Donor-specific antibodies detected by single antigen beads alone can help risk stratify patients undergoing retransplantation across a repeat HLA mismatch.

Whether reexposure to mismatched HLA antigens (RMM) in the setting of a negative crossmatch is associated with increased immunological risk remains an area of uncertainty. This is due to evidence derived predominantly from registry data, which lacks comprehensive information on alloantibody and rejection. In this study, we analyze the impact of low-level preformed donor-specific antibodies (DSA) against an RMM on transplant outcomes. From 1988 consecutive renal transplant recipients, we analyzed 179 patients undergoing retransplantation, of whom 55 had a RMM. All patients were crossmatch negative and preformed DSA were detected by single antigen beads alone. Multivariate analysis revealed that patients with preformed DSA against an RMM were independently at risk of antibody-mediated rejection (HR 8.70 [3.42-22.10], P < .0001) and death-censored allograft loss (HR 3.08 [1.17-8.14], P = .023). In addition, prior transplant nephrectomy (HR 2.04 [1.00-4.17], P = .0495) was also associated with allograft failure, whereas receiving a retransplant that was matched at HLA class II was associated with a favorable outcome (HR 0.37 [0.14-0.99], P = .047). In the absence of preformed DSA, an RMM was not associated with de novo DSA development, rejection, or allograft loss. In conclusion, an RMM portends increased immunological risk only in the presence of a preformed DSA in patients undergoing retransplantation.

Monitoring native HLA-I trimer specific antibodies in Luminex multiplex single antigen bead assay: Evaluation of beadsets from different manufacturers.

Luminex single antigen bead (SAB) assay utilizes beadsets coated with a set of cloned and purified HLA molecules, for monitoring serum anti-HLA antibodies. Particularly, the level of serum IgG against native HLA-I trimers (heavy chain (HC) and ß2-microglobulin (ß2m) with a peptide), expressed in allograft tissues is correlated with graft failure. In addition to native trimeric HLAI, the beadsets may carry HC only or the dimeric variants, peptide-free HC with ß2m and ß2m-free HC with or without peptides. Currently, three different HLA-I coated beadsets have been produced commercially. The HLA antigen density on one beadset was reported to be approximately 50% of that present on another beadset as evidenced by the binding of an anti-HLA-I mAb W6/32. To date, no efforts have been made to compare the relative distribution of HLA-I variants in these three beadsets. In this study, using monoclonal antibodies (W6/32, HC-10 and TFL-006) that can distinguish the structural variants based on their epitope specificities, the nature of the variants in the three beadsets were comparatively evaluated. One beadset (Beadset A, see Materials and methods for Brand and Manufacturer's names) (W6/32+/HC-10+/TFL-006+) carried at least three variants, while beadset B (W6/32+/HC-10+/TFL-006-) carried two (peptide-associated and peptide-free ß2m-HC) and the beadset C (W6/32+/HC-10-/TFL-006-) carried exclusively the HLA-I trimer suggesting its usefulness for specific monitoring native HLA-I trimer antibodies. Because of the salient differences in the variants coated on the different beadsets, it would be warranted to investigate, if these differences are clinically relevant for monitoring serum anti-HLA antibodies in sensitized patients waiting for donor organs and in allograft recipients (274).

Experimental validation of the RATE tool for inferring HLA restrictions of T cell epitopes.

BACKGROUND: The RATE tool was recently developed to computationally infer the HLA restriction of given epitopes from immune response data of HLA typed subjects without additional cumbersome experimentation. RESULTS: Here, RATE was validated using experimentally defined restriction data from a set of 191 tuberculosis-derived epitopes and 63 healthy individuals with MTB infection from the Western Cape Region of South Africa. Using this experimental dataset, the parameters utilized by the RATE tool to infer restriction were optimized, which included relative frequency (RF) of the subjects responding to a given epitope and expressing a given allele as compared to the general test population and the associated p-value in a Fisher's exact test. We also examined the potential for further optimization based on the predicted binding affinity of epitopes to potential restricting HLA alleles, and the absolute number of individuals expressing a given allele and responding to the specific epitope. Different statistical measures, including Matthew's correlation coefficient, accuracy, sensitivity and specificity were used to evaluate performance of RATE as a function of these criteria. Based on our results we recommend selection of HLA restrictions with cutoffs of p-value < 0.01 and RF ≥ 1.3. The usefulness of the tool was demonstrated by inferring new HLA restrictions for epitope sets where restrictions could not be experimentally determined due to lack of necessary cell lines and for an additional data set related to recognition of pollen derived epitopes from allergic patients. CONCLUSIONS: Experimental data sets were used to validate RATE tool and the parameters used by the RATE tool to infer restriction were optimized. New HLA restrictions were identified using the optimized RATE tool.

Anti-HLA Antibodies Testing on Solid Phase: Comparative Evaluation of Different Kit Vendors Through Luminex Technology.

OBJECTIVES: For decades, the detection of anti-HLA antibodies in candidates for solid-organ transplant has been performed with the traditional complement-dependent cytotoxicity method; this assay has been then integrated with the introduction of solid-phase assays. Over the past 20 years, the Luminex assay has become the most widely used in clinical laboratories due to both increased sensitivity and specificity versus enzyme-linked immunosorbent assay. However, even the Luminex technique has shown some critical issues, and choosing the most reliable method still remains challenging. In this study, we verified the concordance of the results obtained in detecting anti-HLA antibodies with 2 kit vendors that provide reagents for the Luminex platform. MATERIALS AND METHODS: We used 314 serum samples from patients on wait lists for solid-organ transplant. Sera were tested with LABScreen Mixed-LSM12 (One Lambda-Thermo Fisher, Canoga Park, CA, USA) and LIFECODES LifeScreen Deluxe-LMX (Gen-Probe-Immucor, Stanford, CT, USA),which we indicated as vendor A and vendor B, respectively. Anti-HLA class I and class II antibody analyses were conducted by verifying the concordance of the results with Cohen kappa coefficient statistics and confidence interval. RESULTS: The kappa coefficient statistics showed "substantial" reliability for class I (0.61; confidence interval, 0.50-0.73) and "moderate" reliability for class II (0.56; confidence interval, 0.43-0.69). There were no considerable differences in results between the 2 kits regarding overall assignment of negativity or positivity of a sample. Discordant data between positive values for a test and negative for the other were found for samples with weak antibody positivity. CONCLUSIONS: Some discordant data were probably attributable to several factors such as the composition of the kits, the antibody titer in the serum, whether sera were diluted, different washing methods, and type of plate used.

HLA genotyping using the Illumina HLA TruSight next-generation sequencing kits: A comparison.

Illumina first introduced their TruSight human leucocyte antigen (HLA) next-generation sequencing (NGS) typing kit in 2015 and subsequently followed up with a new version in 2016. Here we report on our experience comparing the two versions of the Illumina HLA NGS kits.

[Cytotoxicity of natural anti-HLA antibodies in Moroccan patients awaiting for kidney transplantation]. / Cytotoxicité des anticorps anti-HLA naturels chez des patients marocains candidats à la greffe rénale.

The presence of anti-HLA antibodies in the serum of a patient result from an immune response produced during an immunizing event as transfusion, pregnancy or graft. These antibodies can be cytotoxic by activating the complement pathway via C1q and may cause organ rejection during the transplant. Some male patients awaiting kidney transplantation are seropositive for anti-HLA antibodies when they have no immunizing antecedent event. These antibodies are qualified as natural antibodies. Our work is to assess the cytotoxicity of natural anti-HLA antibodies in patients followed at the immunology laboratory of the blood transfusion service and hemovigilance (STSH) as part of the kidney transplant. PATIENTS AND METHODS: We evaluated the cytotoxicity of HLA antibodies detected in male Moroccan patients without immunization history using C1qScreen One Lambda reagent for Luminex™. RESULTS: Non-immunized men were positive for HLA antibodies screening in 25.4%. These antibodies are not cytotoxic. CONCLUSION: Our study showed a positivity rate of natural HLA antibody low than the literature (25.4% against 63%). It appears that these natural antibodies are not cytotoxic and their involvement in renal transplant remains to be determined.

Next generation sequencing with a semi-conductor technology (Ion Torrent PGM™) for HLA typing: overall workflow performance and debate.

Current high resolution HLA typing technologies produce ambiguous results, and it is often necessary to perform additionnal tests to resolve these ambiguities. Next generation sequencing is a promising technology, which can overcome this problem. It is going to usher a new strategy to determine HLA compatibility between donor and recipient. It can lead to non ambiguous results by analysing the full amplified sequence of HLA genes and by eliminating heterozygote phase ambiguities. Instead, as many new techniques, we can face several problems, such as analysis difficulties because of incomplete HLA sequences in the database or errors related to the sequencing instrumentation. Moreover, the clinical relevance of analysing non coding regions of HLA genes is not well understood, but raise questions about the interest of getting HLA full sequence to understand drugs side effects or pathogenesis of infectious or auto-immune diseases. Our objective in this article is to present a commercial workflow for HLA typing by NGS, on Ion Torrent PGM™ sequencer, and to focus attention about pitfalls encountered during the analysis.

HLA genotyping in the clinical laboratory: comparison of next-generation sequencing methods.

Implementation of human leukocyte antigen (HLA) genotyping by next-generation sequencing (NGS) in the clinical lab brings new challenges to the laboratories performing this testing. With the advent of commercially available HLA-NGS typing kits, labs must make numerous decisions concerning capital equipment and address labor considerations. Therefore, careful and unbiased evaluation of available methods is imperative. In this report, we compared our in-house developed HLA NGS typing with two commercially available kits from Illumina and Omixon using 10 International Histocompatibility Working Group (IHWG) and 36 clinical samples. Although all three methods employ long range polymerase chain reaction (PCR) and have been developed on the Illumina MiSeq platform, the methodologies for library preparation show significant variations. There was 100% typing concordance between all three methods at the first field when a HLA type could be assigned. Overall, HLA typing by NGS using in-house or commercially available methods is now feasible in clinical laboratories. However, technical variables such as hands-on time and indexing strategies are sufficiently different among these approaches to impact the workflow of the clinical laboratory.

Determining performance characteristics of an NGS-based HLA typing method for clinical applications.

This study presents performance specifications of an in-house developed human leukocyte antigen (HLA) typing assay using next-generation sequencing (NGS) on the Illumina MiSeq platform. A total of 253 samples, previously characterized for HLA-A, -B, -C, -DRB1 and -DQB1 were included in this study, which were typed at high-resolution using a combination of Sanger sequencing, sequence-specific primer (SSP) and sequence-specific oligonucleotide probe (SSOP) technologies and recorded at the two-field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African-American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixon's target and GenDx's NGSengine. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA-A, -B, -C and -DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs.

[Detection of HLA antibodies by Luminex and kidney transplantation decision making: a case report]. / Détection des anticorps anti-HLA par Luminex et décision de transplantation rénale : à propos d'un cas.

We report the case of a patient planned for living-related donor kidney transplantation. HLA antibodies research, performed by the Luminex method, is positive for all the available sera. Cross match was performed by complement-dependent lymphocytotoxicity technique with three family members. The first donor, her husband, was excluded due to the positivity of the cross-match and the presence of donor-specific anti HLA antibody (DSA). The second one, her daughter, was immediately excluded because the recipient also presented DSA. Despite the negativity of the cross match, the decision to transplant the patient with the third donor, her son, was difficult to take because of the presence of DSA identified by the Luminex method. Consequently, the patient could not be transplanted.

[Pitfalls in interpreting anti-HLA antibodies by Luminex technology]. / Pièges de l'interprétation des anticorps anti-HLA par technologie Luminex.

The Luminex technology has become an important tool for HLA antibody screening and identification. This is the most sensitive technology to detect HLA antibodies for transplant patients and patients on awaiting list, and it has ushered a new strategy to determine HLA compatibility between donor and recipient. Moreover, the clinical relevance of all detected anti-HLA antibodies is not well understood, because this technique was shown to be prone to many artefacts or interferences, leading to a complicated interpretation for biologists and clinicians. Our objective in this article is to provide a careful consideration about this solid phase assay, and to focus attention on raised questions about technical performance and interpretation of the results. We should keep in mind that our results could change the clinical management of sensitized patients, their aptitude to receive a graft, and their follow-up.

HLA typing by next-generation sequencing - getting closer to reality.

Next generation sequencing (NGS) denotes novel sequencing technologies that enable the generation of a large number of clonal sequences in a single sequencing run. NGS was initially introduced for whole genome sequencing and for quantitation of viral variants or genetic mutations in tumor tissues; more recently, the potential for high resolution HLA typing and high throughput analyses has been explored. It became clear that the complexity of the HLA system implicates new challenges, especially for bioinformatics. From an economical point of view, NGS is becoming increasingly attractive for HLA typing laboratories currently relying on Sanger based sequencing. Realizing the full potential of NGS will require the development of specifically adapted typing strategies and software algorithms. In the present review, three laboratories that were among the first to perform HLA-typing using different NGS platforms, the Roche 454, the Illumina Miseq and the Ion Torrent system, respectively, give an overview of these applications and point out advantages and limitations.

Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing.

BACKGROUND: A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing. RESULTS: We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week. CONCLUSIONS: The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.

High density FTA plates serve as efficient long-term sample storage for HLA genotyping.

Storage of dried blood spots (DBS) on high-density FTA(®) plates could constitute an appealing alternative to frozen storage. However, it remains controversial whether DBS are suitable for high-resolution sequencing of human leukocyte antigen (HLA) alleles. Therefore, we extracted DNA from DBS that had been stored for up to 4 years, using six different methods. We identified those extraction methods that recovered sufficient high-quality DNA for reliable high-resolution HLA sequencing. Further, we confirmed that frozen whole blood samples that had been stored for several years can be transferred to filter paper without compromising HLA genotyping upon extraction. Concluding, DNA derived from high-density FTA(®) plates is suitable for high-resolution HLA sequencing, provided that appropriate extraction protocols are employed.

[Evaluation of high resolution Luminex technology for specific HLA-DRB1* typing in the Moroccan population]. / Évaluation de la technologie Luminex de haute résolution pour le typage HLA-DRB1* spécifique dans la population marocaine.

The aim of this study is to evaluate the possibility of using routinely Luminex high definition technology for the specific HLA typing of donors and recipients of hematopoietic stem cells. 340 HLA-DRB1 *, all from Moroccan individuals were first tested at the generic level and then at the specific level by PCR-SSO Luminex high definition. Alleles identified correspond to those originally found with the generic typing. The specificity could be determined only in 41.5% of cases. The percentage of specific alleles identified for DRB1 * 04 was 78.7% and varies between 17.6% and 34.6% for other alleles. Of the eight haplotypes tested, blanks obtained by PCR-SSP were all resolved. Our results confirm the reliability of the method, since we confirm the alleles identified at the generic level. Luminex high definition technology can be used for HLA-DRB1 * 04 typing, and for the resolution of ambiguities and blanks. However, it must be completed by other techniques (PCR-SSP, SBT) in the context of hematopoietic stem cells.

Significance of panel reactive antibodies in patients requiring kidney transplantation.

Presence of antibodies against human leukocyte antigen (HLA) molecules, which may be may be directed against HLA class I or/and class II antigens, is a known risk factor for acute rejections and graft loss. Pre-transplantation panel reactive antibody (PRA) estimation is done to identify sensitized patients prior to solid organ transplantation and also forms the basis of cadaver organ allocation. The aim of this study is to evaluate the PRA in 52 patients awaiting first renal transplant, identify various factors contributing to high PRA, and observe its influence on graft survival. This was a case control study performed in a tertiary care hospital. Eighty-five samples including 63 from 52 patients with end-stage renal disease (ESRD), 10 from healthy volunteers, and 12 from presumed sensitized individuals were tested for class I and/or II PRA by enzyme-linked immunosorbent assay (ELISA) using Quik ID ® GTI kits. PRA for both class I and II was zero in all healthy controls and 19/46 (37%) patient samples; while individually, PRA class I and II were zero in 32/60 (53%) and 39/45 (86.3%) samples, respectively. PRA exceeded 10% in 16 samples from 12 patients with peak class I and II PRA of 100% and 46%, respectively. Post-transplantation, 27/31 patients are doing well, while four died with a functioning graft. Patient reactivity to antigen stimulation is the most important factor determining the PRA levels, and class I PRA is more relevant for detection of sensitization in first-time recipients and adversely affects the graft outcome.

Evaluation of the blood compatibility of materials, cells, and tissues: basic concepts, test models, and practical guidelines.

Medicine today uses a wide range of biomaterials, most of which make contact with blood permanently or transiently upon implantation. Contact between blood and nonbiological materials or cells or tissue of nonhematologic origin initiates activation of the cascade systems (complement, contact activation/coagulation) of the blood, which induces platelet and leukocyte activation. Although substantial progress regarding biocompatibility has been made, many materials and medical treatment procedures are still associated with severe side effects. Therefore, there is a great need for adequate models and guidelines for evaluating the blood compatibility of biomaterials. Due to the substantial amount of cross talk between the different cascade systems and cell populations in the blood, it is advisable to use an intact system for evaluation. Here, we describe three such in vitro models for the evaluation of the biocompatibility of materials and therapeutic cells and tissues. The use of different anticoagulants and specific inhibitors in order to be able to dissect interactions between the different cascade systems and cells of the blood is discussed. In addition, we describe two clinically relevant medical treatment modalities, the integration of titanium implants and transplantation of islets of Langerhans to patients with type 1 diabetes, whose mechanisms of action we have addressed using these in vitro models.

[The analysis of detection rate of ambiguity of loci under HLA-typing by SSO technology].

The article deals with the results of HLA-typing of 1829 patients on loci HLA-A, HLA-B, HLA-DRB1 using kits of LABType SSO reagents (One Lambada, USA). The indicators of each locus ambiguity are determined: locus HLA-A - 4.43%, locus HLA-B--4.43%, locus HLA-DRB1--0.16%. The list of "rare" alleles was applied to analyze 13 types of determined ambiguities on locus HLA-A, 27 types on locus HLA-B, 3 types on locus HLA-DRB1.

Predictive value of the Luminex single antigen panel for detecting flow cytometry cross-match positivity.

Anti-human leukocyte antigen (HLA) antibodies are a major cause of allograft loss. Solid-phase immunoassays, notably Luminex technology, have lately begun to replace traditional techniques for detecting these antibodies. This platform, however, carries some restrictions in the type of antibodies it detects. For this reason, results using these new technologies must be correlated with results using traditional techniques that have proven clinical significance. We have correlated flow cytometry cross-match (FCXM) outcomes with results from Luminex assays. Serum samples from patients awaiting transplantation who had known anti-HLA antibodies as detected by Luminex were incubated with lymphocytes expressing (a) 1 of the HLA antigens detected by the sera or (b) several of them. Of the 169 T-cell FCXMs we performed, in 92 cases the target cell expressed only 1 of the HLA antigens detected by the serum. The results obtained correlated well with Luminex data (r = 0.84). A cutoff mean fluorescence intensity value of 6,500 for the Luminex single antigen assay yielded a sensitivity of 85% and specificity of 82% for detecting a positive FCXM. In the other 77 cases, the target cell expressed 2 or more of the HLA antigens detected by the serum. In this situation, the same cutoff proved a useful tool for differentiating negative from positive FCXMs.