Positive Flow Crossmatch Research Articles

Successful Combined Liver and Kidney Transplant with Positive Flow Crossmatch and Multiple High Donor Specific Antibody Levels

Successful Combined Liver and Kidney Transplant with Positive Flow Crossmatch and Multiple High Donor Specific Antibody Levels

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Aim A 58/F/C kidney candidate with negative auto-B XM and CPRA = 35% appeared on a UNOS matchrun. The donor typed as A2,A31;B35;Cw4, lacking the B8, B64, B65, B39, B18,and B78 unacceptable antigens previously identified by both single antigen ELISA and luminex assays. However, the T-AHG CDC XM was positive with 5 months of current sera. The T-3wash and B-37 CDC XMs were negative. Retrospective T and B flow XMs were strong positive. Methods Frozen T cell panel with AHG showed 26% PRA with no specificity. Cells bearing B8, B64, B65, B39, and/or B18 were mostly negative. By considering “2” rxns, a PRA of 60% with possible weak anti-A3, B7, B58, and A31 was seen. The HLA-A31 was a mismatched donor antigen leading us to conclude that this was a weak alloantibody not detected by solid phase testing. The patient’s spouse typed as HLA-A3,A32;B14,B-,Bw6, accounting for her anti-A3 and B14 alloantibodies. Results Further examination showed positive XMs with 5 prior deceased donors. Three of these donors had HLA-B7, two of whom also had A3. All of these donors were T XM positive with and without AHG, and one was also B-37 CDC XM positive. The two other donors were positive only by T-AHG and did not have any of the unacceptable antigens seen by solid phase or frozen cell tray. They did both have the B38 antigen, which might be crossreacting with the patient’s anti-B39 antibody. A sixth donor was negative for all CDC crossmatches and had none of the above antigens. Based on Schnaidt et al (Transplantation 2011;92:510) we retested the serum on the luminex single antigen panel following heat inactivation, addition of EDTA, dilution, and removal of IgM to see if a prozone effect or steric blocking was occurring. No additional antibody specificities were uncovered, and none appeared stronger. A C1q assay was negative except for a very weak anti-B44. Conclusions Thus, using solid phase single-antigen testing for prediction of crossmatch can miss antibodies that can cause positive CDC and flow crossmatches.

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Aim Low level antibodies may not always produce a positive flow crossmatch and increase the risk for transplant. The aim of this study was to investigate the impact of increasing the MFI cutoff for listing the unacceptable antigens (UA) in UNET. UA are use in virtual crossmatches and exlude recipients without performing crossmatches. Methods Antibody detection was performed by One Lambda LABScreen Single Antigen HLA Class I and II beads. The normalized MFI was used to calculate the cutoff. The MFI cutoff for a positive result was increased from 1,500 to 3,000. T and B cell crossmatches were performed by flow cytometry with pronase treated cells and reported as MCS. The T cell cutoff for positive was 61 and B cell was 93 which is 3 standard deviations above our negative control serum. The ten highest ranking recipients on the kidney runs from the first 8 deceased donor workups following the change were studied. Results Fifty six of the 126 actively listed patients from CTHH had UA entered in UNET. Low level UA were removed from 50/56 recipients. After the modification, eight recipients fell below and 21 stayed above 80% CPRA. The remainder had a CPRA range from 5-73%. 48 of the top 80 recipients on recent kidney match runs were from CTHH and further studied. 14/48 (29%) had low level antibodies present. 3/14 (21%) were flow T and B cell negative and the DSA was to DQ7 in all three crossmatches. Eleven were T and/or B cell positive. Four of the positive crossmatches were due to Cw, DQA, or DP antibodies which are not reported as UA. Three were due to low levels of multiple antibodies producing a cumulative effect. One was due to Bw6 antibody that reacted with all Bw6 beads on the Luminex assay but reacted below 3,000 MFI. The remaining four positive crossmtaches were due to historically higher MFI values. Conclusions Low level antibodies should not be added as unacceptable antigens and automatically rule out a recipient for kidney transplantation. Some crossmatches will be negative.

607 Which T-Cell Crossmatch Method Best Predicts Allograft Loss in Pediatric Heart Transplant Recipients?

Purpose: Several methods are currently available for performing a T-cell crossmatch (CM). No national cohort studies have explored the relationship between T-cell CM, CM methodology, and allograft survival after pediatric heart transplantation. Methods and Materials: All children 18 years old transplanted between 1/1999 and 12/2010 with an interpretable T-cell CM result, either by compliment dependent cytotoxicity (CDC) or flow cytometry, were identified using OPTN data. Children with a CDC-positive CM (N 83) and those with a CDC-neg/flow-pos CM (N 37) were compared with CDCneg/flow-neg controls (N 1225). Cox proportional hazard analysis was used to identify multivariable risk factors for time to graft loss. Results: During the past decade, utilization of CDC-CM has decreased significantly whereas the use of flow CM has increased (both P 0.01). Currently 1 in 10 pediatric heart transplants are performed in the context of a positive T-cell CM. Compared to CDC-neg/flow-neg controls, patients with a CDC-pos CM were more likely to be listed UNOS status 1A, have a pre-transplant diagnosis of congenital heart disease or re-transplant, and have a positive PRA (both class I and II). Utilization of pre-transplant mechanical circulatory support was not associated with CM result. CDCpos CM was associated with an increased prevalence of treated allograft rejection by 1 year compared to CDC-neg/flow-pos patients and CMnegative patients (48% vs. 19% and 23%; P 0.001). In multivariable analysis, patients with a CDC-pos CM were more likely to have graft loss than controls (HR 1.9; CI 1.3-2.6) while those with a CDC-neg/flow-neg CM did not show increased risk (HR 0.9; CI 0.4-2.0). Conclusions: Amongst a population of pediatric heart transplant recipients, CDC-pos T-cell CM was strongly associated with an increased risk of death or allograft loss. While the utilization of flow based assays has become more common, a positive flow CM may perform less well in identifying patients at risk for graft loss than the traditional CDC method.

87-P Post-transplant detection of a donor specific (epitope) antibody resulting in a positive flow crossmatch

87-P Post-transplant detection of a donor specific (epitope) antibody resulting in a positive flow crossmatch

220-P Case study: Positive T-cell flow crossmatch (TFXM)/negative B-cell flow crossmatch (BFXM) can be mediated by a Class I antibody

220-P Case study: Positive T-cell flow crossmatch (TFXM)/negative B-cell flow crossmatch (BFXM) can be mediated by a Class I antibody

Use of Intravenous Immune Globulin and Rituximab for Desensitization of Highly Human Leukocyte Antigen-Sensitized Patients Awaiting Kidney Transplantation

The article by Vo et al. (1) about transplanting highly sensitized patients is one of their most recent contributions in trying to tackle this recalcitrant problem. The authors used a rituximab and intravenous immune globulin desensitization protocol, which is simple to administer. This, however, together with the fact that the Cedars-Sinai group is well respected and that the protocol is proposed for highly human leukocyte antigen-sensitized patients (as shown in the title) is promoting its use worldwide with no results to justify the expense. Their definition of “highly sensitized” patients by flow panel reactive antibody is questionable. This test is sensitive but can give high percentage values with low or no sensitization (in contrast to the traditional cytotoxicity panel reactive antibody where high values usually go together with high titers). In addition, they do acknowledge that a number of their transplanted patients had flow crossmatches of less than 250 mean channel shifts, which they defined as “acceptable positive.” Many centers transplant these patients with increased immunosuppression but no desensitization. The authors mention that in some cases, patients received the treatment but the donor-specific crossmatch with the pretransplant specimen turned out to be low by chance. True, but this is certainly not a case of success because of the protocol. The authors also state that they would not transplant such patients without using their protocol, but they have not shown that patients with a weakly positive flow crossmatch do better with the protocol than without. Thus, it is not clear from the data how many patients would have had acceptable positive crossmatches before desensitization, or how many patients were treated and not transplanted. In addition, the effect of rituximab itself on alloantibody, and its indications in transplant patients, has not been established, as one of the authors acknowledged recently (2). Although a strict quantitative evaluation on the effect of rituximab plus intravenous immune globulin on human leukocyte antigen antibody levels remains to be done, the patients transplanted were obviously not highly sensitized against their donors. Indeed, at the end of the discussion, the authors themselves state that patients with lower donor-specific antibody levels benefit more and that no protocol deals well with highly sensitized patients. Some readers may miss this statement, but no one will miss the title. In summary, it has to be made clear that this protocol does not represent a solution for highly sensitized patients. Shehzad Rehman1 Herwig-Ulf Meier-Kriesche1 Juan Scornik2 1 Department of Medicine University of Florida Gainesville, FL 2 Department of Pathology University of Florida Gainesville, FL

47-P: Titer of Antibodies Can Be Estimated from Median Channel Shifts of Positive Flow Crossmatches

47-P: Titer of Antibodies Can Be Estimated from Median Channel Shifts of Positive Flow Crossmatches

458: Positive Flow Crossmatches Associated with Poorer Survival

Flow crossmatching (FCXM) is a sensitive technique to detect alloantibodies against donor HLA antigens. While used extensively in renal transplantation, the utility of FCXM for heart transplantation is unclear. This report summarizes a single center experience in correlating preoperative FCXM reactivity with long-term outcome.

94-P: Kinetics of DSA in a highly sensitized heart transplant recipient transplanted in the face of positive flow crossmatch and DSA

94-P: Kinetics of DSA in a highly sensitized heart transplant recipient transplanted in the face of positive flow crossmatch and DSA

Acceptable Donor-Specific Antibody Levels Allowing for Successful Deceased and Living Donor Kidney Transplantation After Desensitization Therapy

The aims of this study were to determine the level of donor-specific antibody (DSA) that allows for successful transplantation after desensitization with IVIG and rituximab and to identify patients at risk for antibody-mediated rejection (AMR). Pre- and posttransplant sera from 16 patients with DSA before desensitization were tested. Strength of DSA was determined by single antigen Luminex bead assay and results expressed as standard fluorescence intensity (SFI). T-cell flow crossmatch results were expressed as mean channel shifts (MCS). AMR was determined by biopsy and C4d deposition. Six had negative pretransplant flow crossmatches with a mean DSA of 8,805 SFI. Five had positive flow crossmatches (78-192 MCS) with mean DSA of 55,869 SFI. No patients in either group had AMR. Five had positive flow crossmatches (222-266 MCS) with mean DSA of 118,063 SFI. Three experienced AMR. The MCS and DSA levels for patients with AMR were significantly higher than patients without (P < or = 0.001). For patients without complications (n=7), DSA remained less than 10(5) SFI and usually decreased to approximately 10(4) SFI posttransplant for both class I and II. For patients with AMR (n=3), predominant increases in class II DSA more than 10(5) SFI were observed. All three patients continue to have DSA approximately 10(5) SFI with stable creatinine after treatment for AMR. Approximately 63% of patients were transplanted with a positive flow crossmatch. The results show that patients with DSA more than 10(5) and FCM more than 200 MCS are at higher risk for AMR. Treatment of AMR improves renal function without significant changes in DSA.

Clinical Significance of a Positive Flow Crossmatch on the Outcomes of Cadaveric Renal Transplants

Pretransplantation crossmatching is an integral part of kidney transplantation. Flow cytometric crossmatch (FCXM) is more sensitive than complement-dependent cytotoxic crossmatch (CDC-XM). However, the clinical significance of positive FCXM with negative CDC-XM is controversial. We evaluated FCXM in 455 consecutive deceased donor renal transplants. All had a negative CDC-XM. There were 341 T-cell and B-cell FCXM negative and 38 T-cell and B-cell positive. There was a higher percentage of retransplantations and HLA mismatches (26.3% vs 8.2%, P = .002 and 2.45 vs 1.99, P = .02, respectively) in the FCXM-positive group compared with the FCXM-negative group; 65.8% of the FCXM-positive patients had rejection compared with 49.3% of the FCXM-negative patients (odds ratio [OR] = 1.89, P = .06). FCXM-positive patients had a higher incidence of vascular rejection (28.9% vs 12.6%, OR = 2.68, P = .008). One- and 5-year graft survivals were 84% and 66% in the FCXM-positive group vs 90% and 75% in the FCXM-negative group. Censoring for patient death, 1- and 5-year graft survivals were 84% and 73% in the FCXM-positive group vs 94% and 82% in the FCXM-negative group. There was no difference in renal function between the 2 groups. In conclusion, a positive T-cell and B-cell FCXM transplant with a negative CDC-XM is associated with a higher incidence of rejection, twice the risk of vascular rejection, and a trend toward poorer graft survival.

DP reactive antibody in a zero mismatch renal transplant pair

This study describes molecular basis for positive B-cell flow crossmatch in a zero mismatch (0MM) transplant pair. Our end-stage renal disease patient was B-cell flow crossmatch positive with a 0MM deceased donor. DNA from the donor and patient were further typed by a high-resolution method. The patient's sera were tested for anti-HLA reactivity by single antigen bead using a Luminex platform. The patient and donor were found to be HLA identical except for a single DP allele MM (DPB1*0601). As there was no single antigen bead coated with DPB*0601, analysis of the amino acid residues of reactive and nonreactive DPB1 alleles was conducted. The results showed that all reactive alleles carried the amino acid D-E at residue 55 and 56 of DPB1. The MM allele DPB1*0601 also carries the DE 55-56 epitope. We conclude that positive B-cell flow crossmatch was likely the result of single MM in the DP locus.

Pretransplant HLA Antibodies Are Associated with Reduced Graft Survival After Clinical Islet Transplantation

Despite significant improvements in islet transplantation, long-term graft function is still not optimal. It is likely that both immune and nonimmune factors are involved in the deterioration of islet function over time. Historically, the pretransplant T-cell crossmatch and antibody screening were done by anti-human globulin--complement-dependent cytotoxicity (AHG-CDC). Class II antibodies were not evaluated. In 2003, we introduced solid-phase antibody screening using flow-based beads and flow crossmatching. We were interested to know whether pretransplant human leukocyte antigen (HLA) antibodies or a positive flow crossmatch impacted islet function post-transplant. A total of 152 islet transplants was performed in 81 patients. Islet function was determined by a positive C-peptide. Results were analyzed by procedure. Class I and class II panel reactive antibody (PRA) > 15% and donor-specific antibodies (DSA) were associated with a reduced C-peptide survival (p<0.0001 and p<0.0001, respectively). A positive T- and or B-cell crossmatch alone was not. Pretransplant HLA antibodies detectable by flow beads are associated with reduced graft survival. This suggests that the sirolimus and low-dose tacrolimus-based immunosuppression may not control the alloimmune response in this presensitized population and individuals with a PRA > 15% may require more aggressive inductive and maintenance immunosuppression, or represent a group that may not benefit from islet transplantation.

Biomarkers in transplantation: Prospective, blinded measurement of predictive value for the flow cytometry crossmatch after negative antiglobulin crossmatch in kidney transplantation

This prospective, blinded observational study was conducted to measure the predictive value the of flow cytometric crossmatch for biopsy-proven acute rejection, graft loss, or death following kidney transplantation. Patients were selected for renal transplantation on the basis of a conventional antihuman globulin cytotoxic T-cell crossmatch. Flow crossmatch was performed simultaneously, but the results were not disclosed to the transplant team. A total of 257 kidney transplant recipients were enrolled in the study; 78 patients experienced biopsy-proven rejection in the first post-transplant year, and 41 patients lost their graft or died during the period of follow-up (mean: 2046 days). Kaplan-Meier estimates of rejection, graft loss, or patient death did not differ between subjects with a positive or negative flow crossmatch. Cox analyses showed no influence of the flow crossmatch on the risk of biopsy-proven acute rejection (P = 0.987). The sensitivity and specificity of the flow crossmatch for prediction of biopsy-proven rejection were 0.128 and 0.883, and the positive and negative post-test probabilities were 0.323 and 0.301, respectively. The magnitude of the channel shift did not influence the multivariate Cox regression model. The area under the receiver operating characteristic curve of the flow crossmatch was 0.483 (P = 0.71) and 0.572 (P = 0.38), respectively for the living and cadaver transplant recipients, indicating no discriminative value in this study population. Flow crossmatch appears to have no significant incremental value in predicting biopsy-proven acute rejection, graft loss, or death following kidney transplantation in patients who have a negative antihuman globulin cytotoxic T-cell crossmatch against their donor.

Steroid Avoidance Immunosuppression in Low-Risk Kidney Transplant Recipients

Recent clinical trials have documented the short-term safety of steroid avoidance (SA) in kidney transplant recipients. Since July 2003, we have used a SA immunosuppression protocol for low-risk kidney transplant recipients. Eligibility criteria are age ≥18, primary transplant (living or deceased donor), and tacrolimus started by postoperative day 3. Recipients were excluded if peak/current PRA was >50%/20%, or if they had a positive flow crossmatch, or if they had the recent use of corticosteroids (<6 months). All recipients received induction with rabbit anti-thymocyte globulin, total dose 6 mg/kg, or basiliximab. Recipients received 5 daily doses of corticosteroid and mycophenolate mofetil 1 gm twice daily starting on the day of transplantation. Tacrolimus was started when the serum creatinine level decreased by 20%, or by postoperative day 3. The goal for trough tacrolimus levels was 10–15 ng/mL for the first month, 8–12 ng/mL for months 2–3, and 5–10 ng/mL after month 3. Protocol biopsies (bx) were performed at reperfusion, 1 month, 4 months, and 12 months. Ninety-four kidney transplantations were performed during the study period. Sixty-seven recipients (71%) were eligible and enrolled in SA. Characteristics of the 67 SA recipients: mean age, 53 years (range, 26–70); 41% female; 67% Caucasian; 24% Hispanic; 15% African American; and 5% Native American. Also, 77% received a living donor kidney. The mean follow-up was 180 days (range, 10–360). At last follow-up, 91% remained steroid-free. Biopsy-proven acute rejection (BPAR) occurred in 5 recipients (7.5%). Three recipients (4.5%) had clinical BPAR and 2 had subclinical. One recipient died with pneumonia 4 months following transplantation. Posttransplantation diabetes mellitus (PTDM) occurred in 2 (5%) of 38 recipients. In the initial 41 recipients, 27 had protocol bx at 1 month and 13 at 4 months available for analysis. Chronic allograft nephropathy (CAN) was present on protocol bx in 48% at 1 month and 69% at 4 month. Actuarial (Kaplan-Meier method) patient and graft survival rates at 351 days were 97.8% and 96.8%, respectively. SA with anti-thymocyte globulin induction in low-immunologic risk kidney transplant recipients is safe and is associated with a low risk of BPAR. The incidence of PTDM appears to be lower.

THE RISK OF CORONARY TRANSPLANT VASCULOPATHY IN THE PATIENT BRIDGED WITH A VENTRICULAR ASSIST DEVICE

O343* Aims: A common consequence of left ventricular assist device (LVAD) implantation is the development of anti-HLA alloantibodies. While alloantibodies have been shown to be associated to vascular rejection the ultimate impact on vasculopathy is still ill defined. Previously observed the presence of Class I anti-HLA donor reactive antibodies was associated with significant early post-transplant vasculopathy. To examine this we obtained coronary angiograms from 196 patients bridged with LVAD and compared their post transplant coronary angiograms to a non-LVAD cohort (n=112) examined in a previous study. Methods: Adult patients undergoing orthotopic heart transplant between 1999-2000 were retrospectively studied and compared to our LVAD population. Coronary angiograms were retrospectively reviewed and severity of coronary vasculopathy was categorized as either normal, mild, moderate or severe. Other variables studied included cytotoxic panel reactive antibodies (PRA) against T-cell targets and flow cytometric crossmatching against donor t lymphocytes. Results: As anticipated there was an increase sensitization in those patients receiving a LVAD. Twenty-one percent of LVAD patients had a T-cell PRA greater than 10% at the time of transplant compared to 3.6% of the controls (p<0.0001, Fishers exact). Likewise, 25% of the LVAD patients had positive T-cell flow crossmatches compared to 5.4% of the controls (p<0.0001). Despite this coronary angiograms revealed no significant difference in the degree of coronary vasculopathy between the groups at follow-up. Normal coronary anatomy was detected in 72.5 % of LVAD patients and 64.6% of non-LVAD patients (p=0.79). These results were nearly identical at 2- and 3- year follow-up (71.8% vs. 64.6% and 58.3% vs.59.7%). Conclusions: While preoperative LVAD use is associated with a risk of recipient sensitization these patients develop transplant vasculopathy at the same rate as those not receiving a LVAD.

Transplant: immunology and treatment of rejection

Transplant: immunology and treatment of rejection

Flow cytometry beads rather than the antihuman globulin method should be used to detect HLA Class I IgG antibody (PRA) in cadaveric renal regraft candidates

HLA Class I antibody screening can be performed by flow cytometry using a mixture of 30 distinct bead populations each coated with the Class I antigen phenotype derived from different cell lines. In this study we compared the efficacy of Class I antibody screens done by flow cytometry beads with the antihuman globulin (AHG) method for patients awaiting cadaveric renal retransplantation. Class I panel reactive antibody (PRA) screening by flow cytometric beads of 21 regraft serum samples that had all been found to be negative by AHG DTT Class I PRA, revealed that 57.1% (12 of 21) had a flow Class I PRA of > or = 10%. Furthermore, when five regraft sera with an intermediate PRA were screened (mean AHG DTT PRA = 33.2 +/- 13%) the mean flow Class I PRA almost doubled (mean flow PRA = 72.4 +/- 10.2%) (p < 0.01). When active UNOS waiting list regraft candidates, after several months of screening the Class I PRA by flow beads, were divided into the three PRA categories based on their peak flow Class I PRA value (0-20%, 21-79% and > or = 80%), the incidence of a positive flow cross-match was 0%, 72% and 85% and the incidence of retransplantation was 60%, 22% and 10%, in each of these groups, respectively. These data provided our histocompatibility laboratory with the rationale to stop performing the AHG PRA and perform only the flow Class I PRA method for regraft candidates.

C4d staining of perioperative renal transplant biopsies1

Deposition of C4d in peritubular capillaries (PTCs) has been shown to be a sensitive marker for antibody-mediated (humoral) rejection in renal transplant biopsies. Some studies also suggest that C4d in PTCs is specific for humoral rejection or, at least, for the presence of donor-specific antibodies. However, in other studies, PTC C4d deposits were noted in more than 40% of renal transplant biopsies performed for graft dysfunction and capillary C4d deposition in heart transplants may result from ischemic injury. To test the specificity of C4d staining as a marker for acute humoral rejection ACR in renal allografts, indirect immunofluorescence using a monoclonal anti-C4d antibody and a fluorescein-isothiocyanate-conjugated secondary antibody was performed on cryostat sections of 90 renal transplant biopsies, including 35 pairs of preimplantation and 1-hr postreperfusion biopsies of the same graft, postreperfusion biopsies of 12 additional grafts, and 8 positive controls (biopsies with known C4d-positive AHR). Eighteen grafts were cadaveric, 17 grafts were liviing-related, and 12 grafts were living-unrelated (excluding controls). Included in these grafts were 13 grafts that developed AHR 3 to 34 days posttransplantation. Only 2 of 82 perioperative biopsies showed C4d staining in PTCs. Both perioperative biopsies were postreperfusion biopsies of grafts diagnosed with AHR 5 and 34 days posttransplantation, respectively, and, in each case, the recipient had been treated with plasmapheresis before transplantation because of a positive crossmatch (cytotoxic and flow cytometric) and continued to have a weakly positive flow crossmatch at the time of transplantation. In one biopsy, C4d staining was focal, and in the other biopsy, it was diffuse; in both biopsies, C4d staining was relatively mild (1+ on a 0-4+ scale). No C4d staining was noted on preimplantation biopsies of each graft. All biopsies that contained glomeruli showed linear capillary loop or blotchy mesangial staining, or both, which was similar in prereperfusion and postreperfusion biopsies. All positive controls showed diffuse C4d staining in PTCs. C4d staining in PTCs may be seen as early as 1 hr posttransplantation in some recipients with low levels of antidonor antibodies. However, this was not observed as a feature of ischemic or ischemia-reperfusion injury in perioperative renal transplant biopsies, including those of cadaveric grafts with cold ischemia times of as long as 41 hr.