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High resolution typing of the HLA-DPB1 locus for patient who requested for hematopoietic stem cell transplantation (HSCT) workup has recently become mandatory by the National Marrow Donor Program (NMDP) in order to facilitate matching between donors and recipients for better outcomes. The likelihood of identifying HLA matched donors in Hong Kong, on top of the existing HLA-A, -B, -C, and -DRB1 loci, is revisited in this study. HLA-A, -B, -C, -DRB1 and -DPB1 genotypes of 5,266 volunteer unrelated Chinese donors from the Hong Kong Bone Marrow Donor Registry (HKBMDR), were included in this study. Matching models were employed to determine the matching probabilities for 10/10(DPB1) and 9/10(DPB1) HLA match. The matching probabilities are 20% at 10/10(DPB1) HLA match and 55% at 9/10(DPB1) match, based on the existing 130,000 donors in the HKBMDR. The likelihoods of match become 27% and 65% respectively, by increasing the registry to 250,000. However, if DPB T-cell-epitope (TCE) model is considered in the matching, the probability will increase to 46% at 10/10 DPB1 permissive mismatching. Our findings provide vital information about the future planning on the targeted recruitment size, HLA typing and search strategies of the donor registry and arose the transplant physicians’ acceptability to 9/10(DBP1) or 10/10(DBP1) HLA match. Nevertheless, the marrow donor registry has planned for increasing the registry size and bringing down the age of recruited donors which will ultimately enhance patient outcome.

Nanopore sequencing, enabled initially by the MinION device from Oxford Nanopore Technologies (ONT), is the only technology that offers portable, single-molecule sequencing and ultralong reads. The technology is ideal for the typing of human leukocyte antigen (HLA) genes for transplantation and cancer immunotherapy. However, such applications have been hindered by the high error rate of nanopore sequencing reads. We developed the workflow and bioinformatic pipeline, Athlon (accurate typing of human leukocyte antigen by Oxford Nanopore), to perform high-resolution typing of Class I HLA genes by nanopore sequencing. The method features a novel algorithm for candidate allele selection, followed by error correction through consensus building. Here, we describe the protocol of using Athlon packaged in a VirtualBox image for the above application.

A novel MICA allele, MICA*078, has been identified during HLA/MICA high resolution typing of Moroccan patients with celiac disease. MICA*078 shows an uncommon variation at a highly conserved nucleotide position (nt 493, G -> A), resulting in one amino acid change at codon 142 (V -> I) of MICA gene (compared to MICA*002:01), located in the alpha 2-domain, in which V142 is the common residue. (C) 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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The aim of the study was to evaluate the presence of nonfrequent, rare and very rare alleles among Croats and to estimate whether they are associated with specific alleles at other human leukocyte antigen (HLA) loci. This retrospective study included the typing results from the last 10 years; total number of individuals included was approximately 45,000. Among 17 alleles so far observed only once in our population, 6 (A*24:41, B*07:02:28, B*35:03:03, B*39:40N, DRB1*13:23 and DRB1*14:111) belong to very rare alleles, 2 (B*44:16 and DRB1*01:31) belong to rare alleles according to the ‘Rare Alleles Detector’ tool ( www.allelefrequencies.net), while for the B*35:101:01 allele published data exist only in the IMGT/HLA database. The remaining eight HLA alleles observed only once among Croats are considered as frequent according to the ‘Rare Alleles Detector’. Those 17 HLA alleles are not declared as common well defined (CWD) alleles in the CWD allele catalogue 2.0.0. Haplotype analysis of nonfrequent alleles detected in our sample supports the idea that different populations, although similar in some aspects regarding HLA allele and haplotype distribution, still have some unique characteristics. This is the case for A*01:02, B*39:10 and DRB1*13:32 which form haplotypes unreported to date among our subjects.

Donor-specific alloantibodies (DSA) to HLA-DP may cause antibody-mediated rejection (AMR), especially in re-transplants. We describe the immunization history of a patient who received 3 kidney transplants; the 3rd kidney was completely matched except at DPA1 and DPB1. Prior to the 3rd transplant, single antigen bead analysis (SAB) showed DSA reactivity against DPA1 shared by the 1st and 3rd donors, but B and T flow crossmatch (FXM) results were negative. Within 11 days the 3rd transplant underwent acute C4d+ AMR which coincided with the presence of complement (C1q)-binding IgG1 DSA against donor DPA1 and DPB1. Using HLAMatchmaker and SAB, we provide evidence that eplet (epitope) spreading on DPA1 and eplet sharing on differing DPB1 alleles of the 1st and 3rd transplants was associated with AMR. Since weak DSA to DPA1/DPB1 may induce acute AMR with negative FXM, donor DPA1/DPB1 high resolution typing should be considered in sensitized patients with DP-directed DSA.

In this issue of EBioMedicine, Kohl and colleagues describe the development of a new core genome MLST scheme (cgMLST) for Mycobacterium tuberculosis complex strains based on a set of 2891 genes. Here, the application of the scheme to a number of tuberculosis surveillance studies is examined.

Objectives Repeated infections of Chlamydia trachomatis may be new infections or persistent infections due to treatment failure or due to unresolved infections in sexual partners. We aimed to establish the value of using high-resolution multilocus sequence typing (CT-MLST) to discriminate repeated C trachomatis infections. Methods Paired C trachomatis positive samples (baseline (T0) and after 6 months (T1)) were selected from two Dutch screening implementation studies among young heterosexual people. Typing with six CT-MLST loci included the ompA gene. The uniqueness of strains was assessed using 256 reference CT-MLST profiles. Results In 27 out of 34 paired cases, full sequence types were obtained. A multilocus (13 cases) or single locus variant (4 cases) was seen, indicating 17 new C trachomatis infections at T1. The ompA genovar was identical for 5 of 17 discordant cases. The 10 cases with concordant typing results were categorised as treatment failure (5 cases) versus persistent or recurrent infections (5 cases). Surprisingly, these concordant cases had C trachomatis strains that were either unique or found in small clusters. The median time between T0 and T1 did not differ between the concordant and discordant cases. Conclusions High-resolution typing was superior in discriminating new infections compared with only using ompA genovar typing. Many cases (37%) showed exactly the same C trachomatis strain after 6 months. CT-MLST is not conclusive in distinguishing recurrent infections from treatment failure.

HLA testing is an essential part of the process to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow, peripheral blood or cord blood and the DNA typing in high resolution is now recommended as the Scientific Societies also describe in their standards. Recently the new PCR-Luminex HLA typing method, based on the reverse sequence specific oligonucleotide probes coupled with a microsphere beads in an array platform, has been well established. We report the data from 146 samples previously typed to a four digits level and used to evaluate the accuracy, sensitivity and performance of the new high definition DRB1 by PCR-Luminex kit. One hundred and forty-six samples from unrelated healthy donors, haematological patients or external proficiency tests were used in this study. The Luminex high definition DRB1 typing represents a versatile method and may be easily introduced in the routine, particularly when the technical team has already acquired experience on the technique. Only few HLA allelic combinations need an additional typing by PCR-SSP or SBT to solve the ambiguous results thus reducing the time necessary to produce a final report.

Methods for estimating the cord blood (CB) inventory size required vary according to the ethnic diversity of the HLA, degree of HLA matching and HLA-typing resolution. We estimated the CB inventory size required using 7190 stored CB units (CBU) and 2450 patients who were awaiting or underwent allogeneic hematopoietic stem cell transplantation. With high-resolution typing of HLA-A, B and DRB1, 94.6% of Korean patients could find CBUs in 100 000 CBUs with a 5/6 match, and 95.7% could find CBUs in 5000 CBUs with a 4/6 match. With low-resolution typing of HLA-A and B and high-resolution typing of leukocyte antigen-DRB1, 95% of patients could find CBUs in 50 000 CBUs with a 5/6 match, and 96.7% could find CBUs in 3000 CBUs with a 4/6 match. With additional high-resolution typing for HLA-A and B, which could improve transplantation outcome, the size of the CB inventory would need to increase twofold for Koreans.

This mini-review briefly describes some of the issues that are important for a transplant physician preparing a patient for an unrelated donor (UD) stem cell transplant. It is primarily directed at referring hemato-oncologists and trainees in transplantation and aims to provide them with the information they require to deal with these patients appropriately and efficiently. It discusses search strategies, the need for urgency and early identification of the major problems that will determine the outcome of the transplant. Excellent communication with the histocompatibility and immunogenetics laboratory and the referring physician are emphasized. Many of the recommendations are derived from personal experience; there is a need for more of an evidence base to aid decision-making.

We investigated Chlamydia trachomatis strains from Nanjing, China, and whether these strains differed from Amsterdam, the Netherlands. C. trachomatis type was determined with multilocus sequence typing. Most strains were specific to Nanjing, but some clustered with strains from Amsterdam. This demonstrates a geographical variation in C. trachomatis previously left undetected.

Nucleotide sequence of HLA-C*07:208 allele was different from that of HLA-C*07:02:01:01 by a single-nucleotide substitution at position 475 G>C.

A set of robust PCR-SSP reactions were developed for each of the five polymorphic sites that define the five alleles of the HLA class Ib gene, HLA-E. This method was developed using 28 homozygous cell lines and further tested in a sample of African-Americans, a sample of Japanese, and a core panel of cell lines compiled for the 13th International Histocompatibility Workshop. Three alleles were found in each of these four sample groups, HLA-E*0101 (64.29, 50.00, 32.00 and 56.58%, respectively), *01031 (5.36, 20.65, 39.00 and 18.42%) and *01032 (30.35, 29.35, 29.00, and 25.00%). HLA-E*0102 was not detected in any of these samples nor in the cell line, LCL 722.221, in which this allele was originally described. HLA-E*0104 was not found either. This latter allele was originally reported in Japanese at a frequency of 1/22 (4.5%), which should have been high enough to have resulted in multiple occurrences of the *0104 allele in the samples tested in this study. We propose that the existence of the HLA-E*0102 and E*0104 alleles should be questioned.

Aim Next Generation Sequencing (NGS) is rapidly becoming the method of choice for HLA typing for Hematopoietic Stem Cell Transplantation. Jeevan Stem Cell Foundation, Chennai, India validated the full gene HLA typing using Immucor’s MIA FORA FLEX kits from conceptualization to implementation in ninety days. This implementation, in line with the Indian Government’s policy of “Make in India”, enabled Jeevan to establish a cost effective high resolution HLA typing to international standards in India to support the transplant physicians and registries in the region and prevent Indian DNA being shipped to other countries. Methods Reference DNA used for validation were previously typed by SBT included samples from UK NEQAS, FRED HUTCHINSON and Jeevan’s registry samples. DNA was extracted using the Qiacube. HLA typing for A, B, C, DRB1 and DQB1 were performed. All workflows were performed according to the supplier’s instructions. NGS sequencing was done using the mid output flow cell kit from Illumina and sequenced on the Illumina MiniSeq platform. Immucor’s proprietary software was used to analyse the data. Results Results were evaluated per allele on the 2nd field consensus compared to the reference, the number of allelic ambiguities, genotype ambiguities and allelic drop outs were measured. The second field resolution was used because higher resolution typing was not available for all samples. MIA FORA NGS software provided high resolution typing for all samples. There results from NGS typing compared to SBT typing are shown in Table 1. Conclusions The implementation of the MIA FORA FLEX HLA typing kit at Jeevan is the first outside USA. With the MIA FORA kits, the turnaround time will depend upon the number of samples. In our experience reports for 48 samples can be delivered within 72 h. There were no ambiguous results and no allelic dropout. The proprietary software of Immucor was easy to use and significantly reduced the analysis time. Download high-res image (213KB) Download full-size image

Aim After a year of implementing HLA typing by NGS, we present our most relevant achievements and challenges. Methods Class I (full HLA-A, -B, and -C genes) and HLA Class II (Exon 2 thru intron 3 of HLA-DR and HLA-DQ, and Exon 2 thru 3’UTR of HLA-DP) amplified with NXType™ Class I and Class II primer sets. Automated template preparation and chip loading were performed in the Ion Chef™ system and run on Ion S5™. Data was analyzed with TypeStream™ v1.1.0.11 software. Results Having typed over 1000 samples by NGS, with 50% from bone marrow programs and 50% from solid organ programs, our workflow has changed significantly: Threefold increase in capacity with simultaneous decrease in turn-around-time (TAT, on average 10 days) for high resolution typing. Our current capacity is up to 96 HLA typings in 3–4 days vs. minimum of 16 days for the same volume with SBT by Sanger. Resolution of most ambiguities (98.7%), minimizing the need for reflex testing.Repetitions due to technical failures and ambiguities decreased from 4.3% with SBT-Sanger to 0.3% with NGS. In spite of positive workflow changes, there remain challenges: Automation of library preparation has not been implemented.No merging of library preparation reagents into a single kit.Smaller runs increase the number of reads, leading to artifacts and high background generated by the software, interfering with analysis and limiting flexibility for STAT sample processing.For solid organ typings, generating high resolution results while reporting Serological Equivalents for UNOS, poses logistical difficulties.TypeStreamTM software not interfaced with Fusion, requiring continuous need to run SSOP for DSA analysis.Unresolved ambiguities (1.3%), due to incomplete exon coverage and phase ambiguities. Resolution may be obtained by increasing gene coverage, length of reads and with additional testing methods. Conclusions We have confirmed that NGS is both a suitable and faster method to obtain high resolution HLA typing. We have seen a significant decrease in TAT along with a significant cost savings due to fewer repetitions, fewer reagents, and most significantly, reduced time from our most valuable asset, our technologists.

A state-of-the-art overview of molecular Chlamydia trachomatis typing methods that are used for routine diagnostics and scientific studies. Molecular epidemiology uses high-resolution typing techniques such as multilocus sequence typing, multilocus variable number of tandem repeats analysis, and whole-genome sequencing to identify strains based on their DNA sequence. These data can be used for cluster, network and phylogenetic analyses, and are used to unveil transmission networks, risk groups, and evolutionary pathways. High-resolution typing of C. trachomatis strains is applied to monitor treatment efficacy and re-infections, and to study the recent emergence of lymphogranuloma venereum (LGV) amongst men who have sex with men in high-income countries. Chlamydia strain typing has clinical relevance in disease management, as LGV needs longer treatment than non-LGV C. trachomatis. It has also led to the discovery of a new variant Chlamydia strain in Sweden, which was not detected by some commercial C. trachomatis diagnostic platforms. After a brief history and comparison of the various Chlamydia typing methods, the applications of the current techniques are described and future endeavors to extend scientific understanding are formulated. High-resolution typing will likely help to further unravel the pathophysiological mechanisms behind the wide clinical spectrum of chlamydial disease

Predisposition to Crohn disease (CD) seems to be genetically determined but, though several reports on the matter, the association between HLA antigens and the disease is still controversial. PCR-SSP high resolution typing in 107 CD patients, and in subgroups selected according to clinical features, showed a positive association with the rare haplotype DRB1∗07, DQB1∗0303 both in the overall patients ( p = 0.002; pc = ns) and in the subgroup of nonfistulized patients ( p = 0.0008; pc = 0.032). Moreover, the protective role of the haplotype DRB1∗03, DQB1∗0201 ( p = 0.029) was confirmed also in Italian patients, whereas no strong association with HLA class I alleles has been found. In addition, variability of the HLA alleles frequency in CD subgroups was observed, supporting the hypothesis of a genetic heterogeneity of the disease and suggesting that HLA alleles distribution in selected groups may allow to identify patients with probably different prognosis or associated complications.

Leptospirosis is a worldwide zoonosis which is responsible for the typical form of Weil's disease. The epidemiological surveillance of the Leptospira species agent is important for host prevalence control. Although the genotyping methods have progressed, the identification of some serovars remains ambiguous. We investigated the multispacer sequence typing (MST) method for genotyping strains belonging to the species Leptospira interrogans , which is the main agent of leptospirosis worldwide. A total of 33 DNA samples isolated from the reference strains of L. interrogans serogroups Icterohaemorrhagiae, Australis, Canicola, and Grippotyphosa, which are the most prevalent serogroups in France, were analyzed by both the variable-number tandem-repeat (VNTR) and MST methods. An MST database has been constructed from the DNA of these reference strains to define the MST profiles. The MST profiles corroborated with the VNTR results. Moreover, the MST analysis allowed the identification at the serovar level or potentially to the isolate level for strains belonging to L. interrogans serovar Icterohaemorrhagiae, which then results in a higher resolution than VNTR (Hunter-Gaston index of 0.94 versus 0.68). Regarding L. interrogans serogroups Australis, Canicola, and Grippotyphosa, the MST and VNTR methods similarly identified the genotype. The MST method enabled the acquisition of simple and robust results that were based on the nucleotide sequences. The MST identified clinical isolates in correlation with the reference serovar profiles, thus permitting an epidemiological surveillance of circulating L. interrogans strains, especially for the Icterohaemorrhagiae serogroup, which includes the most prevalent strains of public health interest.

The aim of the study was to evaluate the presence of non-frequent, rare and very rare alleles among Croats and to estimate whether they are associated with specific alleles at other HLA loci. Haplotype analysis of non-frequent alleles detected in our sample supports the idea that different populations, although similar in some aspects regarding HLA allele and haplotype distribution, still have some unique characteristics. This is the case of A*01:02, B*39:10 and DRB1*13:32 which form haplotypes unreported to date among our subjects. Among 16 alleles so far observed only once in our population, six (A*24:41, B*07:02:28, B*35:03:03, B*39:40N, DRB1*13:23, and DRB1*14:111) belong to very rare alleles, two (B*44:16 and DRB1*01:31) belong to rare alleles according to the “Rare Alleles Detector” tool, while for the B*35:101:01 allele published data exist only in the IMGT/HLA database. There is no data available for any of those 16 alleles in the CWD allele catalogue 2.0.0.

The novel HLA-DQB1*06:43 allele differs from HLA-DQB1*06:01:01 in one nucleotide substitution at codon 73 in exon 2.

HLA-C*14:52 has one nucleotide difference from HLA-C*14:02:01 at position 427G>C in exon 3.

Aim Next Generation Sequencing (NGS) appears to be the future of HLA high resolution as it can deliver allele level typing without ambiguities. However, NGS is still new in its development and may require a high volume of samples to be cost effective for most clinical labs. The existing well-established Sanger sequencing may give us the allele level typing to meet the clinical needs if a proper strategy is implemented. Our lab has used a group-specific amplification/sequencing approach for over a decade. The percentage of the ambiguities in Sequence-Based Typing (SBT) has increased over the years due to continuous discovery of new alleles. To resolve DRB1 SBT ambiguities, we have evaluated an approach of adding an additional sequencing step. Methods HLA DRB1 typing was performed by using a set of group-specific primers (Protrans DRB1 S4). Ambiguities observed in S4 SBT were then resolved by performing an additional amplification and/or sequencing with selected primers (Protrans Domino Stone). Sequencing data were analyzed and allele level typing was assigned by using SBT software (SeqPilot, JSI). Results Six samples that were initially typed with ambiguities using DRB1 S4 reagent were sequenced with Domino Stones primers. Multiple samples were typed with the same HLA-DRB1 ambiguity. All the ambiguities were resolved with an exception for DRB1*12:01 and 12:10 which are different from each other in exon 1 (Table 1). The final typing assignments can be obtained within 3 days. Conclusions Our typing results suggest that the new approach of adding an additional sequencing step may resolve most of the DRB1 typing ambiguities and deliver the allele level typing needed by transplant clinicians. Studies in resolving ambiguities of HLA DQB1 with Domino Stone primers are under evaluation. Download : Download full-size image

Aim Based largely on the success of virtual crossmatching (vXM), the OPTN implemented a new kidney allocation process. Specifically, transplant candidates with cPRA values of 9899% and 100% now have priority for deceased donor (DD) kidneys. Recent studies revealed that the majority of these highly sensitized patients (HSPs) had HLA-DP antibodies, meaning that vXM was not possible unless the corresponding donor HLA-DP type was provided. A similar situation occurs if patients possess allele specific HLA antibodies and donors with the corresponding antigen are not allele level typed. Here again, vXM is not possible. In this study, we evaluated the distribution and frequency of allele specific HLA antibodies among HSPs. The allelic antibodies represented on SAB products are shown in the table below. Methods Luminex SAB assays were performed with sera from all listed kidney candidates with cPRA > 98% (n = 242). An antibody was considered positive when the MFI was > 2000. Results Of 189 black, 44 white and 9 Hispanic HSPs, 85% (n = 160), 89%(n = 39) and 78% (n = 11), respectively, had at least one allele specific antibody.The average number of allele specific antibodies per patient was 1.28 ± 0.71 (Class I) and 2.6 ± 1.59 (Class II). The most prominent allele specific antibodies are shown below (shaded). MFI values for the allele specific antibodies ranged from 2000– > 25000. Conclusions These data clearly show that allele specific HLA antibodies are common in HSPs. They are likely the tip of the iceberg since the only detectable allelic antibodies are those represented on the SAB panel. The “real” number of allelic antibodies is likely much greater. High resolution typing of DDs, at least for those alleles to which antibodies are commonly detected, needs to be provided in a timely fashion in order to perform accurate vXM for HSPs with allele specific antibodies. Download : Download full-size image

Aim NMDP high resolution (HR) testing programs require typing for common or well documented (CWD) null alleles in alignment with Mack et al., Tissue Antigens. 2013: 81(4): 194–203 (CWD 2.0). The aim of this study was to determine the frequency of CWD 2.0 Class I null alleles in NMDP HR typing programs and whether these alleles occur outside of their expected haplotypes. Methods Labs performing HR typing routinely test exons 2–4, then additional regions depending on where a CWD null allele polymorphism is located. Typing methods include SSO, SBT, and NGS. HR Class I typing results from historic adult donor registry samples and CBU samples performed in 2013–2015 were queried for reports of CWD null alleles. Results Four different Class I CWD null alleles were reported a total of 35 times in 41,864 tests. B∗51:11N is expected in the A∗02∼DRB1∗04 haplotype but was observed in A∗03:01∼C∗07:02∼DRB1∗04:02. C∗04:09N was observed with its most common haplotype A∗23:01∼B∗44:03∼DRB1∗07:01 in 53% of cases and with B∗44 in 100% of cases. The common A∗02:53N was not reported; it occurs in up to 0.10% of Chinese populations. The remainder of the CWD nulls were not observed. G group frequency is the null allele frequency within the two digit G group typing. Conclusion Although the frequency of Class I CWD null alleles in the queried HLA results is low, they may warrant continued routine typing. Failure to identify a null allele results in a hidden mismatch between donor and patient. However, lab costs increase due to the additional typing required to detect polymorphisms located outside of exons 2–3. As exon 7 is not routinely tested, typing for C∗04:09N only in the presence of B∗44 may be a future consideration for NMDP HR contracts and policies. Download : Download full-size image

Definition of sensitization to class II antigens based on its β chain may be problematic for DQ due to the polymorphism of both α & β chains in DQ antigens and can wrongly assigned DSA that may decline immunologically compatible transplant. The patient presented here highlights this problem A 23-year-old male referred for kidney transplantation. HLA-typing for him and three living related potential donors are summarized below. A B C DRB1 DQA1 DQB1 Pt 02, 33 49, 51 07, 15 17, 16 05:01, 01:02 02:01, 05:02 D1 11, 68 35, 51 04, 15 04, 11 03:02, 05:05 8, 7 D2 01, 03 51 06, 16 04 03:01 8 D3 01, 31 35, 51 06, 07 17, 04 03:01, 05:01 8, 02:01 HLA-antibodies by SAB were positive for DQ7–9. Flow XM with donor 1&2 (D1, D2) were positive for B-cells but negative with donor 3 (D3). A closer analysis of SAB data (Fig. 1) revealed positive reactivity of all DQ8 coated beads except bead no. 82. DQA1 ∗ 03:02 was thought then contributing to the positivity. High resolution typing (HR) for DQ8 confirmed sharing of DQA1 ∗ 03:02, DQB1 ∗ 03:02 between D1&2. Reactivity analysis for bead no. 42 and 43 could not exclude positivity to DQ8 β (B ∗ 03:02) component due to negative reaction of the corresponding α components. Furthermore, the combination DQA ∗ 03:01, DQB ∗ 03:02 coated beads (present in D3) reacted strongly. Flow PRA class II bead on BD analyzer, showed positive DQA1 ∗ 03:02 homozygous bead only. DQA1 ∗ 03:02 homozygous present in D2 & a single copy of this allele present in D1, while D3 does not carry this allele, so we concluded that actual antigenic epitopes is located in DQA1 ∗ 03:02, possibly the homozygous conformation increased its expression & contributed to the higher channel shift of the FXM with D2. Due to uncertainty in assessing the immunological risk the transplant was denied several times before the patient underwent uneventual transplant from D3 and continued enjoying good kidney function up to one month post transplant. Download full-size image

Strict HLA-match using high resolution typing for HLA-A,B,C,DRB1 improves unrelated volunteer donor (URD) hematopoietic stem cell (HSC) transplant outcome but limits the number of suitable donors. In contrast, CB transplantation (CBT) is associated with a greatly reduced HLA-match requirement which may extend the donor pool. Therefore, we prospectively evaluated donor availability in 309 patients [median age 46 (range 1–71 years)] with high-risk hematologic malignancies between 10/05–5/08 with the hypothesis that CB would extend transplant access. Adequate donor-recipient HLAmatch was ≥/10 HLA-A,B,C,DRB1,DQ alleles for a T cell depleted or ≥9/10 alleles for an unmodified URD graft. Adequate CB units were ≥4/6 HLA-A,B antigen, DRB1 allele matched and ≥1.5 × 107/nucleated cells/kg/unit and double unit grafts were used to augment engraftment. URDs had priority as HSC source; CB was chosen if no suitable URD was available in the required time period. While 28 patients with suitable URDs had no CB search, the results of the 281 patients with both URD and CB formal search/confirmatory typing are shown in Table 1. This combined search group had highly diverse ancestry with only 60 (21%) of northwestern (NW) European origin and 99 (35%) were non-European. 9–10/10 URDs were available for the majority of NW, Eastern and mixed Europeans but not for Southern or non-European patients. Notably, CB extended HSC availability to all and especially Southern European, Asian, African, and Hispanic patients with no Asian patient being without a CB graft. Table 1: Comparison of Formal Search for Both URD and CB by Patient Ancestry Best URD NW Europe (n=60) East Europe (n=40) South Europe (n=39) Mix: Europe (n=43) Asian (n=19) African (n=33) Hispanic (n=36) Middle East (n=6) Mix: Non-Europe (n=5) 9–10/10 53 (88%) 33 (82%) 23 (59%) 31 (72%) 4 (21%) 14 (42%) 16 (44%) 5 (83%) 3 (60%) ≤8/10 7 (12%) 7 (18%) 16 (41%) 12 (28%) 15 (79%) 19 (58%) 20 (56%) 1(17%) 2 (40%) Best CB 5–6/6 48 (80%) 30 (75%) 20 (51%) 30 (70%) 14 (74%) 19 (58%) 22 (61%) 4 (67%) 4 (80%) 4/6 11 (18%) 8 (20%) 13 (33%) 12 (28%) 5 (26%) 6 (18%) 10 (28%) 2 (33%) 0 (0%) No CB 1 (2%) 2 (5%) 6 (16%) 1 (2%) 0 (0%) 8 (24%) 4 (11%) 0 (0%) 1 (20%) The ancestry of the 201 patients who were transplanted and the 15 not transplanted due to lack of suitable graft is shown in Table 2. The remaining 93 patients were not transplanted for other reasons. Table 2: Patient Ancestry if Transplanted or No Graft 8–10/10 URD (n=149) 4–6/6 CB (n=52) No graft (n=15) NW Europe 47 (32%) 4 (8%) 1 (7%) East Europe 27 (18%) 6 (12%) 0 (0%) South Europe 15 (10%) 8 (15%) 3 (20%) Mix:Europe 27 (18%) 8 (15%) 1 (7%) Asian 6 (4%) 7 (13%) 0 (0%) African 11 (7%) 10 (19%) 6 (40%) Hispanic 10 (7%) 8 (15%) 3 (20%) Middle East 5 (3%) 1 (2%) 0 (0%) Mix:Non-Europe 1 (1%) 0 (0%) 1 (7%) URD transplant recipients were predominantly (68%) NW, Eastern, or mixed European with Asian, African and Hispanic patients combined accounting for only 18%. In contrast, only 4 (8%) of CBT recipients were NW European. While these patients received CB due to urgency (n=1), patient preference (n=1), or MD preference over mismatched URD (n=2), the remaining 48 (92%) of CBT recipients including 15% Southern Europeans and 49% non-Europeans (13% Asian, 19% African, 15% Hispanic, 2% Middle Eastern) did not have other donor options. Of 15 patients (5% of the 309 total) not transplanted due to lack of any HSC source 10 were non-European including 6 of African ancestry. Notably, the median weight of this “no graft” group [87kg (range 66–151)] was significantly higher than CBT [70kg (range 13–109)] recipients (p

Aim The ability to identify suitably matched (10/10 or 9/10) adult unrelated donors (URD) impacts the patients’ search time and costs. A simple scoring system that provides a prognosis of the patient (Pt) search success could be a useful tool for transplant physicians and would provide a surrogate measure of search productivity. We aimed to determine if Pt genotype frequency (GF) can predict whether a Pt is likely to have a potential 10/10 and/or 9/10 URD in the Be The Match (BTM) Registry. Methods A test dataset contained 516 Caucasian (CAU) Pts who initiated a search of the BTM Registry with 5 locus (HLA-A, B, C, DRB1, DQB1) intermediate/high resolution typing. Pt HLA was analyzed in the haplostats.org application and the output of GF for the most common predicted haplotype pair was recorded. Pt BTM URD search results output was then recorded for URD who had a HapLogic predicted match of > 50% 10/10 and >50% 9/10. GF ranges were defined to identify pts with search prognosis categories of: (1) “productive” (>15 10/10 and > 2 9/10), (2) “good” (between 3 and 14 10/10 and > 2 9/10), (3) “fair” (⩽2 10/10 and >2 9/10), or (4) “poor” (No 10/10 and ⩽2 9/10). A second cohort (N= 1126) was used to validate the precision of using GF. Results The table below shows the categories and precision. For example, patients with GF ⩾ 2x10ˆ-6, 93.5% were category 1, 5.5% were category 2, and 1% category 3. [ Table 1 ] Conclusions This study demonstrates a categorization scheme for CAU Pts using GF as a predictive measure for the productivity of an URD search. When GF did not correspond with the productivity category, it was most often in a bordering category. Although no replacement for running an unrelated search for a Pt, this is a quick way for physicians to get an indication of the search productivity.

Aim The aim of this study is to test the feasibility and compare the analytical performance of HLA typing of clinical and proficiency testing samples using next-generation sequencing against standard molecular HLA typing methods in a medium-sized clinical HLA laboratory without prior sequencing experience. Methods We amplified HLA-A, -B, -C, -DRB1 and –DQB1 genes, from clinical samples, by long-range PCR using primers developed at our laboratory. Long-range PCR product identity was verified by Luminex SSO. HLA gene products were pooled and processed to prepare a sequencing library using the Nextera XT sample kit followed by sequencing on the Miseq instrument. Data was analyzed using the Omixon Target HLA software to assign HLA types. HLA assignment by next-generation sequencing was compared against results previously obtained by standard molecular typing methods. Results We report the concordance of HLA typing results obtained by next-generation sequencing and analysis compared with standard molecular typing methodologies. Conclusions Importantly, the reagent cost of performing high-resolution typing by next-generation sequencing is similar to molecular methods currently used. Next generation sequencing can be a viable approach to high resolution HLA typing in medium-sized clinical HLA laboratories. Berces: Omixon: Employee; Stockholder. Major: Omixon: Employee. Hague: Omixon: Employee.

Aim Sequencing Based Typing (SBT) is the gold standard for high resolution typing, as the technique analyses the actual DNA sequence of the relevant HLA regions. However, Sanger SBT strategies often show ambiguous results, caused by sequencing PCR fragments with a mix of two alleles. Additional sequencing reactions are required to resolve the ambiguities. This can be overcome with Next Generation Sequencing (NGS) technology. One of the unique features of NGS is that reads originate from a single molecule, thus automatically determining the two alleles separately. This allows for development of an HLA genotyping system that yields typing results without ambiguities. In addition, NGS technology is developing fast and has come to a point where it can be applied routinely. Methods We developed NGSengine®, a software package to perform HLA typing based on NGS data. NGSengine® is an integrated approach which includes determining HLA locus, applying a sequence reference, aligning reads, phasing and typing. Since HLA is highly polymorphic, each of these steps contains HLA-specific features. In this updated study we show that HLA typing by NGS can be routinely applied using data of different NGS systems. Results Data was generated on Roche 454, Ion Torrent PGM, PacBio, Illumina MiSeq and HiSeq. All data available from these platforms could be analyzed by NGSengine®. In a single analysis the expected HLA typings were obtained. In case PCR fragments were generated with whole gene amplification (NGS-go®) no ambiguities were found since alleles were effectively separated, and the complete gene was covered by the reads. Conclusions NGSengine®: · performs reliable unambiguous HLA allele assignment based on NGS data · enables HLA-typing of NGS data in a single procedure · can be applied on data from different NGS platforms · can be applied on data generated with different amplification strategies · determines parts of allele sequences not yet present in the IMGT/HLA database · is easy to use by ‘single button analysis’. Kooter: GenDx: Employee. Ruzius: GenDx: Employee. Penning: GenDx:Employee. Mulder: GenDx: Employee; Stockholder. Rozemuller: GenDx:Employee; Stockholder.

Aim As fewer sibling marrow donors are available due to decreasing birthrates, alternatives are more frequently being considered when 8/8 matched allele donor for HLA-A,B,C,DRB1 are not available & clinically, recipient needs urgent transplant. Our current Bone Marrow Policy includes consideration of single mismatch & single haplotype matched donors. Antibodies directed against donor specific antigens (DSA) are avoided to reduce the risk of graft failure. This case demonstrates the need for additional typing beyond policy when antibodies were identified. Methods Patient: 59 Y/O female, AML Initial typing performed by SSP at HLA-ABCDRB1, no related donors, high resolution typing performed and unrelated search initiated. No 8/8 matches identified. Two potential 7/8 unrelated registry donors were selected, samples sent to us for high resolution and DQB1 typing. Antibody ID was performed using Luminex with OneLambda Single Antigen on recipient’s serum. Results Antibodies were identified in the recipient’s serum to Class I & II antigens. No DSA’s were identified to either of the mismatched class I donor antigens. DRB3∗01:01 & DRB3∗03:01 Class II antibodies were identified. Additional high resolution typing of the recipient & donor’s DRB3∗ (not normally required) was performed to determine DSA status. Recipient & donors typed as DRB3∗02:02, therefore the antibodies were not DSA. Donor was selected & transplant booked for April 19, 2013. Conclusions When a fully matched donor is not available for a patient and the clinical status of the patient indictes the need to continue to transplant then alternatives must be considered. Our transplant program is moving in the direction of related single haplotype matched transplants and single mismatch antigen/allele unrelated donors while ensuring that DSA is not identified against the mismatched antigen(s)/alelle(s). Additional urgent laboratory testing & investigation beyond our standard testing process is required to ensure the most suitable donor is selected.

Aim Next Generation Sequencing (NGS) has entered routine in HLA sequencing laboratories. We implemented a short amplicon-based NGS workflow for high-throughput donor registry HLA typing of 6 loci at high resolution. An elaborate primer design was key for this cost-efficient approach. Methods Potential primer binding sites are restricted by the read lengths of NGS systems. For most amplicons this requires a mix of primers that has to be optimized for uniform amplification of all alleles. In our case primers were designed to amplify under identical PCR conditions for all amplicons to facilitate the use of the 48.48 Fluidigm Access Array. Using the Illumina MiSeq 2x250bp system we chose primer binding sites to achieve full exon coverage with an overlap of 50 to 200 bases of exons 2 and 3 for HLA loci A, B, C, DRB1, DQB1 and DPB1. Self-developed Perl and Python scripts were used for analysis of each primer’s performance, as individual NGS reads can be classified and quantified. Primer sets were assessed for the ratio of target reads between amplicons and the ratio of target reads to off-target reads. Off-target reads were classified in three categories. Uniformity of amplification was assessed with the minimum requirement of a 1:5 ratio of target reads between alleles in a heterozygous sample. Based on this approach over 700 primers were screened and a final set of 66 primers was chosen. Results In three MiSeq runs 1140 samples (6840 loci) were typed and compared with previous Sanger sequencing results. All typings were in full concordance. In average 80.2% of the allocated reads corresponded to target HLA alleles, with a range from 63.9% (HLA-B exon 2) to 89.2% (HLA-A exon 2). Conclusions The validated primer set was launched in December 2012 and yields 98.7% high resolution typing. In an automated workflow, this approach allows very cost-efficient high-throughput (currently about 1500 donors per day) HLA donor registry typing.

Aim Strong allele-specific antibodies are frequently detected by Luminex single antigen bead (SAB) assays. However, these cannot be reported as unacceptable antigens in the UNOS database. The problem compounds when a patient expresses the same antigen and the non-self-allele specific antibody cannot be documented in the system. We report herein a case of a 60 year-old Hispanic female patient listed in UNOS for a deceased kidney donor who expressed the B62 antigen and whose sera were positive for strong anti-B*15:01(B62) antibody detected by positive B*15:01 beads in both Luminex PRA and SAB assays. Methods HLA typing was performed by SSP (Olerup). HLA antibodies were detected by Labscreen PRA and SAB (One Lambda). Flow cytometry and standard CDC crossmatches were performed. Results Strong anti-B62 antibody against her “self” B62 antigen was detected by both PRA and SAB panels in the patient’s pre-transplant sera. High resolution typing of the patient revealed B*15:15(B62), while B62 antigens beads on both PRA and SAB panels are B*15:01. B*15:15, a common allele in the US, is not represented in the PRA and SAB panels. Thus, non-self allele-specific B*15:01(B62) antibody was present. A deceased donor shared A, B, DR and DQ antigens with the patient was offered. However, strong positive T- and B-cell flow cytometry crossmatches and positive B-cell CDC crossmatches against this donor were observed. Auto-crossmatches were negative. The positive allo-crossmatch resulted from the strong allele donor-specific antibody against B*15:01which differs from the recipient’s B*15:15 at one amino acid at position 63 (B*15:01 = E63 vs. B*15:15 = N63). Conclusions High resolution typing is necessary for some HLA antigens in solid organ transplantation. The ability to report allele-specific unacceptable antibodies in the UNOS will further assist organ allocation and prevent unnecessary delays. In addition, lack of beads representing common alleles in the SAB assays can result in false-negative virtual crossmatch results.

SUMMARY We describe HLA-DRB1 typing using polymerase chain reaction-based microtitre plate hybridization (PCR-MPH), which can process large numbers of samples. MPH typing is similar to an enzyme-linked immunosorbent assay (ELISA), in which a tandemly ligated sequence-specific oligonucleotide is immobilized on microtitre wells. The typing procedure consisted of two steps. In the first, PCR-MPH with 16 probes was performed to determine the specificities of the serological levels (DR1, DR2, DR3, DR4, DR11, DR12, DR13, DR14, DR7, DR8, DR9 and DR10) after generic amplification (‘low resolution typing’). In the second step, DR1, DR2, DR4, DR 12/8 and DR3/11/13/14 were group-specifically amplified based on the results of the first PCR-MPH, and microtitre plate hybridization proceeded in a similar manner to the first step (‘high resolution typing’). Low resolution typing was completed within 2 h after generic amplification, and the results of high resolution typing were obtained in another 3.5 h after amplification. The allelic types classified using PCR-MPH were completely concordant with those obtained by PCR- single-strand conformation polymorphism or PCR-restriction fragment length polymorphism.

We report herein the identification of a new HLA-DRB1 allele, DRB1*0707. This new allele was seen in a volunteer bone marrow donor (ID#118069) belonging to the German bone marrow donor registry (DKMS). HLA-DRB1*0707 was detected while performing HLA-DRB1 high resolution typing by sequence based typing. This novel allele differs from DRB1*070101 by a single nucleotide substitution at position 163 (C-->T) in exon 2.

Aim A prospective typing project to confirm HLA alleles with an unconfirmed status in the IMGT/HLA database identified sequence errors for 2 HLA-C alleles, C ∗ 03:12 and C ∗ 15:20. C ∗ 03:12 was described in November 1999 on a reference cell from a Be The Match Registry volunteer. C ∗ 15:20 was described in February 2007 on a reference cell typed through a NMDP prospective high resolution typing project. Methods A sample initially reported as C ∗ 03:12 was re-typed by two independent labs, who both confirmed the presence of C ∗ 03:03. Because of the discrepancy, the IMGT/HLA reference cell was evaluated at an NMDP contract lab by SBT to confirm the presence of C ∗ 03:12. Concurrently, the IMGT/HLA reference cell carrying C ∗ 15:20 re-typed as C ∗ 15:27. Since the reference cell sequence contained a likely error, it was sent for cloning and sequencing of the putative C ∗ 15:20 in isolation. A second recently recruited sample with C ∗ 15:20 was identified and re-typed by SBT. Results The IMGT/HLA reference cell reported to carry C ∗ 03:12 was corrected to C ∗ 03:19, whereas the other sample with C ∗ 03:12 retyped as the common C ∗ 03:03, indicating two discreet typing errors. C ∗ 03:12 differs from C ∗ 03:19 at nucleotide (nt) 343 (A to G) at the end of exon 2. C ∗ 03:12 and C ∗ 03:19 are further differentiated from C ∗ 03:03:01 at nt 419 and nt 420 in exon 3. C ∗ 03:19 was described in February of 2005 and has a confirmed status in the IMGT/HLA database. Both the IMGT/HLA reference cell and the other sample with C ∗ 15:20 retyped as C ∗ 15:27. C ∗ 15:20 differs from C ∗ 15:27 at nt 259 (G to A) and nt 261 (G to C) in exon 2. C ∗ 15:27 was described in January 2010 and has a confirmed status in the IMGT/HLA database. Conclusions As a result of the resequencing results of the reference cells, which were found to contain errors, C ∗ 03:12 and C ∗ 15:20 will be deleted from the IMGT/HLA database. This project highlights the importance of confirming IMGT/HLA database allele sequences to validate existence of reported alleles.

Aim The completeness and resolution of the HLA typing of registered volunteer donors is usually not sufficient to determine their suitability for a concrete patient. Therefore, knowing the probability for each ambiguity in a donor to turn out to be a match is indispensable for planning the search strategy at large and for deciding each single step. Methods Since the update to version 2 in late 2009, OptiMatch ® has been using HLA-A, -B, -C, -DRB1, -DQB1 high resolution haplotype frequencies (HF) to predict the probability that each donor will be a 10/10 or 9/10 match as well as the probability of identity for each of the five loci individually. The HF used had been derived from over 145.000 active donors of ZKRD typed for the five loci at intermediate or high resolution. We retrospectively validated the predictions performing the same calculation for over 38,500 CT samples requested between January 2000 and March 2011 where no high resolution information was available for HLA-A, -B, -C, -DRB1 and -DQB1 at the time of request, but was revealed by the CT result. We grouped predictions in intervals of 5%, then compared the expected and observed number of matches per interval and performed a weighted linear regression. Results The R2 were >.995, regression coefficients >.94 and absolute intercepts Download full-size image Conclusions The predictive power of the OptiMatch ® algorithm exceeds our own expectations. With an adequate base of haplotype frequencies it is an excellent tool for speedy, economic and tailored unrelated donor searches.