Flow Cytometry Crossmatch Research Articles

59-P Effect of adsorption with fetal calf serum on unexpected positive flow cytometry crossmatch results

59-P Effect of adsorption with fetal calf serum on unexpected positive flow cytometry crossmatch results

Anti-Cw Donor-specific Alloantibodies Can Lead to Positive Flow Cytometry Crossmatch and Irreversible Acute Antibody-Mediated Rejection

Anti-Cw Donor-specific Alloantibodies Can Lead to Positive Flow Cytometry Crossmatch and Irreversible Acute Antibody-Mediated Rejection

Flow Cytometric Crossmatching and Panel-Reactive Antibodies in Chronic Renal Failure Patients

Aim Anti-donor antibodies, denoted as “panel-reactive antibodies” (PRAs), are one of the most important factors influencing graft survival after renal transplantation. PRA is generally analyzed with enzyme-linked immunosorbent assay or flow cytometry (FC), which identify the HLA antigen specific for the preformed antibody. Patients and methods We tested 66 patients for FC crossmatch (FCXM) when they were called for cadaveric renal transplantation. Thirty of 66 patients were FCXM-positive; 36 were FCXM-negative. Among the FCXM positive crossmatches, 21 were T- and B-cell positive; seven B positive; and two T positive. The HLA antibodies in the sera of FCXM-positive patients were reanalyzed using flow-PRA. Results We detected HLA antibodies in 28/66 sera with flow PRA. The sera of 16/21 T-/B-, FCXM-positive patients contained both class I and II anti-HLA antibodies, five had only class I anti-HLA antibodies. One out of seven B-cell FCXM-positive patients had class I and class II anti-HLA antibodies, three, class I and 1 class II anti-HLA antibodies; the other two were negative. Class I and class II HLA antibodies were observed in two T-cell FCXM-positive patients. Four of 36 patients who were FCXM-negative were flow PRA positive: one had both class I and class II HLA antibodies and three, only class I HLA antibody. The comparison of FCXM and flow PRA results was significant ( P = .001). Conclusion FCXM results may be confirmed by flow PRA tests, an important method to differentiate HLA versus non-HLA antibodies.

Outcome of Patients with Preformed Donor-Specific Antibodies Following Alemtuzumab Induction and Tacrolimus Monotherapy

It has been shown that low-level preformed donor-specific antibodies (DSAbs) detected by luminex beads in the setting of a negative CDC and flow cytometry crossmatch (CDC/FCXM) are associated with inferior allograft outcomes. The relevance of preformed DSAbs in patients receiving alemtuzumab induction and tacrolimus monotherapy has not been studied. Four hundred and eighty renal transplant recipients with a negative CDC/FCXM had their pretransplant sera retrospectively screened for DSAbs. 45/480 (9.4%) of patients were found to have preformed DSAbs. Females and patients receiving regrafts were more likely to have a DSAb (p = 0.008 and p < 0.0001, respectively). Patients with DSAbs had inferior allograft survival (p = 0.047), increased incidence of antibody-mediated rejection (p < 0.0001) and inferior allograft function at 6 months posttransplant (p = 0.017). Patients with HLA class I DSAb (alone or in combination with a Class II DSAb) with high mean fluorescence intensities (MFIs) were at highest risk. We conclude that patients with preformed DSAb are at high risk of adverse outcomes when receiving a minimal immunosuppressive regime incorporating alemtuzumab induction. Patients found to have a preformed DSAb despite a negative crossmatch might benefit from augmented immunosuppression.

XM-ONE Detection of Endothelium Cell Antibodies Identifies a Subgroup of HLA-Antibody Negative Patients Undergoing Acute Rejection

Background Kidney transplant recipients exhibiting antibodies (Ab) against either HLA or non-HLA antigens undergo frequent episodes of rejection and exhibit decreased long-term graft survival. The novel flow cytometry crossmatch kit XM-ONE, detects Abs to HLA antigens plus those directed to Tie-2–positive precursor endothelial cells (anti-endothelial cell antibodies, AECA). We studied the clinical importance of these lesser known antibodies. Methods We retrospectively analyzed 208 sera from 160 recipients of deceased donor grafts for AECA using non-donor peripheral blood endothelial progenitor cells as targets and Luminex methodology for HLA antibodies. Results AECA were detected in 64 patients (40%). A significantly higher proportion of patients showing a positive endothelial crossmatch experienced rejection (31 AECA-positive among 43 rejection cases, 72%) compared with those without rejection (33/117, 28.2%). Immunoglobulin M(IgM) predominated (66%) over IgG (14%) and IgG plus IgM (20%). HLA antibodies positively and significantly associated with rejection as expected. Of special interest were the 19 patients who presented with acute rejection episodes along with restricted AECA positivity. The relative-risk for an acute rejection episode with either AECA or HLA—13.87 and 2.43, respectively—was significant. When HLA was already positive, the relative risk for AECA was 1.24, a non-significant increase. Conclusions Our data identified AECA-positive patients that showed an increased risk to develop an acute rejection episode early after transplantation. Moreover, restricted AECA-positive patients with acute rejection are an important subgroup which otherwise may be wrongly labeled as non-humoral rejection. Among HLA-negative cases, AECA conferred a significantly greater risk for rejection.

Limitations of Rituximab/IVIg desensitization protocol in kidney transplantation; Is this better than a tincture of time?

A plasmapheresis-free protocol for desensitization of donor kidney transplant candidates with high Calculated Panel Reactive Antibody (CPRA) was initiated at our center. The protocol was adopted from previously published work by Vo, Jordan et al. Five patients with CPRA of 94 ± 18%, awaiting kidney transplant from living or deceased donors received rituximab (1 g × 2 doses) and intravenous immunoglobulin (IVIG 2 g/kg × 2 doses) without plasmapheresis. Levels of donor specific antibodies (DSA) and T/B cell crossmatches were followed using solid phase flow cytometry. Three out of 5 patients were sensitized only to Class II HLA antigens. All patients had very high levels of alloantibodies before initiation of the treatment. All of the candidates initially demonstrated reduced levels of HLA antibody, but statistical significance was only obtained in one patient Class II antibody and in another only for Class I. Depletion was transient with observed antibody rebound. Rituximab effectively depleted CD20 cells in peripheral blood. None of the patients were transplanted due to persistently high levels of antibody and strong positive flow cytometry crossmatches. Under this protocol, reduction of HLA antibodies in patients with high levels was insufficient. Highly allo-sensitized patients with a CPRA above 85% may not benefit from a combination of rituximab-IVIG alone. The previously published protocol does not help all patients achieve an acceptable crossmatch. An individualized approach to the treatment of highly sensitized patients is still required.

The Clinical Importance of Flow Cytometry Crossmatch in the Context of CDC Crossmatch Results

Background The complement-dependent microcytotoxicity crossmatch (CDCXM) is a standard method for evaluating the presence of preformed antibodies before transplantation. The flow cytometry crossmatch (FCXM) is more sensitive, but there is controversy regarding translation of its increased sensitivity to clinically relevant graft outcomes. Methods We analyzed Organ Procurement and Transplant Network registry data for living and deceased donor kidney transplants performed in 1995 to 2009 after both CDCXM and FCXM testing. Transplants with negative CDCXM (CDCXM −) and with T-cell positive (T +), T-cell negative/B-cell positive (T −B +), or T- and B-cell negative (T −B −) FCXM results were included. Graft survival according to crossmatch results was compared by survival analysis. Results Among patients transplanted with negative CDCXM (CDCXM −), deceased and living donor graft recipients with T + FXCM experienced significant absolute reductions in 5-year graft survival of 11.5% and 8.8% compared to those with T − FCXM ( P < .0001). Compared to patients with FCXM −/CDCXM − deceased and living donor recipients with T −B + FCXM/CDCXM − had absolute reductions in 5-year graft survival of 9.6% and 7.6%, respectively ( P < .0001). Upon multivariate adjustment with Cox regression, T + FCXM/CDCXM − deceased donor transplantation was associated with 51% higher adjusted relative risk of 1-year graft loss than FCXM −/CDCXM −. Relative risks were more marked at 1 year for the T + groups but stronger in the 1- to 5-year interval for the T −B + groups. Conclusion Positive FCXM has important prognostic implications even when CDCXM is negative. Thus, positive FCXM should not routinely be dismissed as “overly sensitive” when CDCXM is negative.

The strength of donor-specific antibody is a more reliable predictor of antibody-mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients

The aim of this study was to evaluate the utility of donor-specific antibodies (DSA) and flow cytometry crossmatch (FCCM) as tools for predicting antibody-mediated rejection (AMR) in desensitized kidney recipients. Sera from 44 patients with DSA at the time of transplant were reviewed. Strength of DSA was determined by single antigen Luminex bead assay and expressed as mean fluorescence intensity (MFI). T- and B-cell FCCM results were expressed as mean channel shift (MCS). AMR was diagnosed by C4d deposition on biopsy. Incidence of early AMR was 31%. Significant differences in the number of DSAs (p = 0.0002), cumulative median MFI in DSA class I (p = 0.0004), and total (class I + class II) DSA (p < 0.0001) were found in patients with and without AMR. No significant difference was seen in MCS of T and B FCCM (p = 0.095 and p = 0.307, respectively). The three-yr graft survival in desensitized patients with DSA having total MFI < 9500 was 100% compared to 76% with those having total MFI > 9500 (p = 0.022). Desensitized kidney transplant recipients having higher levels of class I and total DSA MFI are at high risk for AMR and poor graft survival. Recipient DSA MFI appears to be a more reliable predictor of AMR than MCS of FCCM.

53-OR: Disconnect Between Luminex MFI and Flow Cytometry Crossmatch (FCXM) Results for Bw4 and Bw6 Antibodies

53-OR: Disconnect Between Luminex MFI and Flow Cytometry Crossmatch (FCXM) Results for Bw4 and Bw6 Antibodies

10-P: Prediction of Flow Cytometry Crossmatch Results of Deceased Donors With Luminex Single Antigen Bead Assays: The University of Michigan Experience

10-P: Prediction of Flow Cytometry Crossmatch Results of Deceased Donors With Luminex Single Antigen Bead Assays: The University of Michigan Experience

High negative predictive value of an amplified flow cytometry crossmatch before living donor kidney transplantation

Living kidney donation provides the advantage of assessing immunologic risk in detail before transplantation. It was our aim to analyze how results of an amplified flow cytometry crossmatch (FCXM) predict antibody-mediated rejection. T-cell–specific antibodies were analyzed in 92 donor/recipient pairs (164 datasets, average of 1.8 datasets per pair) with negative lymphocytotoxic crossmatch. FCXM results were expressed as delta values of the median fluorescence intensity (MFI), i.e., donor-specific MFI minus negative control. Based on receiver operating curve analysis, positive FCXM results were defined as delta MFI > 22. Delta MFI values in recipients with vs. without antibody-mediated rejection were significantly higher ( n = 6 vs n = 86, p < 0.005). Recipients were grouped into those with reproducibly negative, varying, and reproducibly positive results ( n = 69, n = 12, and n = 11, respectively). Only one of 69 patients with reproducibly negative results displayed antibody-mediated rejection, yielding a negative predictive value of 99%. Furthermore Fisher's exact test showed that the relative risk of a single positive FCXM result for antibody-mediated rejection is 10.1. Thus a negative T-cell FCXM before kidney transplantation is an excellent predictor of the absence of antibody-mediated rejection.

Subtypes of immunoglobulin (Ig)-G antibodies against donor class II HLA and cross-match results in three kidney transplant candidates

Preexisting donor-specific antibodies (DSA) play a critical role in the success of solid-organ transplantation. Cross-match (CM) between donor lymphocytes and recipient serum is a pivotal methodology for detecting these antibodies. Luminex platform based solid-phase methodology for anti-human leukocyte antigen (HLA) antibody analysis has revolutionized the approach to antibody detection and HLA specificity identification. In this study, we have reported three cases of successful living donor kidney transplantations performed against strongly positive B lymphocyte flow cytometry (FC) CM owing to highly reactive DSA directed to HLA class II. IgG solid-phase subtype analysis showed that more than 50% of these antibodies were represented by non-complement binding IgG2/IgG4 subtypes. These findings account for antibody mediated rejection (AMR) free long-term post-transplant course in these patients despite, the high level of DSA. Thus, we conclude that routine application of single HLA-coated beads (SAB) IgG subtype assay may provide new insights regarding transplantation or desensitization of patients presenting with negative B-cell complement dependent cytotoxic (CDC) and positive FC CM.

Mechanism of Acute Antibody-Mediated Rejection in ABO-Incompatible Kidney Transplantation: Which Anti-A/Anti-B Antibodies are Responsible, Natural or de Novo?

Mechanism of Acute Antibody-Mediated Rejection in ABO-Incompatible Kidney Transplantation: Which Anti-A/Anti-B Antibodies are Responsible, Natural or de Novo?

Immunologic study of the donor-receptor couple

The objective of the study is to evaluate the risk of graft failure. The presence of donor specific alloantibodies and the HLA incompatibilities between donor and receptor must be identified. There are several methods to identify alloandibodies that has different sensitivity and different Prognostic Value. Some define a high risk of hyperacute rejection, others an increase in the risk to loss the graft in defined subgroups. First steps of the pretransplant study identify: a) HLA typing of the receptor and available donors; b) alloantibodies by Complement Dependent Cytotoxicity against Panel (PRA-CDC) and screening of alloantibodies against HLA by Solid Phase; c) in sensitized receptors it can be useful to identify acceptable incompatibilities using Single Antigen Solid Phase technique and to evaluate the &laquo;Virtual Crossmatch&raquo;. Pretransplant study (10 days): a) crossmatch by Citotoxicity (CM-CDC) between receptor and donor; b) crossmatch by Flow-Cytometry (FCCM) between receptor and donor specially indicated in the retransplant. Useful also to discard IgM auto-antibodies. Receptors desensitization: the necessity and success probability of desensitization should be evaluated before treatment. Post-Transplant Monitoring: identify alloantibodies for: a) the differential diagnostic of corticorresistant rejection episodes with humoral component, and b) as a marker of long term reduced graft survival probability in the long term. Final remarks: Evaluation should consider the allosensibilization history of the receptor. The cytotoxicity crossmatch indicates a high risk of hyperacute rejection and is considered a contraindication. The Flow Cytometry crossmatch indicates an increase in the probability to loss the graft in the first year that is low for first transplants (>10%) but higher for retransplantation (>30%). The virtual crossmatch by solid phase indicates an increase in the probability to have an antibody mediated rejection (from 5% to 55%) but did not contraindicate always the transplant.

Immediate Antibody-Mediated (Hyperacute) Rejection in Small-Bowel Transplantation and Relationship to Cross-Match Status and Donor-Specific C4d-Binding Antibodies: Case Report

Background The role of preformed donor-specific antibodies (DSAs) as a barrier to isolated intestinal transplantation (ITx) remains ambiguous; thus, a positive cross-match has not been a contraindication to ITx. Objective To report the case of a patient with Crohn's disease who underwent ITx and developed immediate antibody-mediated rejection on reperfusion of the allograft. Methods Percent reactive antibody testing was performed using pretransplantation serum samples and at transplantation using bead-based assays (Luminex, Luminex Corp, Austin, Tex) and flow cytometry solid-phase assays (FlowPRA single-antigen beads (One Lambda, Inc, Canoga Park, Calif). Serologic tests, flow cytometry cross-matching, and flow cytometry assays of C4d-binding serum antibodies were also performed. Histologic and immunofluorescent analysis of biopsy specimens was performed. Results HLA typing revealed no sharing of class I or II antigens between donor and recipient. Pretransplantation donor-specific antibodies (DSA) were present at transplantation. Cross-matching (performed during surgery) was positive for class I and II by serologic testing and flow cytometry. After reperfusion, the graft immediately developed severe ischemic injury and arteritis on mucosal biopsy specimens, with immunoglobulin deposition. The DSA C4d binding antibodies were also present. After intense immunosuppression and plasmapheresis, the graft and the biopsy histologic findings showed marked improvement (day 2). By day 7 posttransplantation, patient and graft status were stable. The patient has remained clinically stable for more than a year after transplantation. Conclusions Pretransplant DSA in ITx can be a risk factor for immediate (hyperacute) but potentially reversible antibody-mediated rejection. Thus, pretransplantation DSA and cross-match results are critical components to be considered in patients awaiting or undergoing ITx.

Continuous Monitoring of Donor Specific Anti-HLA Antibody in Kidney Transplantation Patients

Background: A positive reaction at flow cytometry crossmatch (FCXM) has been highlighted by its predictive value for clinical outcome in kidney transplantation after accumulation of large clinical data. The detection of de novo development of anti-HLA antibodies after transplantation is associated with increased rejection and decreased graft survival. In this study, we report the experience for the detection of anti-donor specific antibody (DSA) by more sensitive FCXM methods in renal transplantation patients. Methods: T and B cell FCXMs were performed on 11 pretransplant and 51 posttransplant sera from 11 patients who received renal grafts between 2004 and 2005. The posttransplant sera were collected in specific and regular intervals from posttranspant 1 week to 1 year. Results: Among 62 sera, four (7.8%) from 2 patients showed positive FCXM. In one patient, pretransplant serum which was negative at previous CDCXM, and 2 consecutive sera collected at 1 week and 1 month after transplantation were positive at FCXM. And the antibody identified was B51 which was specific for one of donor alleles (DSA). In another patient, FCXM became positive 1 week after transplantation although pretransplant serum had negative results at both CDCXM and FCXM. Both patients had experienced more than one rejection episodes. Conclusions: Detection of DSA with more sensitive technique such as flow cytometry based method clearly displayed a beneficial effect for prediction of clinical outcome as a part of pretransplant compatibility test, and also as a posttransplant monitoring test to identify the de novo production of clinically significant DSA.

ABO incompatible living donor kidney transplantation: a dream come true. Experience of Hospital Clínic of Barcelona

During the last years the number of patients on waiting list for kidney transplantation has been stable. Living donor kidney transplantation is nowadays a chance to increase the pool of donors. However, there are a group of patients with ABO incompatibility, making impossible the transplant until now. The aim of the present study is to describe the experience of Hospital Clinic Barcelona on ABO incompatible living transplantation. A retrospective- descriptive study was made based on 11 living donor kidney recipients with ABO incompatibility in Hospital Clinic of Barcelona from October'06 to January'09. Selective blood group, antibody removal with specific immunoadsortion, immunoglobulin and anti- CD 20 antibody were made until the immunoglobulin (IgG) and isoaglutinine (IgM) antibody titters were 1/8 or lower. Immunosuppressive protocol was adjusted to particular recipient characteristics. Isoaglutinine titters were set before, during and post desensitization treatment and two weeks after transplant. Immunological, medical and surgical evaluation was the standard in living donor kidney transplant program. Medium age of donors and recipients were 47.8 +/- 12.4 and 44.4 +/- 14.1 years, respectively. 90% of donors were females and 73% of recipients males. Follow-up time was 10.2 +/- 10.2 months. Siblings and spouses were the most frequent relation (n=4, 36.4%, respectively). Chronic glomerulonephritis, adult polycystic kidney disease and Alport syndrome, the most frequent cause of end-stage renal disease. All the patients acquire appropriate isoaglutinine titters pre transplant (< 1/8), requiring 5.54 +/- 2.6 immunoadsorption sessions pretransplant and 2.82 posttransplant. One patient didn t need any immunoadsorption session (incompatibility blood group B) and another patient plasma exchange instead of immunoadsorption for being hypersensitized with positive flow cytometry crossmatch. Posttransplant isoaglutinine titters remained low. Two patients had cellular acute rejection episode (type IA and IB of Banff classification) with good response to corticosteroid treatment. Patient and graft survival were 91% at first year and remain stable during the follow-up. A graft lost by death of patient in relation to haemorrhagic shock developed within the first 72 hours after transplantation. Renal graft function at first year was excellent with serum creatinine of 1.3 +/- 0.8 mg/dl, creatinine clearance of 62.6 ml/min/1.73 m2 and proteinuria of 244.9 mg/U-24h. ABO incompatible living donor kidney transplantation represent an effective and safe alternative in certain patients on waiting list for renal transplant, obtaining excellent results in patient and graft survival, with good renal graft function.

74-P: The influence HLA expression level on virtual and flow cytometry crossmatches

74-P: The influence HLA expression level on virtual and flow cytometry crossmatches

127-P: Development and type of donor specific antibody (DSA) by flow cytometry crossmatch (FCXM) following living donor related kidney transplantation (LDRKT) may be predictive of rejection and graft prognosis

127-P: Development and type of donor specific antibody (DSA) by flow cytometry crossmatch (FCXM) following living donor related kidney transplantation (LDRKT) may be predictive of rejection and graft prognosis

56-P: Prediction of flow cytometry and AHG-CDC crossmatch results by multiplex bead array MFI

56-P: Prediction of flow cytometry and AHG-CDC crossmatch results by multiplex bead array MFI