Paul P.J. Dunn

Human leucocyte antigen typing: techniques and technology, a critical appraisal

Methods for the identification of Human Leukocyte Antigens (HLA) have changed significantly since this group of polymorphic proteins were first characterized by serological reagents in the 1960s and 1970s. The invention and development of the Polymerase Chain Reaction (PCR) has been key in the progress of methods for HLA genotyping. As the complexity of HLA polymorphism has unravelled so it has exposed the weaknesses in techniques such as PCR – Restriction Fragment Length Polymorphism (RFLP) and Reference Strand Mediated Conformation Analysis (RSCA), which are no longer in use today. Methods which have been considered routine laboratory tools in recent years, such as Sequence‐Specific Primer – PCR and Sequencing Based Typing (SBT) are now also threatened with extinction, not only because of the depth of HLA variation but also because of the rapid development of Next Generation Sequencing and technologies which will follow this. This review describes the merits and disadvantages of current technologies available to HLA Typing laboratories, future trends and the problems posed by new alleles.

Human platelet antigens frequencies in Maori and Polynesian populations

Allele frequencies of human platelet antigens (HPA) reflect population history and possibility of platelet‐specific alloimmunization. Here, we report on screening of variants at HPA loci for Polynesian and Maori subjects.

16th IHIW: Global distribution of extended HLA haplotypes

This report describes the project to identify the global distribution of extended HLA haplotypes, a component of 16th International HLA and Immunogenetics Workshop (IHIW), and summarizes the initial analyses of data collected. The project aims to investigate extended HLA haplotypes, compare their distribution among different populations, assess their frequency in hematopoietic stem cell unrelated donor registries and initiate an international family studies database and DNA repository to be made publicly available. HLA haplotypes compiled in immunogenetics laboratories during the evaluation of transplant candidates and related potential donors were analysed. Haplotypes were determined using the pedigree analysis tool publicly available from the National Marrow Donor Program (NMDP) website. Nineteen laboratories from 10 countries (11 laboratories from North America, five from Asia, two from Latin America and one from Australia) contributed data on a total of 1719 families comprised of 7474 individuals. We identified 10 393 HLA haplotypes, of which 1682 haplotypes included high‐resolution typing at HLA‐A, B, C, DRB1 and DQB1 loci. We also present haplotypes containing MICA and other HLA loci and haplotypes containing rare alleles seen in these families. The project will be extended through the 17th IHIW, and investigators interested in joining the project may communicate with the first author.

Molecular approaches to transfusion medicine in Polynesians and Maori in New Zealand

In recent years, with the application of genotyping technology, there has been a substantial increase in the number of reported blood group alleles. This survey was designed to evaluate new molecular blood group genotyping methods and compile reference blood group data sets for Polynesian and Maori subjects. Subsequent analyses of these results were used to calculate probability of random match, to trace Polynesian ancestry and migration patterns and to reveal past and present episodes of genetic admixture. Genomic DNA samples from Maori and Polynesian subjects were drawn from the Victoria University of Wellington DNA Bank and genotyped using combination of commercial PCR‐SSP kits, hybridization SNP assay services or sequence‐based typing. This survey also involves compilation of serological ABO and Rhesus blood group data from RakaiPaaka Iwi tribal members for comparison with those generated during our molecular blood group study. We observed perfect consistency between results obtained from all molecular methods for blood group genotyping. The A, O, DCcEe, DCCee, MNs, K−k+, Jk(a+b−), Jk(a+b+), Fy(a+b−), Fy(a+b+), Di(a+b−), Co(a+b−) and Do(a−b+) were predominant blood group phenotypes in both Polynesians and Maori. Overall, our survey data show only small differences in distributions of blood group phenotypes between Polynesian and Maori groups and their subgroups. These differences might be associated with selection, population history and gene flow from Europeans. In each case, we estimate that patients with certain blood groups have a very low probability of an exact phenotypic match, even if the patients were randomly transfused with blood from donors of their own ethnicity. The best way to avoid haemolytic transfusion reaction in such cases is to perform a pretransfusion cross‐match and recruit increased numbers of donors with rare phenotype profiles. The conclusion of this study is that application of molecular method covering as many known variants as possible may help to improve the accuracy blood group genotyping and potentially conserve the routine requirements of transfusion centres.

Identification of seven novel HLA class I alleles in New Zealand

Seven new HLA class I alleles have been identified in the New Zealand population in the process of routine HLA typing and they are described here. Unusual bead positivity in Luminex typing identified potential new alleles in a bone marrow registry donor (B*40:285) and two HIV patients prior to abacavir prescription (B*14:02:09, B*41:29). In addition, four new class I alleles were identified through class I sequencing‐based typing (SBT) outside of exons 2 and 3. One mutation was identified in exon 4 (new allele C*12:125) and three have been found in exon 5, an exon rarely sequenced. Two stem cell transplant recipients (B*07:02:45, C*03:279) had novel mutations in exon 5 and one was found in exon 5 of a potentially matched unrelated donor from DKMS, previously thought to be B*40:02:01 (B*40:303).

Recent Developments in Transplantation and Transfusion Medicine

Transplantation and transfusion are related and clinically important areas of multidisciplinary expertise, including pre-operative treatment, donor recruitment, tissue matching, and post-operative care. We have seen significant developments in these areas, especially in the late 20th and early 21st century. This paper reviews the latest advances in modern transplantation and transfusion medicine, including several new genetic markers (e.g., major histocompatibility complex class I chain-related gene A, killer cell immunoglobulin-like receptor, and human platelet antigens) for donor and recipient matching, genotyping platforms (e.g., next-generation sequencer and Luminex technology), donor recruitment strategies, and several clinical applications in which genotyping has advantages over agglutination tests (e.g., genotyping of weakly expressed antigens and determination of blood groups and human leukocyte antigen types in multi-transfused patients). We also highlight the roles of population studies and international collaborations in moving towards more efficient donor recruitment strategies.

Advanced technology for storage and transport of purified DNA from umbilical cord blood prior to use in human leukocyte antigens typing and whole-genome microarray analysis.

Two technologies for dry-state, ambient temperature transport of biospecimens were evaluated in this study. Umbilical cord blood (UCB) samples from 4 individuals were transported at ambient temperature using GenPlates, and the DNA recovered was compared with DNA purified directly from granulocytes of the same UCB samples. GenTegra™ DNA tubes were then used to transport the DNA from California to North Carolina and New Zealand, either immediately after drying or following 30 days of storage at 25°C and 76°C. The integrity of the recovered DNA was thoroughly tested using 2 human leukocyte antigens (HLA)-typing techniques (bead array and sequencing), as well as microarray-based whole-genome scanning. HLA-typing results were the same for all samples whether the DNA had been stored for 3 days during transport or 30 days at either 25°C or 76°C. There were no differences in the HLA-typing results of DNA recovered from UCB samples stored in GenPlates compared with DNA extracted directly from granulocytes. Moreover, the microarray analysis revealed call rates of >99.5% for every sample, regardless of storage method, with a statistical concordance of 99.99% between the UCB samples stored in GenPlates compared with DNA extracted directly from granulocytes. These results indicate that both GenPlates and GenTegra are viable methods of storing and transporting UCB (stem cell) biospecimens in a dry state. The quality and quantity of DNA recovered using both technologies are sufficient for complex genotyping using a number of different methods.

Corrigendum to "Diversity in exon 5 of HLA-C*04:01:01G is significant in anthropological studies" [Hum. Immunol. 77 (2016) 426-428].

http://dx.doi.org/10.1016/j.humimm.2016.06.008 0198-8859/ 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. DOI of original article: http://dx.doi.org/10.1016/j.humimm.2016.03.007 ⇑ Corresponding author at: Transplant Laboratory, Leicester General Hospital, Gwendolen Road, Leicester LE12 7BR, United Kingdom. E-mail addresses: paul.dunn@nzblood.co.nz, paul.dunn@uhl-tr.nhs.uk (P.P.J. Dunn). Paul P.J. Dunn a,⇑, Gareth Lamb , Caroline Selwyn , Jillian Compton , Edward Yang , Martin Maiers , Marcelo Fernandez-Vina e

Diversity in exon 5 of HLA-C∗04:01:01G is significant in anthropological studies

Polymorphisms in Human Leukocyte Antigen (HLA) class I genes are generally considered to be relevant only if they reside in exons 2 and 3 or if they affect the expression of the allele. HLA-C(∗)04:82 differs from the common HLA-C(∗)04:01:01 by having a 9 nucleotide, or 3 amino acid duplication, in exon 5. Having observed HLA-C(∗)04:82 in a New Zealand Maori stem cell patient, we have attempted to examine the prevalence of this allele in different ethnicities. Although our studies are in a limited number of patients and donors, they have revealed that, in the Pacific region, HLA-C(∗)04:82 appears to be the most common allele of the HLA-C(∗)04:01:01G group of alleles, notably in Filippinos and in Maori/Polynesians. In these populations this allele has characteristic HLA-ABCDRB1 haplotypes. Thus, our studies have shown that polymorphisms outside of the clinically important exons can be considered to be relevant in anthropological studies.

RNA-Isolierung aus Blut für die HLA-Typisierung mittels Sequenzierung

ZusammenfassungIn einem Forschungsprojekt wurde mit einem Laborroboter aus Blut Gesamt-RNA isoliert, daraus cDNA synthetisiert und diese als Template-DNA zur HLA-Sequenzierung verwendet. Die Ergebnisse wurden mit aktuellen Methoden der HLA-Typisierung verglichen.AbstractIn a recent research project a laboratory robot was used to isolate total RNA from blood. The cDNA was synthesized and used as a template for HLA sequencing. The results of this sequencing-based HLA typing were compared to state-of-art techniques.