Thomas Bachelet

Interpretation of Positive Flow Cytometric Crossmatch in the Era of the Single-antigen Bead Assay

Background. Prognosis of renal transplants with positive flow cytometric crossmatch (FCXM) remains controversial. Methods. We analyzed the outcome of these kidney transplant recipients by human leukocyte antigen (HLA) donor-specific antibodies (HLA-DSA) using single-antigen bead (SAB) assays in major histocompatibility complex classes I and II. We compared them with controls with a negative FCXM. Results. Forty-five patients consecutively transplanted with a positive FCXM had significantly more acute rejection episodes than the control patients (33.3% vs. 8.9%, P=0.002). Risk of acute rejection was increased with day 0 (D0) positive T-cell FCXM (odds ratio [OR]=9.04, P=0.002), D0 positive B-cell FCXM (OR=7.43, P=0.02), and D0 HLA-DSA identified by SAB assay (OR=6.5, P=0.03). The 21 patients with D0 positive FCXM and D0 HLA-DSA had more acute rejection (62%, P=0.0001) and a lower estimated glomerular filtration rate 1-year posttransplantation (P=0.0001), when compared with controls. Mainly anti-Cw and anti-DP HLA-DSA were found in patients displaying acute rejection. The remaining FCXM-positive patients displayed short-term outcomes similar to controls. The presence of HLA-DSA detected only by the SAB assay in the context of a negative FCXM crossmatch was not associated with increased risk of acute rejection. Conclusion. Identification of HLA-DSA in D0 sera by the two sensitive techniques FCXM and SAB assay indicates which patients are at highest risk of subsequent acute allograft rejection and chronic allograft dysfunction.

High incidence of anticytomegalovirus drug resistance among D+R- kidney transplant recipients receiving preemptive therapy.

Anti‐cytomegalovirus (CMV) prophylaxis is recommended in D+R− kidney transplant recipients (KTR), but is associated with a theoretical increased risk of developing anti‐CMV drug resistance. This hypothesis was retested in this study by comparing 32 D+R− KTR who received 3 months prophylaxis (valganciclovir) with 80 D+R− KTR who received preemptive treatment. The incidence of CMV infections was higher in the preemptive group than in the prophylactic group (60% vs. 34%, respectively; p = 0.02). Treatment failure (i.e. a positive DNAemia 8 weeks after the initiation of anti‐CMV treatment) was more frequent in the preemptive group (31% vs. 3% in the prophylactic group; p = 0.001). Similarly, anti‐CMV drug resistance (UL97 or UL54 mutations) was also more frequent in the preemptive group (16% vs. 3% in the prophylactic group; p = 0.05). Antiviral treatment failures were associated with anti‐CMV drug resistance (p = 0.0001). Patients with a CMV load over 5.25 log10 copies/mL displayed the highest risk of developing anti‐CMV drug resistance (OR = 16.91, p = 0.0008). Finally, the 1‐year estimated glomerular filtration rate was reduced in patients with anti‐CMV drug resistance (p = 0.02). In summary, preemptive therapy in D+R− KTR with high CMV loads and antiviral treatment failure was associated with a high incidence of anti‐CMV drug resistance.

Denatured class I human leukocyte antigen antibodies in sensitized kidney recipients: prevalence, relevance, and impact on organ allocation.

Background Single antigen flow beads assays may overestimate sensitization because of the detection of supposedly irrelevant antibodies recognizing denatured class I human leukocyte antigens (HLAs). Methods Sera of 323 HLA-sensitized kidney transplant candidates positive with a class I HLA single antigen flow beads assay were retested after acid treatment of the beads. Denatured HLA antibodies were identified according to ratio between the measured fluorescence intensity for treated and nontreated beads. T-lymphocyte flow cytometry crossmatches were performed to characterize the ability of these antibodies to recognize HLA on normal cells as a surrogate of their potential clinical relevance. Their impact on organ allocation was evaluated through a calculated panel reactive antibody. The utility of single antigen flow beads largely devoid of denatured HLA (iBeads) was also evaluated. Results Denatured HLA antibodies were detected in 39% of the patients. They provided much less positive flow cytometry crossmatches than anti-native HLA antibodies (16% vs. 83%, P<0.0001). Removing the HLA-A and HLA-B antigens targeted by denatured HLA antibodies from unacceptable antigens lowered the calculated panel reactive antibody for 90 patients, sometimes dramatically. The iBeads assay demonstrated nearly the same ability to predict crossmatch results than the acid treatment assay. Conclusion Denatured class I HLA antibodies are common, but the antigens they target should not be considered as unacceptable in most cases, because they negatively impact access to a transplant while predominantly providing negative sensitive crossmatches. The iBeads assay seems to be a valuable alternative to better define unacceptable antigens.

The complement interference phenomenon as a cause for sharp fluctuations of serum anti-HLA antibody strength in kidney transplant patients.

The single antigen flow bead (SAFB) assay greatly improves the identification of antigenic specificity of anti-HLA alloantibodies. However, it may underestimate or miss high titer antibodies due to the prozone phenomenon caused by a competition between the fluorescent anti-IgG conjugate and serum complement, for the alloantibody. We explored this effect in our cohort of transplant candidates and transplanted recipients. Among a total of 292 and 269 patients with at least three different sera tested with class I and/or II SAFB assays respectively, we identified 9 patients (6 in class I and 3 in class II) who displayed a profound drop (≥ 75%) followed by a subsequent rise (≥ 100%), in strong (mean fluorescence intensity >8000) antibody levels, across an 18-month period. We postulated that such abrupt fluctuations were not explainable by naturally occurring transient desensitization. Sera were analysed with the SAFB assay using EDTA-treated serum and direct complement C1q staining, and with complement-dependent cytotoxicity and flow cytometry crossmatches (CDCXM and FCXM respectively). The prozone phenomenon was involved in all cases. Because it relies on complement activation, the CDCXM was not sensitive to this phenomenon, but the FCMXM was not either, although it resembles in its principle to the SAFB assay. Four additional anti-human conjugates targeting the IgG Fc fragment or the light chains did not circumvent the SAFB drawback. Therefore, a quick decrease in antibody strength must alert against a potential risk for recipients at the time of the transplant, using virtual crossmatch strategies. A prospective pre-transplant crossmatch still remains an ultimate safeguard.

Surveillance of γδ T Cells Predicts Cytomegalovirus Infection Resolution in Kidney Transplants

Cytomegalovirus (CMV) infection in solid-organ transplantation is associated with increased morbidity and mortality, particularly if a CMV mutant strain with antiviral resistance emerges. Monitoring CMV-specific T cell response could provide relevant information for patient care. We and others have shown the involvement of Vδ2(neg) γδ T cells in controlling CMV infection. Here, we assessed if Vδ2(neg) γδ T cell kinetics in peripheral blood predict CMV infection resolution and emergence of a mutant strain in high-risk recipients of kidney transplants, including 168 seronegative recipients receiving organs from seropositive donors (D+R-) and 104 seropositive recipients receiving antithymocyte globulins (R+/ATG). Vδ2(neg) γδ T cell percentages were serially determined in patients grafted between 2003 and 2011. The growing phase of Vδ2(neg) γδ T cells was monitored in each infected patient, and the expansion rate during this phase was estimated individually by a linear mixed model. A Vδ2(neg) γδ T cell expansion rate of ˃0.06% per day predicted the growing phase. The time after infection at which an expansion rate of 0.06% per day occurred was correlated with the resolution of CMV DNAemia (r=0.91; P<0.001). At 49 days of antiviral treatment, Vδ2(neg) γδ T cell expansion onset was associated with recovery, whereas absence of expansion was associated with recurrent disease and DNAemia. The appearance of antiviral-resistant mutant CMV strains was associated with delayed Vδ2(neg) γδ T cell expansion (P<0.001). In conclusion, longitudinal surveillance of Vδ2(neg) γδ T cells in recipients of kidney transplants may predict CMV infection resolution and antiviral drug resistance.

Preemptive Therapy Versus Valgancyclovir Prophylaxis in Cytomegalovirus-positive Kidney Transplant Recipients Receiving Antithymocyte Globulin Induction

International consensus guidelines on the management of cytomegalovirus (CMV) infections in kidney transplantation recommend the use of universal prophylaxis over preemptive therapy for the highest risk kidney transplant recipients (KTR), namely donor+/recipient - CMV serostatus. However, no universal recommendations have been made for R+ KTR undergoing antithymocyte globulin (ATG) induction. In this retrospective study, we compared 1-year outcomes among 24 R+ KTR who received 3 months of valgancyclovir prophylaxis with 72 R+ KTR who were subjected to a preemptive strategy. All subjects received ATG induction. The incidence of CMV infection was significantly higher among the preemptive subjects versus the prophylaxis group (78% versus 38%, respectively; P = .0003), whereas the incidence of CMV disease was low and did not differ significantly between the cohorts (8% versus 7% respectively, P = .8). Late-onset CMV infections were only observed in the prophylaxis group (25% versus 0%, P = .0001). Finally, the rate of opportunistic infections, acute rejection episodes, and graft/patient survivals at 1 year were also similar between the two groups. In light of this study, preemptive therapy and universal prophylaxis were almost equally effective to prevent CMV infection among R+ KTR receiving ATG induction.

Distal Angiopathy and Atypical Hemolytic Uremic Syndrome: Clinical and Functional Properties of an Anti–Factor H IgAλ Antibody

Abnormal regulation of the alternative pathway of the complement system is a well-described trigger of microangiopathy leading to atypical hemolytic uremic syndrome (aHUS). However, the involvement of complement dysregulation in distal angiopathy has not been reported in adults. We describe the clinical course of a patient with severe distal angiopathy (amputation of all fingers and toes) followed 3 years later by aHUS with end-stage renal disease. This course was attributed to a circulating monoclonal immunoglobulin A λ light chain (IgAλ) with unusual properties: it bound complement factor H (CFH) and impaired CFH-glycosaminoglycan interaction and cell-surface protection. Local complement activation with distal angiopathy and microvascular injury was suggested by deposition of IgA, C4d, and C5b-9 in limb and preglomerular arteries. We therefore postulated that the monoclonal IgAλ inhibited activity of endothelial cell-bound CFH, which led to local activation of complement, vasoconstriction (distal angiopathy), and aHUS. While the patient was dependent on dialysis and plasma exchange, treatment with the anti-C5 antibody eculizumab induced remission of distal angiopathy and aHUS. During eculizumab treatment, kidney transplantation was performed. The patient had normal kidney function at the 3-year follow-up. We suggest that the association of distal angiopathy and aHUS in this patient is clearly linked to anti-CFH properties of the monoclonal IgAλ.

Reassessment of T Lymphocytes Crossmatches Results Prediction With Luminex Class I Single Antigen Flow Beads Assay.

Background In virtual crossmatch (XM) strategies, a correct anticipation of XM results is required for appropriately allocating organs. We reassessed the ability to predict T lymphocyte flow cytometry and complement dependent cytotoxicity XM results with the mean fluorescence intensity (MFI) in Luminex class I single antigen flow beads (SAFB) assay, after correction of complement interference and exclusion of antidenatured HLA antibodies. Methods Among 432 XM with T lymphocytes (T-XM), 407 were analyzed after exclusion of antidenatured HLA antibodies. Only ethylenediaminetetraacetic acid-treated serum MFI was considered. Only 1 cellular target HLA antigen for the serum was expressed in 238 cases, 209 and 29 being heterozygous and homozygous for this antigen, respectively. For the remaining 169 cases, at least 2 antigens were recognized. Single antigen flow bead MFI thresholds allowing XM positivity to be predicted were calculated with receiver operating characteristic curves. Results T-XM results were tightly associated with SAFB MFI values. Anti-HLA-A and anti-HLA-B antibodies behaved similarly. Prediction and sensitivity SAFB MFI thresholds were determined, respectively, assessing the highest sensitivity/specificity ratio and at least 95% sensitivity for predicting T-XM positivity. Both were slightly lower for HLA-B than for HLA-A, whereas anti-HLA-Cw antibodies induced random XM results. Both thresholds were only slightly diminished for homozygous and for multiple HLA targets, considering the immunodominant, but not the sum, of antibodies MFI. Conclusions Antibodies against HLA-A, -B, or -Cw behave differently. A homozygous HLA target does not trigger a twice higher XM positivity. Multiple antibodies are better evaluated through the immunodominant DSA MFI than through the sum of DSA MFI.

Anti-HLA donor-specific antibodies are not created equally. Don't forget the flow…

1 Clinique Saint Augustin-CTMR, Bordeaux, France 2 Department of Nephrology, Transplantation, and Dialysis, Bordeaux University Hospital, Bordeaux, France 3 Immunology and Immunogenetics Laboratory, Bordeaux University Hospital, Bordeaux, France 4 National Center of Scientific Research, Mix Unit of Research 5164, Bordeaux, France 5 Bordeaux University, Bordeaux, France *Present address: Immunology and Histocompatibility Laboratory, Saint-Louis Hospital, APHP, Paris, France e-mail: jeanuc.taupin@aphp.fr

Body composition in 98 patients awaiting kidney transplantation

OBJECTIVE Recent data suggest that the nutritional status of patients who are on the waiting list for kidney transplantation, influence outcomes after renal transplantation. Body composition (BC) analysis is rarely included in pretransplant evaluation. The aim of this study was to determine how alteration of the BC of these patients could influence pretransplant and post-transplant care. METHODS We compared the BC of French patients on a waiting list for kidney transplantation to a sex- and age-matched healthy, European control population. Patients were included when listed for kidney grafting in a prospective longitudinal study (CORPOS). Biological nutritional parameters, fat free mass (FFM) and fat mass (FM) estimated by dual-energy x-ray absorptiometry (DXA) were assessed on the day of wait-list registration. FFM and FM index (FFMi - FMi) are the ratio of FFM and FM to height squared. Results are expressed as median (range). These indexes were compared with previous study values used as a normal range in nutritional assessment and clinical practice. RESULTS The study included 28 women and 70 men aged 25.3 to 65.9 y. Body mass index ranged from 16.8 kg/m² to 39.4 kg/m². Compared with controls, FMi was higher in women (10.6 kg/m² [3.7-18.6 kg/m²]) than in men (8.1 kg/m² [3.5-13.3 kg/m²] in M) and FFMi was lower in women (14.3 kg/m² [11.8-21.4 kg/m²]) than in men (17.9 kg/m² [13.9-24.2 kg/m²]) (P < 0.01), reflecting an abnormal distribution of body compartments. All biological parameters were within the normal range. CONCLUSION BC abnormalities, which can only be detected with the use of DXA, are present in patients on a kidney transplantation waiting list. Detection of these abnormalities could influence the post-transplantation survey in order to prevent the frequent risk for developing metabolic complications after the procedure.