The advent of next generation sequencing (NGS) has altered the face of genotyping the human leukocyte antigen (HLA) system in clinical, stem cell donor registry, and research contexts. NGS has led to a dramatically increased sequencing throughput at high accuracy, while being more time and cost efficient than precursor technologies. This has led to a broader and deeper profiling of the key genes in the human immunogenetic make-up. The rapid evolution of sequencing technologies is evidenced by the development of varied short-read sequencing platforms with differing read lengths and sequencing capacities to long-read sequencing platforms capable of profiling full genes without fragmentation. Concomitantly, there has been development of a diverse set of computational analyses and software tools developed to deal with the various strengths and limitations of the sequencing data generated by the different sequencing platforms. This review surveys the different modalities involved in generating NGS HLA profiling sequence data. It systematically describes various computational approaches that have been developed to achieve HLA genotyping to different degrees of resolution. At each stage, this review enumerates the drawbacks and advantages of each of the platforms and analysis approaches, thus providing a comprehensive picture of the current state of HLA genotyping technologies.

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Bioinformatics Strategies, Challenges, and Opportunities for Next Generation Sequencing-Based HLA Genotyping.

Currently, stem cell donor registries include more than 35 million potential donors worldwide to provide HLA-matched stem cell products for patients in need of an unrelated donor transplant. DKMS is a leading stem cell donor registry with more than 9 million donors from Germany, Poland, the United States, the United Kingdom, India and Chile. DKMS donors have donated hematopoietic stem cells more than 80,000 times. Many aspects of donor registry work are closely related to topics from immunogenetics or population genetics. In this two-part review article, we describe, analyse and discuss these areas of donor registry work by using the example of DKMS. Part 1 of the review gives a general overview on DKMS and includes typical donor registry activities with special focus on the HLA system: high-throughput HLA typing of potential stem cell donors, HLA haplotype frequencies and resulting matching probabilities, and donor file optimization with regard to HLA diversity.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/iji.12471

Immunogenetics in stem cell donor registry work: The DKMS example (Part 1).

The COVID-19 pandemic has serious implications also for patients with other diseases. Here, we describe the effects of the pandemic on unrelated hematopoietic stem cell donation and transplantation from the perspective of DKMS, a large international donor registry. Especially, we cover the development of PBSC and bone marrow collection figures, donor management including Health and Availability Check (HAC), transport and cryopreservation of stem cell products, donor recruitment and business continuity measures. The total number of stem cell products provided declined by around 15% during the crisis with a particularly strong decrease in bone marrow products. We modified donor management processes to ensure donor and product safety. HAC instead of confirmatory typing was helpful especially in countries with strict lockdowns. New transport modes were developed so that stem cell products could be safely delivered despite COVID-19-related travel restrictions. Cryopreservation of stem cell products became the new temporary standard during the pandemic to minimize risks related to transport logistics and donor availability. However, many products from unrelated donors will never be transfused. DKMS discontinued public offline donor recruitment, leading to a 40% decline in new donors during the crisis. Most DKMS employees worked from home to ensure business continuity during the crisis.

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Stem cell donor registry activities during the COVID-19 pandemic: a field report by DKMS

PURPOSEUltrahigh resolution (UHR) HLA matching is reported to result in better outcomes following unrelated donor hematopoietic cell transplantation, improving survival and reducing post-transplant...

https://ascopubs.org/doi/pdfdirect/10.1200/JCO.20.03643

Impact of Previously Unrecognized HLA Mismatches Using Ultrahigh Resolution Typing in Unrelated Donor Hematopoietic Cell Transplantation.

New challenges, new opportunities: Next generation sequencing and its place in the advancement of HLA typing.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for severe blood diseases. According to the EBMT activity survey, unrelated stem cell donors provided 52% of all allogeneic stem cell products in 2015 [1]. Unrelated donor registries inform, recruit and administer potential donors and have built up a global pool of currently more than 32 million donors since the 1970s [2]. As an essential part of the donor recruitment process, the potential donor’s HLA genotype is determined. The specification of typing scope (number of loci, resolution) and typing method (e.g., Sanger sequencing, next-generation sequencing (NGS)) is made by donor registries under consideration of limited resources for donor registry operations. NGS technologies offer high-throughput high-resolution HLA typing of new donors at unprecedented low costs [3–6]. The process leading to stem cell donation currently includes a mandatory confirmatory typing (CT) (sometimes also named verification typing (VT)) step. Main purpose of the CT step is the confirmation of the potential donor’s HLA typing result by typing a new donor sample, generally at the same lab that executed the prospective transplant recipient’s HLA typing. In many, albeit not all, cases of discrepancies between the original and confirmatory typing results, the donor registry administering the respective donor and providing the CT sample is informed accordingly. At DKMS, we resolve cases with at least one discrepant HLA locus, e.g., by collecting a third donor sample and mandating a “neutral” HLA lab (different from the labs carrying out the original typing and the CT) to type the sample. Based on the result, either the original typing result or the CT result is classified as erroneous. Discrepancies without relevance for donor-patient HLA matching (e.g., silent mutations outside exons coding for the antigen recognition domain) sometimes remain unresolved. For the analyses in this letter, only samples and loci were considered for which recruitment typing and CT typing results were available. Apart from confirmation or falsification of the donor’s original HLA typing result, the CT step also comprises a phone contact with the potential donor including the provision of detailed information on the two stem cell donation methods, the clarification of the donor’s continued interest to donate, the analysis of infectious disease markers (IDM), and a questionnaire-based health screening. In urgent cases, the CT step can be merged with the following step in the process leading to stem cell donation, the so-called donor work-up (WU). Donor WU includes, for example, the donor’s final medical clearing at the collection center. In 2016, DKMS Germany received 27,232 CT requests, 6825 work-up requests and 412 combined requests (named “CT/work-up parallel requests” (CWP requests) in the following). Of the CT requests, 4924 (18.1%) finally led to a donation while further 116 requests (0.4%) were still open at the time of data retrieval. Furthermore, 227 CWP requests (55.1%) resulted in a donation with no requests open anymore. In this letter, we present real-life data from the DKMS Germany donor file regarding discrepancies between HLA typing results at donor recruitment and CT results. Primarily, we focus on donors who were typed by NGS-based methods at the DKMS Life Science Lab (Dresden, Germany) where NGS had been established as high-throughput method for donor typing at recruitment in 2013 [3, 6]. Until 31 March 2017, 2,055,028 newly registered DKMS German donors * Jürgen Sauter sauter@dkms.de

Very low error rates of NGS-based HLA typing at stem cell donor recruitment question the need for a standard confirmatory typing step before donor work-up.

Heike Fischer

Papers

Very low error rates of NGS-based HLA typing at stem cell donor recruitment question the need for a standard confirmatory typing step before donor work-up.