06:03 (2510 MFI) and anti A2 (891 MFI), B41 (1631 MFI) respectively. CDC and flow crossmatch were highly positive for both sera despite low Median Fluorescent Intensity (MFI) of the identified DSA. The same sera were tested with IC single antigen panel; patients (RH) and (AM) showed anti DQB1*06:03 (17946 MFI) and anti A2 (10500 MFI) respectively. Furthermore, both sera were diluted 1:4 and re-tested with OL single antigen panel. The identified DSA strength in the diluted sera for both patients showed higher MFI values for A2 (3048 MFI), B41 (2863 MFI) for AM patient and DQB1*06:03 (10503 MFI) for RH. Conclusion Significantly, higher prozone effect was observed with OL single antigen panel than in IC. Serum to bead-volume ratio may have contributed to the lower correlation between OL versus IC. Testing patients sera by more than one manufacturer is an advantageous in determination of antibody strength and improves correlation with the crossmatch results.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

Liver Allograft Provides Immunoprotection for the Cardiac Allograft in Combined Heart-Liver Transplantation.

When transplanted simultaneously, the liver allograft has been thought to have an immunoprotective role on other organs; however, detailed analyses in simultaneous heart-liver transplantation (SHLT) have not been done to date. We analyzed patient outcomes and incidence of immune-mediated injury in 22 consecutive SHLT versus 223 isolated heart transplantation (IHT) recipients between January 2004 and December 2013, by reviewing 3912 protocol- and indication-specific cardiac allograft biopsy specimens. Overall survival was similar (86.4%, 86.4%, and 69.1% for SHLT and 93.3%, 84.7%, and 70.0% for IHT at 1, 5, and 10 years; p = 0.83). Despite similar immunosuppression, the incidence of T cell-mediated rejection (TCMR) was lower in SHLT (31.8%) than in IHT (84.8%) (p < 0.0001). Although more SHLT patients had preexisting donor-specific HLA antibody (22.7% versus 8.1%; p = 0.04), the incidence of antibody-mediated rejection was not different in SHLT compared with IHT (4.5% versus 14.8%, p = 0.33). While the left ventricular ejection fraction was comparable in both groups at 5 years, the incidence and severity of cardiac allograft vasculopathy were reduced in the SHLT recipients (42.9% versus 66.8%, p = 0.03). Simultaneously transplanted liver allograft was associated with reduced risk of TCMR (odds ratio [OR] 0.003, 95% confidence interval [CI] 0-0.02; p < 0.0001), antibody-mediated rejection (OR 0.04, 95% CI 0-0.46; p = 0.004), and cardiac allograft vasculopathy (OR 0.26, 95% CI 0.07-0.84; p = 0.02), after adjusting for other risk factors. These data suggest that the incidence of alloimmune injury in the heart allograft is reduced in SHLT recipients.

Comparison of non-magnetic and magnetic beads in bead-based assays

Multiplex bead-based flow cytometry is an attractive way for simultaneous, rapid and cost-effective analysis of multiple analytes in a single sample. Previously, we developed various bead-based assays using non-magnetic beads coated with Staphylococcus aureus and Streptococcus pneumoniae antigens for the detection of antibodies. Here, we compared the performance of the assay using non-magnetic beads with one based on the newly developed magnetic beads. We optimized the magnetic beads' coupling procedure and antibody detection assays for S. aureus and S. pneumoniae antigens and we measured IgG in human pooled serum against a series of S. aureus and S. pneumoniae-derived antigens in a singleplex and in a multiplex assay, respectively. For the multiplex assay, the comparison between magnetic and non-magnetic beads showed: i) in the majority of the cases (13 of the 17 tested S. pneumoniae antigens) significantly higher Median Fluorescence Intensity (MFI) values, ii) lower detection limits, iii) lower coefficient of variation (CV: 12% vs. 7% for non-magnetic vs. magnetic beads), so lower inter-assay variation and hence higher reproducibility. Magnetic bead coupling is cost effective, as we used 25% of the normal amount of antigen and only 50% of the beads in comparison to the non-magnetic beads. This optimized magnetic-based assay, which combines ease of use with an improved assay performance, allows detection of antibodies with a low titer that are potentially missed with the non-magnetic-based assay.

The Effect of Root, Shoot and Seed Extracts of The Iranian Thymus L. (Family: Lamiaceae) Species on HIV-1 Replication and CD4 Expression.

The genus Thymus L. is a cushion plant that was previously used for the treatment of bronchitis and rheumatism. The present investigation was carried out to study the effects of root, shoot, leaf and seed extracts of five Thymus species and subspecies on peripheral blood mononuclear cells (PBMCs) toxicity and HIV-1 replication. In this experimental study, the activity of the Thymus extracts on HIV-1 replication and lymphocytes population were examined respectively using HIV-1 p24 Antigen kit and flow-cytometer. The Thymus species effect was investigated, including Thymus kotschyanus, Thymus vulgaris, Thymus carmanicus, Thymus daenensis subspecies lancifolius and Thymus daenensis subspecies daenensis. The effect of root methanol extracts of all species on PBMCs proliferation was significantly higher than the other extracts. The intensity of CD4, CD3 and CD45 were decreased in the presence of all root extracts. Although the average median fluorescence intensity (MFI) values of CD19 were increased in the cells treated with these extracts. All methanol extracts showed anti-HIV-1 activity at high concentrations (200 and 500 µg/ml). Anti-HIV-1 activity of Thymus daenensis subspecies daenensis was significantly more than the other species. These results demonstrated that root extracts of Thymus species might be a good candidate to investigate anti-HIV infection in vivo.

Induction of Unconventional T Cells by a Mutant Mycobacterium bovis BCG Strain Formulated in Cationic Liposomes Correlates with Protection against Mycobacterium tuberculosis Infections of Immunocompromised Mice.

Earlier studies aimed at defining protective immunity induced by Mycobacterium bovis BCG immunization have largely focused on the induction of antituberculosis CD4(+) and CD8(+) T cell responses. Here we describe a vaccine consisting of a BCGΔmmaA4 deletion mutant formulated in dimethyl dioctadecyl-ammonium bromide (DDA) with d-(+)-trehalose 6,6'-dibehenate (TDB) (DDA/TDB) adjuvant (A4/Adj) that protected TCRδ(-/-) mice depleted of CD4(+), CD8(+), and NK1.1(+) T cells against an aerosol challenge with M. tuberculosis These mice were significantly protected relative to mice immunized with a nonadjuvanted BCGΔmmaA4 (BCG-A4) mutant and nonvaccinated controls at 2 months and 9 months postvaccination. In the absence of all T cells following treatment with anti-Thy1.2 antibody, the immunized mice lost the ability to control the infection. These results indicate that an unconventional T cell population was mediating protection in the absence of CD4(+), CD8(+), NK1.1(+), and TCRγδ T cells and could exhibit memory. Focusing on CD4(-) CD8(-) double-negative (DN) T cells, we found that these cells accumulated in the lungs postchallenge significantly more in A4/Adj-immunized mice and induced significantly greater frequencies of pulmonary gamma interferon (IFN-γ)-producing cells than were seen in the nonvaccinated or nonadjuvanted BCG control groups. Moreover, pulmonary DN T cells from the A4/Adj group exhibited significantly higher IFN-γ integrated median fluorescence intensity (iMFI) values than were seen in the control groups. We also showed that enriched DN T cells from mice immunized with A4/Adj could control mycobacterial growth in vitro significantly better than naive whole-spleen cells. These results suggest that formulating BCG in DDA/TDB adjuvant confers superior protection in immunocompromised mice and likely involves the induction of long-lived memory DN T cells.

Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens

Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI.

Simultaneous detection of three lily-infecting viruses using a multiplex Luminex bead array

A Luminex bead array was applied to detect multiple-virus coinfection in lily plants exhibiting typical symptoms, and the efficiency of this detection system was assessed. Specific primer sets for the simultaneous detection of 4 targets in virus-infected lily plants were constructed and used for reverse transcription (RT)-polymerase chain reaction (PCR), and specific probes were used for Luminex-based assay. Each of the 4 targets was amplified, and the amplicons were used for Luminex bead array experiments. A Luminex bead array analysis of lily-infecting viruses was performed using the quadruplex RT-PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescence signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescence intensity (MFI) values. Detection of the different target elements was found to be very specific to the corresponding viruses in lilies, and coinfection with multiple viruses was specifically detected via the MFI signals. Therefore, the use of a Luminex bead array for the detection of co-infected multiple viruses in lily plants can be an improved system for screening and analyzing multiple-virus infection.

IFN-γ modulates Ly-49 receptors on NK cells in IFN-γ-induced pregnancy failure.

We have previously shown that interferon gamma (IFN-γ) induces aberrant CD49b+ natural killer (NK) cell recruitment by regulating CX3CL1 and eventually provokes foetal loss. In this study, we show that IFN-γ also modulates Ly-49 receptors on NK cells during pregnancy failure. The percentages of Ly-49A+ and Ly-49G2+ NK cells in the uteri of the IFN-γ-treated group were significantly lower than those observed in the control group. Moreover, the median fluorescence intensity (MFI) values of Ly-49A and Ly-49G2 expression on NK cells in the uteri of the IFN-γ-treated group were significantly lower than those of the control group. Using isolated spleen leucocytes, we further found that IFN-γ significantly reduced the percentage of Ly-49A+ NK cells in vitro. However, CX3CL1 was not involved in the modulation of Ly-49 receptors, and the expression of CX3CR1 was not regulated by IFN-γ in spleen leucocytes. Collectively, our data indicate that IFN-γ can modulate Ly-49 receptors on NK cells and this process may play a role in IFN-γ-induced pregnancy failure. Thus, we provide a new line of evidence correlating the deleterious effects of IFN-γ with its role in regulating NK cell Ly-49 receptors during pregnancy failure.

Assessing the correlation between C3d-binding de novo donor specific HLA antibodies and rejection in kidney transplant recipients

Aim Solid phase assays have the ability to detect even low levels of HLA antibodies, but potentially lack the ability to distinguish between clinically relevant or irrelevant DSA. Here we test the clinical significance of a novel C3d binding assay. Methods Serum samples from 85 kidney transplant recipients were retrospectively analyzed using the Immucor© Single Antigen immunoassays, to detect either IgG or C3d binding HLA antibodies. Inclusion criteria included patients that developed de novo DSA by Luminex One Lambda (OL) single antigen assay and had a biopsy obtained at the same time point as the serum sample. Median fluorescence intensity (MFI) value of 1000 was arbitrarily used to assign positive responses. Results Of the 85 patients: 21 had AMR, 12 cell-mediated rejection (CMR), 11 mixed rejection (AMR + CMR), 4 with pathologic finding other than rejection, such as BK virus, and 41 had normal biopsies. C3d DSA were detected in 16/85 patients with the majority having AMR in 11/16 patients (69%) compared to 1/16 patients (6%) with CMR, 2/16 patients (12.5%) with a mixed rejection, and 2/16 patients (12.5%) with negative biopsies. These 2 negative biopsy patients had strong MFI values for Immucor IgG DSA and strong titers on the OL platform; moreover, both of these patients went on to have AMR later on. Immucor IgG assay detected 29/85 patients with DSA, in which, the majority of C3d positive DSA (16/16) being Immucor IgG class II DSA-positive. Immucor IgG positive DSA with negative C3d results had lower MFI (Fig. 1). All the C3d positive assays corresponded to moderate to high titer OL results (titers ⩾ 1 : 64 ), although three samples had class II specificities with titers ⩾ 1 : 128 were not C3d positive. Conclusion C3d-binding antibodies seem to be associated with stronger antibodies with at least moderate level titer and rejection. Further studies are required to substantiate our results. Download : Download full-size image

Conundrum: A negative crossmatch in a patient with high level donor specific antibody

With the advent of sensitive luminex single antigen bead (SAB) antibody testing, virtual crossmatches have become reliable ways to predict and eliminate incompatible donor-recipient pairs. In our laboratory, luminex SAB testing (One Lambda) has repeatedly shown that patient serum containing antibody levels with normalized median fluorescence intensity (MFI) values greater than 5000 will yield a positive flow crossmatch almost 95% of the time. We present a patient (EP) with high level Donor Specific Antibody (DSA) to DQ7 who had a negative crossmatch with her DQ7 positive donor. EP and her living donor (AH) did not match for any HLA antigens. Most of the crossmatching in our lab is performed prior to HLA typing, so the initial negative crossmatch between this pair was not noteworthy. However, following low resolution SSP typing by RT-PCR (Linkage Biosciences), donor AH typed phenotypically for DQ7. Since EP was known to have DQ7 antibodies by both luminex ID and SAB (MFI = 8241) assays, it was predicted that this crossmatch would be positive. The flow crossmatch between EP and AH, however, was B and T cell negative, with a B cell delta channel shift (DCS) of only 13. Higher resolution typing by SSP and SSO (One Lambda) was then performed to confirm and elucidate the class II, specifically DQB, typing. Interestingly, AH was shown to be DQB1∗03:03, DQB1∗03:19 which is phenotypically DQ9, DQ7 or DQ9, – (presumably no serological equivalent or weakly expressed antigen). The SAB assay has five DQ7 beads, all of which contain antigens isolated from DQB1∗03:01 cells, but with different DQA associations. With this information, it was not surprising that this pair’s crossmatch was negative. Additional surrogate crossmatches were then performed with EP sera against known DQB1∗03:01 and DQB1∗03:19 typed cells. Two cells expressing DQB1∗03:01 had positive B cell crossmatches with DCS of 106 and 120, while two cells which typed as DQB1∗03:19 had negative B cell crossmatches (DCS = 44 and 9). This particular donor-recipient pair emphasizes that virtual crossmatches, while valuable, cannot be considered accurate for every donor and recipient. Further studies need to be performed to determine the clinical significance of a negative crossmatch in the presence of DSA to closely related, at the allelic level, HLA antigens.

Effect of Different Sensitization Events on HLA Alloimmunization in Kidney Transplantation Candidates

BackgroundHLA alloimmunization is caused by sensitization events (SEs), such as transfusion, pregnancy, or previous organ transplantation, and the effects of particular SEs have not been thoroughly studied. Our aim was to evaluate how each SE affected HLA alloimmunization by considering Luminex assays. MethodsSera from 722 kidney transplantation candidates were screened per protocol by means of Luminex assays to determine the presence of anti–HLA class I/II antibodies; positive sera underwent single-antigen assay to determine the presence of specific antibodies against HLA A, B, C, DR, DQ, DP loci (positivity if median fluorescence intensity values were >1,000). The effect of each SE was analyzed considering only patients exposed to 1 kind of sensitization. ResultsIn the 453 candidates with ≥1 SE, anti–HLA class I positivity rates were significantly higher in patients with previous transfusion (18.9%; P = .014), pregnancy (38.3%; P < .001) or transplant (75%; P < .001) compared with those with no SE (similar results for class II). The strength (median fluorescence intensity) of specific antibodies was significantly higher in patients with previous transplantation than in those with previous transfusion for HLA-A (8,017 vs 2,302; P = .02), HLA-B (7,765 vs 2,901; P = .018), and HLA-DR (9,835 vs 2,060; P = .003). Other anti-HLA antibody strengths were similar between patients with previous pregnancy or transplantation. ConclusionsPresence of any SE analyzed was associated with a higher prevalence of anti-HLA antibodies for class I ± II compared with nonsensitized patients. Transplantation had the strongest immunization effect on both classes, followed by pregnancy and then transfusion.

Epitope-specificities of HLA antibodies: The effect of epitope structure on Luminex technique-dependent antibody reactivity

The search of HLA antibodies is currently more accessible by solid-phase techniques (Luminex) in the immunized patients leading to an expansion of the antibody patterns. The aim of this study was to investigate low median fluorescence intensity value in unexpected reactivity patterns. Here, we performed HLAMatchmaker analyses to evaluate the potential functional epitopes that can elicit HLA-specific alloantibody responses in a pregnancy-sensitized woman with an epitope defined by the 82LR. Surprisingly, in according to the registry of HLA epitopes, we found that 82LR epitope covered all allelic specificities of our unexpected antibody patterns, shared between Bw4-positive HLA-B antigen and HLA-A23, -A24, -A25 and -A32. This finding is consistent with the verification of HLA ABC epitope recorded in the website-based HLA Epitope Registry and addresses the importance of determining HLA antibody epitope-specificities on Luminex technique-dependent antibody reactivity.