Mean Channel Shift Research Articles

Temporary Deceased Donor Splenic Transplant Prior to Intestinal Transplantation: A New Strategy for Desensitization?

Intestinal transplantation is a therapeutic treatment option for patients with irreversible intestinal failure. The presence of donor-specific antibodies (DSAs) has been associated with increased antibody-mediated rejection and allograft loss for recipients of all the solid organ transplants. This case report describes the posttransplant course in the first year of a patient who received a T-cell and B-cell flow cross-match (FXM) and complement-dependent cytotoxicity cross-match positive intestinal transplant in the presence of several class I and class II DSAs who underwent a "temporary desensitization" using the donor spleen. The temporary donor splenic transplant removed several class I and II DSAs as demonstrated by the negative subsequent T-cell FXM, the decreased mean channel shift of the positive B-cell FXM with a significant decrease in DSA mean florescence intensity post temporary splenic transplant. The patient experienced an isolated incidence of acute rejection, which responded to therapy. He had no infectious or cancerous sequelae from the immunosuppression modalities. He was able to discontinue total parenteral nutrition and gained weight after the procedure. Long-term effects are not able to be determined from this approach; hence, further research is warranted to better evaluate the real efficacy of this strategy.

Reassessment of the clinical impact of preformed donor-specific anti-HLA-Cw antibodies in kidney transplantation.

Anti-denatured HLA-Cw antibodies are highly prevalent, whereas anti-native HLA-Cw antibodies seem to lead to random flow cytometry crossmatch results. We aimed to reassess crossmatch prediction for anti-HLA-Cw using 2 types of single antigen flow beads (classical beads and beads with diminished expression of denatured HLA), and to compare the pathogenicity of preformed anti-denatured and anti-native HLA-Cw antibodies in kidney transplantation. We performed 135 crossmatches with sera reacting against donor HLA-Cw (classical beads fluorescence ≥500); only 20.6% were positive. Forty-three (31.6%) were anti-denatured HLA antibodies (beads with diminished expression of denatured HLA fluorescence <300); all were crossmatch negative. The correlation between classical beads fluorescence and the crossmatch ratio was low (ρ=0.178), and slightly higher with beads with diminished expression of denatured HLA (ρ=0.289). We studied 52 kidney recipients with preformed anti-HLA-Cw donor-specific antibodies. Those with anti-native HLA antibodies experienced more acute and chronic antibody-mediated rejections (P=.006 and .03, respectively), and displayed a lower graft survival (P=.04). Patients with anti-native HLA-Cw antibodies more frequently had previous sensitizing events (P<.000001) or plausibility of their antibody profile according to known anti-native HLA-Cw eplets (P=.0001). Anti-native but not anti-denatured HLA-Cw antibodies are deleterious, which underscores the need for reagents with diminished expression of denatured HLA.

OR10 Data-driven modeling of flow cytometric crossmatch: Enhanced virtual crossmatching

Aim Increasingly HLA laboratories are using virtual crossmatching to predict recipient and donor compatibility using HLA antibody data and donor HLA type. However, virtual crossmatch interpretation is based on HLA experience and expertise of individual transplant centers. The purpose of this study is to develop data-driven algorithms (DDA) that predict flow cytometric crossmatch (FXM) outcomes using HLA antibody mean fluorescent intensity (MFI) data and donor HLA typing. These methods are independent of human intuition or experience and may provide insights that are otherwise imperceptible. Methods Two data sets consisting of 222 and 109 FXM with single antigen bead data for both HLA class I and II antibodies were used. Single antigen bead data was compiled against donor HLA antigens using the OneLambda assay and Luminex. The first DDA found the optimal MFI threshold using summation of mean fluorescent intensity data for class I and/or II that predicted either T cell or B cell mean channel shifts (MCS) above FXM cutoff. The second DDA applied a least-squares regression model to the HLA locus-specific data (second data set) to predict the actual T or B cell MCS. Results The threshold method yielded between 84.9% and 91.1% accuracy when using class I donor specific antibody (DSA) data to predict T cell outcome, and class I and II DSA data to predict B cell outcome. Optimal MFI thresholds of 3450 and 7560 were found for T and B cell prediction. For quality assurance, prediction of T cell MCS was attempted using class II data, resulting in 61.9% accuracy. The least-squares model increased accuracy to 93.6% and 97.2% for T and B cell MCS, respectively. Class I DSA influenced T and B cell MCS more than class II. The relative importance of an individual HLA locus on T cell prediction was found to be HLA-B > -A > -C. In the least-squares fitting, B64 had a large positive effect while A34 had a large negative effect on T cell MCS. Conclusions Utilizing DDA can expand accuracy of biologic systems beyond human experience and expertise. We showed how DDA can improve HLA crossmatch by generating high-level accuracy T and B cell FXM outcomes. Further improvements to the algorithm will incorporate HLA antigen expression and HLA antibody avidity.

P126 Comparison study of CDC and flow crossmatch vs. donor specific antibodies in transplantation

Aim Our approach to transplant highly sensitized patients has been to perform CDC crossmatch (XM) when the results of flow xm are above 200 mean channel shifts (MCS) with donor specific antibodies (DSA). The aim of our study was to determine if the flow crossmatch and solid phase antibody testing can be used as the only measures for decisions to transplant. Methods Results of flow xm, CDC xm and solid phase DSA testing were analyzed for 44 donor and recipient pairs. In all cases, the flow xm were positive with greater than 200 MCS values. Undiluted patient sera were tested by Luminex single antigen (LSA) test for HLA antibodies (ABS). The ABS detected by this method were grouped into weak (MFI 2500–5000) moderate (MFI 5000–7500), and strong (MFI 7500-greater than 17000). The CDC xm was performed with and without DTT treatment using the same sera for flow xm. Results In 70% of this group with positive T and/or B CDC xm at least one DSA detected was in the strong binding range above 17,000 MFI. In 30% of cases multiple DSA detected in weak to moderate range below 7500 MFI values. In 50% of neg T cell and 96% of neg B cell CDC crossmatches, the MFI of DSA detected was in moderate to strong range. In 90% of negative CDC xms the MCS values for flow T was below 300 and flow B below 400 MCS. The range of flow MFI values for positive CDC B cells xm was 156–596 and 238–671 for T cells. Conclusions While the CDC xm test is the classical standard to exclude transplant, it is not always a reliable test. This assay is quite subjective and dependent on quality and strength of complement, technical skills of the laboratory technician and donor cell viability. This assay is also not reliable when patient is under desensitization therapy. There is a need to develop this assay using flow cytometer platform. Use of solid phase testing methods and determination of standards for decision to transplant based on number, type and strength of antibodies can be a better method

P149 Anti-angiotensin ii type 1 receptor antibodies are associated with positive flow crossmatches in heart Transplant Candidates

Aim Heart transplant (HTX) candidates with pre-formed anti-HLA antibody (HLA-Ab) have lower transplantation rates and increased waiting time due to positive crossmatches. Anti-angiotensin II type 1 receptor antibody (AT1R-Ab) has recently been associated with allograft loss and antibody-mediated rejection. The aim of the present study was to evaluate: 1) the prevalence of anti-angiotensin receptor 1 antibody (AT1R-Ab) in HTX candidates and 2) the impact of AT1R-Ab on flow cytometry crossmatch (FXM). Methods 115 HTX candidates were tested by ELISA for the presence of AT1R-Ab (U/mL after 1:50 dilution, positive cut-off = 10 U/mL, strong-positive cut-off = 20 U/mL. 43/115 cases were identified with either positive (N = 15) or negative (N = 28) flow crossmatches in the absence of any donor-specific anti-HLA alloantibody (virtual negative crossmatches for HLA antibody). Mean channel shift (MCS) for flow cytometry crossmatches was measured on a 1024 scale. Finally, two index HTX cases with pre-formed AT1R-Ab and positive flow crossmatches are discussed. Results A high proportion of HTX candidates with HLA-Ab exhibited AT1R-Ab (Strongly reactive AT1R-Ab 46/115, 40%; weakly reactive AT1R-Ab 25/115, 21.7%; no AT1R-Ab 44/115, 38.2%). Furthermore, AT1R-Ab levels were significantly higher in patients with a positive versus a negative flow crossmatch (54.81 ± 54.92 versus 9.04 ± 7.36, p = 0.00001, Fig. 1). In addition, MCS was significantly higher in patients with AT1R-Ab versus patients without antibodies (73.95 ± 94.21 versus −51.45 ± 52.07, p = 000003, Fig. 2). In the 15 cases with positive flow crossmatch without HLA-specific antibody, all 15 patients exhibited AT1R-Ab, 11/15 had strong AT1R-Ab, while only 5/25 patients with negative FXM exhibited AT1R-Ab, Chi-square = 24, p = 0.00001). Conclusions More than half of HTX candidates exhibit AT1R-Ab, which can interfere with flow crossmatch interpretation even in the absence of HLA-specific antibodies.

P119 Correlation between the strength of donor specific antibody (DSA) using single antigen bead assay (SAB) and the results of CDC- cytotoxicity and flow cytometric crossmatch

Aim In this study we correlated flow and CDC- crossmatch outcomes with results of Luminex-single antigen bead (SAB). Methods Between 2010–2016 HLA-antibody Sera were tested by screening and SAB class I & II (One lambda) according to manufacturer recommendation. MFI of ⩾1000 were considered positive. These sera were used to perform the following: 1. 80 crossmatches using isolated PB or spleen lymphocytes from healthy deceased or living donors respectively by CDC–crossmatch. Correlation was performed on sera with single antibody or multiple classes I and II antibodies. Titer of CDC was given as the inverse of the highest dilution yielding positive XM. 2.108 FCXM cross using isolated PB donor lymphocytes expressing one HLA- antigen (class I or II) and 78 crossmatches using cells expressing multiple antigens detected by the sera. Analysis performed on FACSCanto II & mean channel shift (MCS) was calculated. The cut off for T &B are 64, 100 respectively. T- Positive, B- negative results in the absence of HLA- antibody were excluded from the study. Results Our results indicate good correlation of FCXM and CDC-XM with LUMINEX -SAB assay. SAB assay has good correlation with T-FCXM (r2 = 0.7) but not with B-FCXM. Fig. 1 A cutoff MFI of 3000 for SAB assay (as single or sum of multiple weak antibodies) had a sensitivity & specificity of 100% in detecting a positive FCXM. The few number of sera with single anti HLA-Cw and DP antibodies did not give positive reaction up to 5000 and 10,000 MFI respectively but were not included in the study. Single anti HLA-A, B, DRB1of ⩾10,000 MFI was associated with positive CDC-XM in most cases and multiple low levels antibody that yield this 10,000 would also give positive reaction. We noticed presence of A2 specificity among multiple anti class I or class II antibodies increased the chance of positive reaction even when sum of MFI is ⩽10000. Conclusions SAB correlates significantly with outcome of both cytotoxic and T-FCXM. Based on the threshold established it is possible to predict correctly results of crossmatch. Download : Download high-res image (164KB) Download : Download full-size image

P154 Variability in the sensitivity of the flow cytometric crossmatch

Aim The sensitivity of the flow cytometric crossmatch (FXM) is often variable across labs in the presence of weak donor specific antibodies (DSA). In addition, inter-laboratory variation to the interpretation of Mean Fluorescence Intensity (MFI) values escalates the potential for discordance. The aim of this study is to compare FXM results among laboratories and correlate the results with DSA strength. Methods The study collected and analyzed data reported by participants in the Flow and Virtual Crossmatch Exchange over a period of 4 years. Data collected included antibody specificities, MFI values, T- and B-cell positive cutoffs, mean channel shifts (MCS), and negative control MC. In addition, the coefficient of variations (%CV) were calculated using the center-specific MFI values to analyze the variability in the reporting of antibody strength among labs. Results A total of 24 cells were tested against 48 sera over 12 Exchanges for a total of 96 T/B flow crossmatches. Among the 96 FXM pairs, 84% of T-cell and 88% of B-cell crossmatches were reported with good agreement (>80%). Positive concordant T-cell crossmatches were observed in 92% (57/62) of cases when class I DSA were reported with MFI >3000. Positive concordant B-cell crossmatches were observed in 89% (49/55) of cases when class I and class II DSA were reported with MFI >5000. Conclusions The data shows there is good consistency in T/B Flow crossmatch outcomes among labs in the presence of moderate or strong DSA. However, when DSA strength is weak, the sensitivity of the flow crossmatch is more widely variable. In addition, center practice, such as cutoff values and the reporting of MFI values may influence FXM outcomes. Thus, educational activities should continue to provide standards for quality control for the FXM.

False-Positive Flow-Cytometric T Cell Crossmatch caused by Blood Type Antigen Expressed on Lymphocytes in ABO Incompatible Kidney Transplantation

Background Flow-cytometric T cell crossmatch (FTXM) uses live donor lymphocytes to detect anti-donor-human leukocyte antigen (HLA) IgG antibodies. Therefore, the results can be affected by individual cell surface molecular expression. Herein we report three cases with FTXM false-positive living-related kidney transplants whose positivity was influenced by the expression of incompatible ABO blood-type antigens. Patients and Methods The kidney recipients were three men with blood type O who received from their wives with blood type B. Their anti-B IgG titers were 1:32, 1:128, and 1:256. Lymphocyte cytotoxic assays were negative in all cases. Median of mean channel shift (MCS) in FTXM was 78.7% (range, 52.8%–85.7%). There were no anti-HLA (either donor-specific or not) class I IgG antibodies in all 3 cases detected by a solid-phase bead assay. We hypothesized that non-HLA antigens expressed on T lymphocytes affect false-positive FTXM results. To examine this, we incubated the patients’ sera with donor-type red blood cells (RBCs) to adsorb anti-ABO antibodies prior to FTXM. Results When the incubated sera were tested in FTXM with donor T lymphocytes, the results were negative in all cases with median MCS 3.75% (0.0%–7.5%). To clarify the expression of ABO antigens on the donor T lymphocytes, we incubated donor T lymphocytes with recipients’ sera and then incubated the cells with acid solution to detach the bound antibodies from the T lymphocytes. The eluates showed agglutination in indirect Coombs test with blood type B RBCs but no agglutination with O blood type RBCs. This indicates that the donor T lymphocytes expressed blood type B antigen and anti-blood type IgG antibodies reacted with the B antigen. These results show that anti-blood type IgG antibodies can bind to ABO-blood type antigens on the surface of T lymphocytes, resulting in positive FTXM. The false-positive FTXM can be excluded by short-term incubation of recipients’ sera with donor-type RBCs. Conclusion In ABO-incompatible kidney transplantation, anti-blood type IgG antibodies can be detected as anti-donor IgG antibodies in FTXM.

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.

Flow cytometric crossmatch results using lymphocytes isolated from donor peripheral blood and spleen tissue on five consecutive days

A negative flow crossmatch (FXM) is considered as a “go ahead” to transplant solid organs at many centers. FXM is a biologic assay where donor lymphocytes are reacted with recipient sera to identify donor specific antibodies (DSA). Lymphocytes can be derived from different sources like peripheral blood (PB), lymph node or spleen. Mix of cellular components from all three sources are different and also may have different HLA expression depending on the age of the sample. We present our data to understand this variability. Lymphocytes were isolated from PB and spleen of the same donor on five consecutive days using a different sample aliquot. Lymphocytes from PB were isolated by Ficoll–Hypaque technique. Lymphocytes from spleen tissue were isolated using a cell separation kit (Stemcell Technologies). Recovered cells were treated with 1mg/2mL pronase and re-suspended for testing in PBS containing 2% fetal calf serum. FXM was performed in a tray after mixing 3×105 cells with 20 μl of serum. After washing, cells were labeled with FITC goat anti-human IgG, anti-CD3 PerCP and anti-CD19 PE then acquired on a FACS Calibur to detect DSA. A comparison of the FXM mean channel shift (MCS) results for PB and spleen with a cutoff of 52 for T-cells and 106 for B-cells were made for 5 consecutive days. The negative control mean fluorescence intensity (MFI) increased with specimen age for PB and spleen (Figure A), which caused a decrease in MCS in the positive control over time for both lymphocyte sources (Figure B). Patient samples with FXM results close to the T-cell and B-cell cutoffs were discrepant based on lymphocyte source and age of specimen (Table 1). Older samples have a higher negative control which results in a decrease in the MCS and may cause borderline positive results to be reported as negative. The isolation of lymphocytes from PB by day 5 proved to be difficult as the prep was contaminated with whole blood components. Our data showed that the interpretation is dependent on the donor sample source and quality.Download : Download full-size image

Are IgM donor-specific hla antibodies (DSA) clinically relevant?

Aim The clinical relevance of IgM alloantibodies (Ab) directed against donor HLA (IgM DSA) in solid organ transplant recipients is not clear. To study this issue, we conducted a retrospective study utilizing flow cytometric crossmatching and solid-phase antibody testing. Methods From 1998 to 2006 we incorporated an anti-IgM reporter antibody into our flow cytometric crossmatch (FCXM) assay. The frozen sera from the patients that had a clearly positive IgM crossmatch ( ⩾ 40 mean channel shift or MCS, 256 scale) and were transplanted (negative IgG crossmatches) were tested for IgM HLA reactivity by a solid-phase screening assay. The sera that were positive on the screening assay were then tested for IgM HLA specificity by single-antigen beads. If IgM antibodies specific for HLA were identified, they were then compared to the HLA type of the transplant donors. Graft/patient outcome was obtained from UNet. Results In 10,150 IgM FCXMs, 2877 resulted in transplant. The IgM FCXM was positive (MCS ⩾ 15 ) in 158 (5.5%) of the cases. In 59 patients with IgM FCXM MCS ⩾ 40 , 29 were positive for IgM HLA Abs in the screening assay. IgM DSA were detected in 18 recipients (3 deceased donor renal, 6 living donor renal, 1 renal/pancreas, 1 pancreas, 3 renal/liver, 1 liver, and 3 lung). The antibodies ranged from weak (1000 MFI) to strong (10,000 MFI). Class I IgM DSA were detected in 15 recipients, Class II IgM DSA were found in one recipient, and 2 recipients had both. There was one case of a lung transplant recipient who died 12 days post-transplant, and the cause of graft loss was listed as acute rejection in UNet. Serum samples obtained at 3 and 11 days post-transplant were negative for IgG DSA, and the IgM DSA were similar to those detected in the pre-transplant serum. All other patients were either lost to follow up ( N = 5, range = 1–6 yr), had a functioning graft at last report ( N = 8, range = 7–14 yr), died with a functioning graft ( N = 3, range = 1 mo–9 yr), and one patient had graft failure due to drug toxicity at 5 yr. Conclusions IgM Abs specific for HLA are relatively uncommon, compared to IgG (data not shown). In the relatively small number of patients that were identified as having IgM DSA, except for a single recipient where we could not exclude the effect, there was no evidence that IgM DSA were detrimental to graft function/survival.

Prospective Iterative Trial of Proteasome Inhibitor-Based Desensitization

A prospective iterative trial of proteasome inhibitor (PI)-based therapy for reducing HLA antibody (Ab) levels was conducted in five phases differing in bortezomib dosing density and plasmapheresis timing. Phases included 1 or 2 bortezomib cycles (1.3 mg/m(2) × 6-8 doses), one rituximab dose and plasmapheresis. HLA Abs were measured by solid phase and flow cytometry (FCM) assays. Immunodominant Ab (iAb) was defined as highest HLA Ab level. Forty-four patients received 52 desensitization courses (7 patients enrolled in multiple phases): Phase 1 (n = 20), Phase 2 (n = 12), Phase 3 (n = 10), Phase 4 (n = 5), Phase 5 (n = 5). iAb reductions were observed in 38 of 44 (86%) patients and persisted up to 10 months. In Phase 1, a 51.5% iAb reduction was observed at 28 days with bortezomib alone. iAb reductions increased with higher bortezomib dosing densities and included class I, II, and public antigens (HLA DRβ3, HLA DRβ4 and HLA DRβ5). FCM median channel shifts decreased in 11/11 (100%) patients by a mean of 103 ± 54 mean channel shifts (log scale). Nineteen out of 44 patients (43.2%) were transplanted with low acute rejection rates (18.8%) and de novo DSA formation (12.5%). In conclusion, PI-based desensitization consistently and durably reduces HLA Ab levels providing an alternative to intravenous immune globulin-based desensitization.

P112 : COMBATING CROSS-REACTIVITY IN HLA-B27 DETECTION BY FLOW CYTOMETRY

Presence of HLA-B27 (associated with ankylosing spondylitis, juvenile rheumatoid arthritis, and Reiter’s syndrome) on leukocytes in whole blood can be detected with monoclonal antibodies by a rapid qualitative two-color direct immunofluorescence flow cytometric method. In this method cross-reacting glycoproteins can provide a positive result; thus, some samples need to be confirmed by an alternate test method. We present an overview of our reagent quality control as well as observations requiring modification to the laboratory process. Whole blood samples are tested with monoclonal antibody GS145.2 [Becton, Dickinson and Company Biosciences (BD), San Jose] as per the manufacturer’s suggested protocol acquired on FACS Calibur (BD) utilizing the manufacturer provided template. Results are displayed as mean channel shift and reported as HLA-B27 negative or positive sample based on the kit decision marker. The kit package insert references a study in which some cross-reactive samples fell within a ten-channel zone above the decision marker. Results that fall within this zone should be confirmed by an alternate method. It is our protocol to utilize a molecular method to confirm this zone above the decision marker as well as below (±10). For reagent quality control it is our practice to select two negative, two ±10, and two positive from the current lot and then re-test with the new lot. The HLA typing for these samples is confirmed using a molecular method. Sample 4 for both comparisons was false positive in our test system. QC summary.SampleNew lot 1 comparisonNew lot 2 comparisonCurrent lot MCF decision marker 147New lot MCF decision marker 150HLA-B typingCurrent lot MCF decision marker 150New lot MCF decision marker 149HLA-B typing188818.441041247.8293848.5172828.3531431567.715715927.6541531617.371501637.4251681738.271631657.27617918527.2716216327.35 QC summary. These reagent quality control results caused us to increase the +10 to +15 above the decision marker for a period of time to monitor the findings. Although this change led to more work and expense in the laboratory, it was justified by patient safety. While following the manufacturer’s suggestions are critical, a stringent internal quality control program can identify samples providing discrepant results.

P027 : DONOR SPECIFIC HLA ANTIBODIES DETECTED BY LUMINEX CANNOT PREDICT THE OUTCOME OF A CDC OR FLOW CYTOMETRIC CROSSMATCH IN AN INDIVIDUAL PATIENT

For clinical purposes it is necessary that before a renal transplantation takes place, the patient is screened for the presence of circulating HLA antibodies that may harm the transplanted kidney. Antibody analysis is nowadays often performed by testing the reactivity towards HLA molecules bound to Luminex beads. The prospective crossmatch, however, is performed either by the complement-dependent cytotoxicity assay (CDC) or by flow cytometric assays. In the current study we compared the outcome of the luminex single antigen (SA) analysis with respect to the presence of donor specific antibodies (DSA) and the crossmatch result of 125 patients. CDC crossmatch results (pos/neg) and flow cytometric T cell and B cell crossmatch results (mean channel shift) were related to anti-class I or anti-class II reactivity in luminex (MFI). Results On the population level, there was a significant association between the sum of the MFI’s of the donor reactive HLA class I and II antibodies and outcome of the CDC cross match. However, when the significance for individual patients was analysed, the situation is less clear. Numerous negative CDC crossmatches were observed in case of high DSA levels in luminex ( Fig. A ). With respect to the flow T cell crossmatch, a higher channel shift was observed when anti-donor luminex reactivity increased but, in general, the correlation was poor. Similar findings were observed for the flow B cell crossmatch ( Fig. B ). In conclusion, despite the presence of a clear correlation between DSA detected by Luminex and the crossmatch result on the population level, for an individual patient one cannot rely on measurement of DSA by luminex only. This observation is very important if one considers the introduction of a virtual crossmatch. A combination of techniques will be necessary in order to predict in a reliable way the outcome of a crossmatch in sensitized individuals.

Antibody-mediated rejection: analyzing the risk, proposing solutions.

Antibody-mediated rejection: analyzing the risk, proposing solutions.

169-P: SIGNIFICANCE OF FLOW CYTOMETRIC CROSSMATCH IN THE REALM OF VIRTUAL CROSSMATCH: A CASE OF ALTERED HLA EXPRESSION ON CELL SURFACE

Aim Refinement and better understanding of solid phase assays (SpA) to detect anti-HLA antibodies has resulted in a good correlation between actual crossmatch (XM) and a virtual crossmatch (VxM). This has lead to a greater reliance on VxM for organ allocation and selection. We present a case of weak positive flow cytometric XM (FxM) that was predicted to be a strong positive FxM on the basis of VxM. Methods Case: 46 year old male with diabetes for second renal transplant. Patient is alloimmunized and demonstrated allele specific antibody to the donor (DSA) HLA-DQB1∗02:01-DQA1∗05:01 (DQ2.5) with mean fluorescence intensity (MFI) of 15413. In our laboratory this would be predicted to result in a strong positive FxM defined as mean channel shift (MCS) >250 (cutoff >106). B-cell FxM (B-FxM) with donor cells was positive with MCS: 173 and presence of an autoantibody. Results Investigations for this case of low positive XM with an autoantibody and DSA MFI 15413 are summarized: 1. Repeat B-FxM MCS: 171 2. B-FxM with index donor cells and serum from three other patients with DQ2.5 antibody around 15000 MFI was low positive:[ Table 1 ] 3. B-FxM using index patients serum and a surrogate donor with DQ2.5 was MCS: 288 (strong positive). 4. HLA-DQ2 expression on donor cells by flow cytometer using monoclonal (Mab) anti-DQ (FM5148) and anti-DQ2 (FD100) antibodies revealed no DQ2 expression on the donor cells.[ Table 2 ]Patient was transplanted (Tx) and monitored post-Tx with SpA. At 2 months post-Tx serum creatinine is 1.0 mg/dL and DQ2.5 MFI: 13276. Conclusions This is a case of low positive FxM in the presence of DSA MFI around 15000 due to altered HLA expression on donor cells. Only a VxM could have resulted in the patient being denied Tx indicating that actual XM is required for organ allocation and selection. Chen: Thermo Fisher Scientific: Employee. Lee: Thermo Fisher Scientific: Employee.

5-OR

Aim Recent literature suggests that the presence of donor specific anti-HLA antibodies (DSA) may result in delayed or failure to engraft in bone marrow transplantation (BMT). We present a case of successful BMT in the presence of DSA. Methods Case: 54 year old female with therapy related myelodysplastic syndrome and excess blast for BMT. Patient had no matched siblings and NMDP search identified two donors that were 9/10 match at HLA-A, B,C,DRB1 and DQB1. She is highly alloimmunized and serum demonstrated multiple anti-HLA antibodies including anti-HLA-DP antibodies using single antigen beads (SAB, One Lambda). Thus both the donors were also typed for HLA-DPB1. Selected donor was mismatched at HLA-DQB1 and DPB1. [ Table 1 ] Results Pre-transplant (Tx) SAB revealed presence of DSA to HLA-DPB1*04:02 that demonstrated a prozone phenomenon and B-cell flow crossmatch with the donor cells was positive with mean channel shift=346 (cutoff : >106). Clinical outcome: Plasmapheresis one month before transplant failed to significantly reduce DSA. As there were no additional therapy options, after discussing the relevant risks with the patient we proceeded to Tx. Pre-Tx conditioning included plasmapheresis on days −3 to 0. Patient engrafted by day 15 post-Tx. To characterize the DSA, patient samples were tested for C1q and IgG subtypes. Results demonstrate a post-Tx decrease in DSA and it is predominantly IgG1 subtype and C1q positive. [ Table 2 ] Conclusions Successful BMT is possible in the presence of DSA. This case demonstrates that mere presence of DSA may not be the cause of delayed or failure to engraft in BMT. Evaluation of additional mechanisms and hypothesis is required. Chen: Thermo Fisher Scientific: Employee. Lee: Thermo Fisher Scientific: Employee.

20-OR

Aim The clinical significance of low strength donor specific antibodies (DSA) is not clearly understood. We evaluated the outcome of 322 renal allograft recipients with various degrees of immunologic risk, transplanted from 2009 to 2012. Methods Antibody screening and identification were performed using the One Lambda Single Antigen Bead (SAB) assay. Patients with DSA mean fluorescence intensity (MFI) lower than 5,000 and flow-cytometry T and B cell cross-match channel shift values lower than 150 and 250, respectively, were accepted for transplantation. All patients with DSA received thymoglobulin (total 6 gram) and IVIG (2 gram/kg). To monitor DSA, the SAB assay was performed at 1, 3 and 12 months post-transplantation. Results Patients with pre-tx DSA (N = 54) had on average 1.67 ± 0.83 DSAs with a mean MFI of 2926 ±2904. Mean channel shift for flow cytometry T and B cell cross-match was 136 ± 50 and 162 ± 60, respectively. During a median follow-up of 37 months, there were no significant differences between the 2 groups in terms of patient and graft survival, acute rejection rate, serum creatinine levels and development of BKV and CMV viremia (Table). Twenty eight patients lost 44 DSA and there was a significant decrease in post-transplant mean number of DSAs (0.69 ± 0.85) and mean MFI (1185 ± 2202) (p Table 1 ]. Conclusions We demonstrate that similar patient and graft survival rates can be achieved in patients with pre-transplant DSA compared to patients without DSA by careful assessment of the immunologic risk prior to transplantation.

Effects of mycophenolic acid on highly sensitized patients awaiting kidney transplant

Background10–30% of dialysis population awaiting renal transplantation is sensitized. Mycophenolic acid (MPA) has been shown to reduce panel reactive antibody (PRA) formation in kidney transplant recipients. Our aim was to investigate whether MPA could effectively reduce anti-HLA antibody levels and allow successful transplantation. MethodsA total of 40 highly sensitized patients were treated orally with MPA. All patients had T-cell PRA values greater than 30% (73% of patients were ≥75%). The PRAs, T-cell/B-cell flow cytometry crossmatch (FCXM) mean channel shift (MCS), patient/graft survival, acute rejection, and serum creatinine (SCr) were recorded. ResultsAll 40 patients showed a decrease in PRA levels. Eighteen of the 40 patients (40%) received a transplant. All four living donor recipients converted to a negative crossmatch. There was a significant decrease in FCXM MCS in all 18 transplanted patients. The mean SCr at 24months was 1.00±0.25mg/dL. Five patients (28%) experienced acute rejection. The overall one year actuarial patient and graft survival were 94% and 88%, respectively. ConclusionsThese findings suggest that MPA therapy is effective in reducing PRAs and increases the likelihood of successful transplantation in sensitized recipients in a potentially simpler and more cost effective manner than the current regimens employed.

Long‐term histopathology of allografts in sensitized kidney recipients

Successful desensitization therapy has brought satisfying short-term outcomes in the recipients with anti-donor antibody. We analyzed the long-term pathology of the allografts in the sensitized kidney recipients. Eleven stable recipients after desensitization against positive flow cytometry T-cell crossmatch (FTXM) were included. They were divided into two groups, based on the protocol biopsies findings at three to eight yr (group 1: subclinical glomerulitis and/or peritubular capillaritis, n = 5 and group 2: no rejection, n = 6). Estimated glomerular filtration rate (eGFR), presence of donor-specific antibody (DSA), mean channel shift (MCS) of FTXM, urine protein levels, acute antibody-mediated rejection (AAMR) episodes, and protocol biopsy findings were compared. Chronic transplant glomerulopathy was found in final biopsy of all group 1 cases. DSA was positive in 60% but C4d was positive in 20% case of the group 1. The history of AAMR was only found in the group 1. There was no difference in eGFR decline or proteinuria. The MCS of FTXM was higher in the group 1. The recipients with AAMR history, high MCS in FTXM, and subclinical microvascular inflammation in the early protocol biopsies have risk for developing chronic rejection in long term.