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.

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.

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.

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.

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.

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.

High-throughput multiplex HLA-typing by ligase detection reaction (LDR) and universal array (UA) approach.

One major goal of genetic research is to understand the role of genetic variation in living systems. In humans, by far the most common type of such variation involves differences in single DNA nucleotides, and is thus termed single nucleotide polymorphism (SNP). The need for improvement in throughput and reliability of traditional techniques makes it necessary to develop new technologies. Thus the past few years have witnessed an extraordinary surge of interest in DNA microarray technology. This new technology offers the first great hope for providing a systematic way to explore the genome. It permits a very rapid analysis of thousands genes for the purpose of gene discovery, sequencing, mapping, expression, and polymorphism detection. We generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. In particular, we set up a universal array approach in combination with a PCR-LDR (polymerase chain reaction-ligation detection reaction) strategy for allele identification in the HLA gene.

[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.

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).

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.

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.

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.

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.

A new method for determining anti-B cell antibodies and their specificity using flow cytometry.

We describe a new flow cytometric B cell crossmatch method with improved sensitivity and specificity. It is based on the coating of B cell surface immunoglobulins with an unconjugated polyvalent anti-human immunoglobulin antibody. The method also provides a means for determining the specificity of anti-B cell antibodies and, potentially, for anti-HLA class II antibody specificity differentiation (DR, DQ or DP) in a binding inhibition test using mouse monoclonal antibodies directed against human HLA class II antigens.

A simple procedure for freezing and storing lymphocyte panels in trays.

Lymphocytes from different donors may be frozen in Terasaki trays by a simple procedure. This method provides the tissue typing laboratory with a readily available panel that can be routinely used to test for lymphocytotoxic antibodies sera from patients waiting for kidney transplant, transplanted patients and polytransfused individuals. The advantage of this procedure is that expensive programmed freezing apparatus is not required.

Use of SEOPF regional crossmatch trays to share kidneys for sensitized patients. Local experience of three centers.

Regional organ procurement (ROP) crossmatch trays are used by members of the South-Eastern Organ Procurement Foundation (SEOPF) to facilitate sharing of cadaver kidneys for highly sensitized patients. ROP trays carry a single representative serum sample from over 900 patients with panel reactive antibody (PRA) levels of greater than or equal to 60%. Trays are centrally prepared periodically and distributed to member laboratories where they are used for preliminary crossmatching against locally obtained donors. Crossmatching results are used in conjunction with the SEOPF computer match program for sharing of kidneys. Proficiency testing studies by the 40-member laboratories show a greater than 90% concordance rate on results with these highly and broadly reactive sera. Data were available from 3 centers on 74 kidneys shared between SEOPF-member institutions on the basis of a remote, preliminary, negative ROP tray crossmatch. Of these, 44 (59%) crossmatched negative locally with the same and other sera, and were thus considered acceptable to be transplanted to the intended highly sensitized patients. Sixteen (22%) donors had a positive crossmatch locally, but with sera other than that present on the ROP tray used for screening. In 14 cases (19%) the same serum as on the ROP tray gave a positive crossmatch. The majority of ROP tray inconsistencies appeared to be due to use of more sensitive crossmatching techniques at the recipient center. Of the 27 patients transplanted at these 3 centers with kidneys received on the basis of ROP tray results, none experienced hyperacute or early irreversible rejection and actual graft survival at 6-48 months is 74%. These studies indicate that regional sharing of patient sera by the existing network of histocompatibility testing laboratories is an effective and reliable mechanism to identify crossmatch-negative donors for highly sensitized patients.

[Computer-assisted histocompatibility assessment in mixed lymphocyte culture (MLC)]. / Computergestützte Verträglichkeitsbeurteilung in der gemischten Lymphozytenkultur (MLC).

Analysis of the results of mixed lymphocyte culture (MLC) for compatibility testing preceding transplantation of bone marrow and other organs has so far required a vast input, both in terms of laboratory staff and work hours. We have developed a computer programme which performs this work rapidly. Graphics of the reaction patterns can be obtained, moreover, and these can prove a helpful tool in interpretation of the results.

The value of HLA-ABC common typing tray in relation to bone marrow transplantation.

Bone marrow transplantation (BMT) is one of the most effective procedures to cure the previously uncured hematologic diseases. However, it is costly and HLA typing to select the compatible donors contributed to its cost. A total of 53 prospective patients for BMT and their 114 siblings were analyzed to evaluate the use of locally prepared HLA-ABC common typing tray (ABCCT) during Mar 1988-Mar 1992. The 16, 9, 7, 5, 5 and 12 patients were diagnosed as aplastic anemia, CML, thalassemia, ALL, ANLL and other blood diseases, respectively. It was found that 18 patients were HLA-identical (HLA-ID) with one of their siblings except one patient had 2 HLA-ID sibs. All of those who appeared to be HLA-ID were further tested for the HLA-ABCDR typings. It was observed that 16 (88.89%) of 18 patients and 17 (89.47%) of 19 sibs were confirmed as HLA-ID. After careful clinical screening, only 13 HLA-ID pairs were able to proceed to the mixed lymphocyte culture and confirmed their status of HLA-ID by this test. Finally, only 6 (46.15%) of 13 patients received BMT with a high rate of success, ie all patients have survived with bone marrow engraftment. Thus, ABCCT is very useful for related BMT. It was highly efficient to exclude HLA-non-ID and haplo-ID yet the cost and workload were greatly reduced.