BSHI guideline: HLA matching and donor selection for haematopoietic progenitor cell transplantation

A review of the British Society for Histocompatibility and Immunogenetics (BSHI) Guideline ‘HLA matching and donor selection for haematopoietic progenitor cell transplantation’ published in 2016 was undertaken by a BSHI appointed writing committee. Literature searches were performed and the data extracted were presented as recommendations according to the GRADE nomenclature.


| INTRODUC TI ON
The infusion (transplantation) of haematopoietic progenitor stem cells (HPC) into a patient with haematological failure due to malignant or nonmalignant causes can result in successful engraftment of donor-derived HPC which undergo haemopoiesis to replace the malfunctioning cells of the patient's immune system.
The effectiveness of these transplants in terms of patient overall survival (OS) and disease-free survival (DFS) has improved with each decade due to accurate histocompatibility matching between donor and patient; use of alternative donors; improved patient conditioning protocols; use of therapeutic agents; prevention and treatment of infections and post-transplant supportive care.
This guideline is an update to the previous published guideline (Little et al., 2016) and describes the selection of donors for allogeneic, that is the donor is not genetically identical to the patient, HPC transplantation (HPCT).

| ME THOD
This guideline was produced by the following actions:

A writing committee (authors of this manuscript) comprising
Histocompatibility and Immunogenetics (H&I) scientists providing an H&I clinical service for related and unrelated donor haematopoietic progenitor cell transplantation was established. Ann-Margaret Little was appointed as the chair of the committee.

| Disclaimer
These recommendations represent consensus opinion from experts in the field of H&I within the United Kingdom. They represent a snapshot of the evidence available at the time of writing. This evidence may become superseded with time. It is recognized that recommendations have been made even when the evidence is weak.
The British Society for Histocompatibility and Immunogenetics (BSHI) cannot attest to the accuracy, completeness or currency of the opinions and information contained herein and does not accept any responsibility or liability for any loss or damage caused to any practitioner or any third party as a result of any reliance being placed on this guideline or as a result of any inaccurate or misleading opinion contained in the guideline.

| RECOMMENDATI ON S AND SUGG E S TIONS
Recommendations and suggestions are summarized in Tables 1 and   2. These evidence-based recommendations expand and adapt previous guidance (Little et al., 2016).

| HL A MATCHING IN HP C TR AN S PL ANTATI ON
Amongst the many factors that contribute to successful transplantation, the most significant is the degree of human leucocyte antigen (HLA) compatibility between donor and patient.
Unlike most genes in the human genome, HLA genes are hyperpolymorphic, that is there are many variations within their gene sequences and each variant is called an allele. Most allele differences are nonsynonymous and the majority of amino acid variations impact on the structure of the peptide-binding domains thus directly influencing how self and non-self peptides are presented to T cells and how they are recognized by natural killer (NK) cells.
In a HPCT, alloreactive cells of the patient can initiate an immune response against non-self antigens expressed by the transplanted donor cells, causing rejection of the donor cells. This is defined as either lack of initial engraftment of donor cells (primary graft failure) or loss of donor cells after initial engraftment (secondary graft failure). Pre-transplant conditioning of the patient with chemotherapy and/or irradiation reduces this HVG response allowing

Supporting text location Section
Level 1 (we recommend) Quality of Evidence: GRADE A (high)

5.1
All laboratories performing H&I testing for allogeneic HPCT in the UK must be accredited by EFI and UKAS 5.2 HLA typing definitions as described by Nunes et al. (2011) and within this document should be used 6 Alternative progenitor cell donors (single mismatched unrelated donor/umbilical cord blood (UCB)/haploidentical) should be considered early in the donor search when a patient is unlikely to have an HLA matched unrelated donor 7 HLA typing of patients and all donors (matched and mismatched, related, unrelated and cord) proceeding to transplant should be carried out at high resolution for HLA-A, B, C (exons 2 and 3 minimum) and DRB1, DQB1 and DPB1 (exon 2 minimum) which identifies polymorphisms within the antigen recognition domain (ARD) 8.3 When selecting an unrelated donor a 10/10 high or UHR/allele resolution HLA-A, -B, -C, -DRB1 and -DQB1 matched donor should be preferentially selected over a mismatched donor 8.3.1 Where a 10/10 matched unrelated peripheral blood stem cell (PBSC) or bone marrow donor is not available a single mismatch at HLA-A, B, C, DRB1 or DQB1 is acceptable with mismatches at DQB1 preferred 8.3.2 When selecting mismatched donors, avoid amino acid mismatches within the ARD 9.2.1 Ensure shortlisted UCB units meet the minimum threshold required for a single UCB transplant (UCBT), (>3 × 10 7 /kg recipient weight). In non-malignant conditions, especially bone marrow failure syndromes, or in cases where HLA match is <6/8, consider increasing the total nucleated cell (TNC) threshold to >5.0 × 10 7 /kg. When the patient's weight indicates that a double UCBT is required, maintain a minimum TNC of >3.5 × 10 7 /kg. The minimum TNC required for each unit is 1.5 × 10 7 / kg, though preference should be given to the best HLA matched UCB with TNC in excess of this minimum threshold, where possible Immunogenetics (EFI) (https://efi-web.org/) and must be undertaken by a laboratory accredited by the United Kingdom Accreditation Service (UKAS) (https://www.ukas.com/) and EFI.

| Definitions of HLA typing resolution
The definitions for low, high and allele resolution typing as compiled by a joint international working party: the Harmonisation of Histocompatibility Typing Terms Working Group, to define a consensual language for laboratories, physicians and registries to communicate histocompatibility typing information are used within this guideline (Nunes et al., 2011). In addition, we accept the following definitions for intermediate resolution, and ultra-high resolution (UHR) typing (Mayor et al., 2019). Official names are assigned to the HLA genes, antigens and alleles by the World Health Organisation (WHO) Nomenclature Committee for Factors of the HLA System . The Immuno Polymorphism Database-ImMunoGeneTics (IPD-IMGT)/HLA Database is the official repository of the HLA allele sequences (Robinson et al., 2015).

| Intermediate resolution
The term intermediate resolution can be applied when high resolution cannot be achieved, and the provided HLA type includes a subset of alleles sharing the digits in the first field of their allele name, for example A*02:01 or A*02:02 or A*02:07 or A*02:20 but not other A*02 alleles due to differences in the second or other fields. There may be cases in which the subset of alleles includes one or more alleles within a group beginning with different digits but these alleles should be the exception, for example A  (Clayberger et al., 1994) which will impact on the assessment of compatibility between a patient and potential donor. Improvements in HLA typing by next-generation sequencing (NGS) and third generation sequencing (TGS) enable near full-gene (allele) sequencing with greater phase determination facilitating the potential matching of HLA genes beyond previously considered criteria, including polymorphisms in noncoding regions and consideration of synonymous nucleotide mismatches (Petersdorf & O'hUigin, 2019).
The terminology 'UHR' was introduced by Mayor et al. (2019) to describe HLA typing resolution achieved using these techniques which give greater than high but not quite achieving complete allele level. Table 3 gives examples of acceptable reporting conventions.

| HLA antibodies
The role of HLA alloreactive antibodies in HPCT outcome is described in Section 10. Laboratories should be able to detect IgG

| S TAG E OF D IS E A S E AND TIME TO TR AN S PL ANT
One of the earliest steps in donor selection is to consider the disease status of the patient. Depending on the disease type and treatment plan, patients will have different timescales to transplant. In acute leukaemia, where the patient's condition can rapidly deteriorate, there may only be a limited window of opportunity to transplant when the patient is in clinical remission, thus limiting the time available for an extended related or unrelated donor search (Cutler et al., 2004). A patient progressing to an advanced disease usually has a higher mortality risk from the disease than the added risk of a transplant from a single allele mismatch donor or alternative donor therapy such as UCB or haploidentical donor transplantation. The impact of the time required to identify an optimum matching donor has to be offset against the potential negative impact of the disease stage and progression and will determine the source of progenitor cells selected for treatment (Brissot et al., 2017;Weisdorf, 2008 (Valcárcel et al., 2011). Haploidentical transplantation is discussed in Section 7.2.
The availability of parental HLA typing data are useful for assignment of haplotypes. These data can usually be derived for paediatric patients but are rarely available for adult patients. Although meeting current EFI standards, HLA-A, HLA-B and HLA-DRB1 typing of siblings (without parents), to identify potential matches, does not allow accurate determination of haplotypes and can lead to wrongly F I G U R E 1 (a) Low resolution typing at five HLA loci identifies both sibling 1 and 2 as having the same HLA type. (b) HLA allele level typing identifies the father as being heterozygous and sibling 1 and 2 have inherited different haplotypes from the father resulting in sibling 1 being an 8/10 match and sibling 2 being a 10/10 match for the patient establishing presumptive matches. This is a particular concern when there is haplotype sharing between parents or apparent homozygosity (at low/intermediate resolution HLA typing) for a parent. This is illustrated in Figure 1. Also, novel alleles and recombination (especially between HLA-DQ and HLA-DP genes) may be undetected if HLA typing is restricted to only HLA-A, B, DRB1 loci. High, UHR or allelic HLA typing resolution is required as low/intermediate resolution typing could mask additional mismatches (Hansen, 2012;Kanda et al., 2012). HLA-DPB1 typing is also useful in identifying fully matched sibling donors when common haplotypes are present.

| Haploidentical family donors
The use of haploidentical donors has had a significant positive impact on the transplant opportunities for patients who do not have an HLAidentical sibling or volunteer unrelated donor option, as most patients will have a haploidentical relative within their family (Fuchs, 2012).
Current protocols include the post-transplant administration of cyclophosphamide (PTCy) to actively destroy proliferating alloreactive lymphocytes, without impairing haematopoietic progenitor cells, thus reducing the risk of severe GVHD caused by HLA mismatches (Chang & Huang, 2014;Reisner et al., 2011). As the clinical outcome of haploidentical transplants is proving to be comparable to matched related donors (Rashidi et al., 2019), an increase in the number of haploidentical transplants has been documented (Passweg et al., 2019). For the majority of paediatric patients, the donor will either be the patient's mother or father. For adult patients, this choice is often impracticable and siblings and children are usually considered, with second degreerelated donors also possible (Elmariah et al., 2018). Preferred donor characteristics, which vary depending on the transplant protocol (Tcell deplete or T-cell replete), have been reviewed and published in the European Society for Blood and Marrow Transplant (EBMT) consensus recommendations (Ciurea et al., 2020). For T-cell deplete haploidentical transplants, the preference is a first degree relative over second degree and when parents are being considered the mother is preferred over the father due to the benefit of NIMA, discussed further in Sections 8.3.6 and 9.2.5.5. For T-cell replete transplants, siblings (NIMA mismatched) and children are preferred over a parent donor

| Final donor selection
Both patient and the selected related donor must be HLA typed using a second independent sample to exclude any sampling or laboratory errors and must take place prior to the initiation of the patient's conditioning protocol.  It is essential that the person interpreting search reports understands the algorithm used by the various different registries to ensure optimum donor selection.

| Factors impacting on the identification of an HLA-matched unrelated progenitor cell donor
Caucasoid patients have a 40%-75% chance of identifying a highresolution matched donor at HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 (10/10 match) and finding this match is highly predictable (Gragert et al., 2014;Hirv et al., 2009;Tiercy et al., 2000Tiercy et al., , 2007. The chance of a 10/10 match in other ethnic groups, with HLA haplotypes that are less well represented on the unrelated donor registries, is significantly lower (Gragert et al., 2014;Heemskerk et al., 2005;Schmidt et al., 2009). Hence, patients are less likely to find a matched donor from an ethnic group differing from their own and patients with parents coming from differing ethnic groups are at increased risk of not finding any match.
The use of 'minimally' mismatched adult volunteer donors and UCB units increases significantly the likelihood of finding a usable donor (Gragert et al., 2014). The frequencies of HLA-B and HLA-C and/or HLA-DRB1 and HLA-DQB1 associations in differing ethnic groups are available for comparison with the HLA type of the patient (Gragert et al., 2013;Kollman et al., 2007;Mack et al., 2013;Maiers et al., 2007;Sanchez-Mazas et al., 2017), and these tools can be used to help predict how likely it will be to find a match for a patient.
The following factors must be considered when searching for a high, UHR or allelic resolution matched unrelated donor.
• Uncommon HLA-B and HLA-C and/or HLA-DRB1 and HLA-DQB1 associations have a negative impact on likely donor identification.
• When search results reveal mainly low-resolution HLA-typed donors, the presence of a low frequency allele (<5%) in the patient

| HLA matching requirements for unrelated donor transplants
Multiple studies have reported optimum transplant outcome is achieved when the patient and donor are matched for HLA-A, HLA-B, HLA-C and HLA-DRB1 at high resolution (Flomenberg et al., 2004;Lee et al., 2007;Mayor et al., 2019;Morishima et al., 2002;Petersdorf et al., 2001Petersdorf et al., , 2004Petersdorf et al., , 2020Woolfrey et al., 2011). mismatch, then an association with poorer survival, albeit not statistically significant was observed. Although HLA-DQB1 mismatching did not reach significance in relation to survival, in a German study , HLA-DQ antigen mismatching achieved a higher hazards risk for survival compared with HLA-DQ antigen matches.
A meta-analysis evaluating data from 36 studies published between 2002 and 2016 concluded that HLA-DQB1 mismatches were better tolerated than other HLA loci for acute GVHD, supporting previous conclusions (Tie et al., 2017).
There are no data to support matching for HLA-DQA1 (and DPA1 discussed in Section 8.3.3). However, as many commercial HLA typing methods include these loci, if transplants are performed that are mismatched for DQB1 and/or DPB1, then mismatches are also likely to be present for DQA1 and DPA1. Testing for these additional loci may inform future matching strategies.
Overall, in the absence of a 10/10 high-resolution matched donor, a donor with a selected single mismatch for HLA-A, HLA-B, HLA-C, HLA-DRB1 or HLA-DQB1 can be acceptable, with multiple mismatches conferring significantly worse outcomes with the effect observed greater when the patient had acute leukaemia and was transplanted early during first remission compared to patients with advanced disease (Crocchiolo, Ciceri, et al., 2009;Tiercy, 2016).
There is no consensus regarding which of the HLA-A, B, C, DRB1 loci are more detrimental to mismatch. HLA-A and HLA-DRB1 mismatching were reported as being less well tolerated compared with HLA-B and HLA-C mismatches in a NMDP study with all mismatches reducing OS at 1 year by 9%-10% (Lee et al., 2007). In contrast, the Japanese registry reported transplants with HLA-A and HLA-B mismatches had worse survival than HLA-C and HLA-DRB1 mismatches  . HLA-C protein expression varies for different HLA allotypes and in this study patients with HLA-mismatched HLA-C allotypes with higher expression were significantly associated with increased risks of aGVHD III-IV; nonrelapse mortality (NRM) and overall mortality but with no impact on relapse. When the higher expressing HLA-C mismatch allotype was present in the donor, an increase in NRM and mortality, but no effect on aGvHD or relapse was observed. In this study, the allele mismatches were predominantly C*07:01/07:02 and C*03:03/03:04, which have low levels of protein expression and this may account for previously reported permissive HLA-C allele mismatches.
Mismatching for lower expressing HLA-C alleles of the patient compared with higher expressing HLA-C alleles may lower the GVH immune response supporting selection of mismatches for lower expressed over mismatches for high expressed alleles and avoidance of HLA-C-mismatched donors for patients with two highly expressed HLA-C. This study also demonstrated a higher risk in double mismatch (8/10) transplants involving a class I and II mismatch compared with double class I or double class II. Other research has highlighted the impact of the HLA-C bearing haplotype on HLA-C expression which could differ in HLA-C-mismatched transplants (Bettens et al., 2014).
Further to this, Bettens et al. (2016) investigated the cytotoxic T lymphocyte response to HLA-C mismatches and identified that specific mismatches were more immunogenic than others. As predicted from earlier work (Fernandez-Vina et al., 2014), HLA-C*03:03/03:04 mismatching did not initiate a significant alloresponse.
One of the problems making it difficult to define an accurate risk estimate for a single locus mismatch is the extensive polymorphism exhibited at HLA loci. Mismatches at any locus may involve 1 versus 10 versus 20 amino acid mismatches, for example a mismatch of A*01:01 with A*02:01 will be different to a mismatch of A*01:01 with A*03:01, which also will differ from a mismatch of A*01:01 with A*23:01. Not all mismatches at a given locus will have equal effects on GVL and GVH immune responses post-transplant. It has been calculated that a database of 11,000 to 1.3 million transplants would be required to provide sufficient statistical power to detect an association between particular HLA allele mismatches and survival (Baxter-Lowe et al., 2009). In reality, having a choice of a particular locus to mismatch is not always available.

| Impact of individual amino acid substitutions on transplant outcome
The impact on transplant outcomes (acute, chronic GVHD [cGVHD], TRM, relapse and OS) of amino acid substitutions at peptide-binding positions 9, 99, 116 and 156, and KIR-binding position at amino acid 77 was studied in a multivariate analysis of a heterogeneous cohort of patients transplanted for haematological malignancies . Individual mismatches at residues 99 and 116 within HLA-C were associated with an increased TRM and severe aGVHD, respectively. A mismatch at residue 9 within HLA-B was associated with an increase in cGVHD. No mismatches studied had an effect on outcome when located within HLA-A.
In the study of Petersdorf et al. (2014), introduced in Section 8.3.1, patients receiving transplants with 'high expressing' HLA-C mismatches that were also residue 116 mismatched had an increased incidence of NRM compared with patients mismatched for higher expressing HLA-C alleles that were residue 116 matched. Similar findings were made for patients receiving high expressing HLA-C mismatches that were also mismatched for the KIR-interacting residues 77 and 80 (C1 and C2 epitopes discussed in Section 11.5.3).
Thus, mismatching for HLA-C allotypes that are highly expressed and contain mismatches at residues 116 and/or 77 and 80 could be The mechanism for this association has yet to be proven and could involve the peptide impacting on HLA-E expression and recognition by NK and T cells or could also be a marker for another linked functional polymorphism. Thus, consideration of the exon 1 sequence of HLA-B-mismatched unrelated donors could improve the risk of aGVHD. The findings of this study remain to be confirmed.
The immunogenicity of mismatches beyond the ARD has also been investigated using cellular assays (Roelen et al., 2018). This study did not identify any class I mismatches that generated an alloreaction suggesting that these mismatches are permissive. This is in contrast to the UHR matching study where better outcomes were observed with greater matching beyond the ARD (Mayor et al., 2019). Weak responses in the presence of HLA-DRB1*14:01 individuals stimulated by DRB1*14:54 mismatches were identified.
The difference between the proteins encoded by these two alleles is a single amino acid residue in the β2 domain. This response was only present in one direction, suggesting that the directionality of the mismatch can also play a role in its immunogenicity. to 5% of otherwise 10/10 matched siblings will also be HLA-DPB1 mismatched, attributed to recombination (Büchler et al., 2002).
HLA-DP-specific T cells have been detected and associated with both GVL (Herr et al., 2017;Rutten et al., 2008Rutten et al., , 2013 and GVHD (Stevanovic et al., 2013) supporting the direct role of HLA-DP proteins in the immune responses occurring between patient and donor cells post-transplant.
Analysis of the impact of HLA-DPB1 matching and mismatching on transplant outcome has been studied in both single-centre and multicentre studies. In an analysis of a heterogeneous international cohort of transplant recipients, allelic HLA-DPB1 mismatches were shown to offer a GVL advantage via a reduction in relapse, but this was also associated with increased aGVHD and a suggestive increase in mortality (Shaw et al., 2007). In an NMDP study, there was no significant association of single or double HLA-DPB1 allele mismatches with survival compared with no HLA-DPB1 mismatches in an otherwise HLA-A, B, C, DRB1-matched group of recipients. An increased risk of TRM and decreased risk of relapse was suggestive in this study albeit not significant (Lee et al., 2007).
In a UK multicentre study, the impact of HLA-DPB1 allele matching was associated with better OS in patients transplanted with early leukaemia but not in patients transplanted with late stage disease (Shaw et al., 2010), supporting other studies where the effect of HLA matching is not as strong in patients transplanted at late stage. HLA-DPB1 mismatches have been assigned as either permissive or nonpermissive based on observed immunogenicity to T-cell epitopes (TCE, Crocchiolo, Zino, et al., 2009;Zino et al., 2004). The effect of dividing HLA-DPB1 mismatches into these two groups has provided evidence of DPB1 mismatching impacting on survival in some but not all studies. In a study of 621 unrelated donor HPCTs, recipients with permissive DPB1 mismatches had a significantly higher 2-year survival than those with nonpermissive DPB1 mismatches (55% vs. 39%). This improved survival was due to a decrease in NRM  the patient is associated with a higher risk of aGvHD (Petersdorf et al., 2015). This SNP is in linkage disequilibrium with the HLA-DPB1 exon 3 sequence which can be used as a marker for two highly diverged allele clades with low or high expression levels (Klasberg et al., 2019;Schöne et al., 2018). A study by Morishima et al. (2018)  HLA-DPA1 is significantly less polymorphic than HLA-DPB1, and certain alleles encoded by the two loci are in linkage disequilibrium. Analysis of the role of HLA-DPA1 mismatches had no effect on transplant outcome observed for the DPB1 permissive and nonpermissive mismatches in an NMDP study of 1,281 10/10 matched unrelated donor transplants (Fleischhauer et al., 2014).

| Use of UCB
UCB is an alternative source of HPCs that can be used to treat patients with both malignant and nonmalignant disorders (Ballen, 2017;Ballen et al., 2013). An early study undertaken by the CIBMTR-Eurocord showed similar survival outcomes comparing paediatric patients receiving HLA-identical UCBT with patients receiving HLA-identical sibling donor transplants. This study highlighted delayed granulocyte and platelet engraftment in UCBT recipients but also demonstrated a reduction in both acute and chronic GVHD (Rocha et al., 2000).
Similarly, a comparison of unrelated HLA-mismatched UCBT to matched unrelated adult donors transplants in paediatric patients demonstrated recipients of the UCBT experienced delayed engraftment but less acute and chronic GVHD with a similar relapse rate, OS and LFS (Rocha et al., 2001). Similar OS and significantly improved cGvHD and relapse-free survival were reported for adult patients with different malignant conditions after UCBT compared with matched sibling PBSC in a single-centre study in Colorado (Sharma et al., 2020).
The use of UCB was initially restricted to children due to the low cell doses obtained and poorer results obtained with adult recipients (Laughlin et al., 2001). However, the selection of UCBs with higher cell doses and the success with infusion of two UCBs to adult recipients (dUCBT) giving comparable results to matched related and matched unrelated donor transplants (Brunstein et al., 2010) together with improved conditioning protocols has led to UCB being a source of HPCs for both children and adults (Barker et al., 2003;Scaradavou et al., 2013).
More recently, UCBT has been shown to provide improved out-  It is suggested that a shortlist of up to 10 UCB units should be produced for each patient. These cords are selected based on the information extracted from the search reports. Minimally, the search results will provide the TNC count and HLA type, with many UCB units also having data on CD34 + cell counts. In the majority of cases, the search report will also contain information about the accreditation status of the relevant CBB.

| TNC
The TNC of the UCB unit is recognized as having a significant impact on the outcome of UCBT. An association between HLA mismatch and TNC with TRM was first published by Barker et al. (2010). Several years later, a large study found that NRM was increased in recipients who received a UCBT with <3 × 10 7 /kg TNC, such that the group recommended prioritizing a minimum pre-freeze TNC of 3 × 10 7 /kg followed by HLA allele match . Recent NMDP/ CIBMTR guidelines confirm the minimum TNC dose of 3 × 10 7 /kg . It has also been found that increasing the infused TNC dose abrogates, to some extent, the presence of HLA mismatches. The UK cord selection guidelines recommend increasing the TNC from a minimum of 3.0 × 10 7 /kg for 8/8 HLA allele-matched UCB to 5.0 × 10 7 /kg for 5-7/8 HLA match (Hough et al., 2016 were not . In addition, the HLA-DPB1 mismatch did not affect the risk of acute GVHD, engraftment or mortality, leading the authors to suggest that HLA-DPB1 mismatch increased the GVL effect without induction of severe acute GVHD or deterioration of survival rate. Further data are required before indicating the selection of an HLA-DPB1-mismatched UCB as the preferable option. The role of allele matching for HLA-DQB1 and HLA-DPB1 will only be conclusively ascertained following large-scale retrospective analysis.
Evidence indicates that inter-unit HLA matching is not required , although this study only considered HLA-A, data. This is also necessary for accurate consideration of the impact of recipient HLA alloantibodies.

Direction of HLA mismatch
The effect of direction of HLA mismatch was investigated in a cohort of 1,202 single UCBTs. Unidirectional mismatches were identified and classified as either GVH or HVG (rejection) mismatches.
Engraftment was faster in patients with GVH unidirectional mismatches compared to patients with single bidirectional mismatches HR = 1.6, p = .003). Other benefits to unidirectional mismatches included lower TRM, lower overall mortality and treatment failures.
The HVG unidirectional mismatches exhibited slower engraftment, higher graft failure and higher relapse rates (Stevens et al., 2011).
However, these findings were not confirmed in a Eurocord study of 1,565 single UCBT for malignant disease. In this cohort, one or two HLA mismatches in the GVH or HVG direction were not associated with NRM and survival (Cunha et al., 2014).
A Japanese study of 2,977 single UCBT for malignant disease did not find any significant association with overall mortality for transplants performed with unidirectional mismatches in either GVH or HVG direction (Kanda et al., 2013). GVH mismatches were associated with a lower incidence of NRM for paediatric recipients only.
The HLA data included in these three studies were not high resolution and HLA-C, HLA-DQ and HLA-DP matching was not considered; therefore, additional mismatches not accounted for in the analysis are likely. The role of HLA alloantibodies was not addressed.
The impact of NIMA matching (discussed in Section 9.2.5.5) was included in the study of Stevens et al. (2011), but not in the others. These studies are also complicated as multiple mismatches are present and not all mismatches (in the same direction) will impact the same biological effect. Further work is required to elucidate the impact of unidirectional mismatches.

Verification typing
All UCB units must receive verification HLA typing (VT) prior to infu-

| CD34 + cell count
The pre-cryopreserved CD34 + cell dose is a marker for haematopoietic progenitor potential post-infusion and is a critical factor to consider for optimal UCB unit selection. Considering TNC dose with no knowledge of the CD34 + cell dose can be misleading. It is possible that some units selected on adequate TNC dose can contain dangerously low CD34 + cell doses, especially if they are not red blood cell (RBC) depleted . The CD34 + cell dose is usually provided together with HLA and TNC data, in the UCB search report, and can be used to aid then ranking of UCB units. The speed of neutrophil, platelet and RBC engraftment has been shown to be significantly correlated with pre-cryopreserved CD34 + cell dose (Konuma et al., 2017). The risk of developing extensive chronic GVHD was associated with the highest CD34 + cell doses, but this did not negatively impact on OS. The pre-cryopreserved CD34 + cell dose can reliably predict the post-thaw CD34 + cell yield in most units and was shown to be an independent predictor of neutrophil engraftment in a study of single UCB transplants (Sanz et al., 2010). This study indicated a pre-cryopreserved CD34 + cell dose of 1.5 × 10 5 / kg of patient weight as a recommended threshold for faster neutrophil engraftment. The same threshold is recommended by NMDP/ CIBMTR . The ASTCT guidelines for UCB unit selection (Politikos et al., 2020) recommend to select, even higher CD34 + cell dose when possible, that is >2.0 × 10 5 /kg for a single UCBT. Eurocord gives a range and recommends 1.0-1.7 × 10 5 / kg of pre-cryopreserved CD34 + cells in single UCBT (Querol & Rocha, 2019). The same findings were demonstrated for dUCBT where pre-cryopreservation and post-thaw viable CD34 + cell doses were independent statistically significant characteristics associated with engraftment, with an arbitrary pre-cryopreservation CD34 + cell dose of ≥0.7 × 10 5 /kg for each unit identified as optimal, and 0.5 × 10 5 /kg as minimum (Purtill et al., 2014). In Eurocord recommendations, the combined CD34 + cell dose in dUCBT is recommended to be in excess of 1.8 × 10 5 /kg (Querol & Rocha, 2019) with the recent NMDP/CIBMTR guideline suggesting 1 × 10 5 /kg minimum for each cord  and >1.5 × 10 5 /kg for each cord where possible (Politikos et al., 2020).
Analysis of the NMDP CBU inventory on the pre- Separately from FACT, the CBB can be accredited by the AABB, which is also a reputable body with a slightly different set of standards. It is recommended to avoid nonaccredited CBBs where possible (Hough et al., 2016;Politikos et al., 2020) or use extra caution in assessing UCB quality and safety information when it is not avoidable. Units from non-NetCord-FACT-accredited CBBs were more likely to have poorer recovery and together with units with cryovolumes outwith 24.5-26 ml had an increased likelihood of having poor post-thaw viability (Purtill et al., 2014). If the accreditation status of the CBB is not marked on the Search Report, this information will be available on the UCB unit report requested from the bank.

| Secondary criteria in UCB unit selection
For each of the UCB units shortlisted, a request should be made to the CBB, via the Anthony Nolan in the UK, for a detailed unit report.
This will give additional information on factors including quality parameters further describing potency and safety of the UCB unit that can be used to aid selection:

Potency of an UCB unit
The ability of the cells in an UCB unit to regenerate the haemat- Cell viability is another important parameter in UCB unit potency evaluation as it indicates the percentage of live cells that are able to divide. There are different methods of viability assessment, the most common being 7-aminoactinomycin D (7-AAD) staining.

Viability of the UCB cells is fundamental for successful engraftment.
It was clearly shown in the dUCBT setting that increasing CD34 + viable cell dose correlated with increased engraftment . NetCord-FACT standards (2020) require TNC viability to be ≥85% post-processing (pre-cryopreservation) and post-thaw CD34 + viability ≥70%. Some CBBs provide Annexin V viability, which can detect apoptotic cells in the UCB unit. When available, it is recommended to consider the results of both 7-AAD and Annexin V methods.

Colony forming unit (CFU) defines the number of viable cells capable
of proliferating in vitro to form colonies in agar. In a multivariate analysis of 435 UCB transplants performed at Duke University Medical Center (USA), CFU was shown to be the best predictor of engraftment.
A threshold of 19.1 × 10 4 total CFU/kg in a fresh sample and 3.3 × 10 4 total CFU/kg in a thawed sample were considered good prognostic factors for UCBT outcome (Page et al., 2011). Although a numerical CFU characteristic gives an opportunity to calculate the UCB unit potency for a particular patient weight, not all CBBs will provide these data, as NetCord-FACT Standards require just the evidence of CFU growth on a post-thaw segment or representative sample as 'growth or positive result for potency'. Some CBBs provide post-processing and even post-thaw CFU data in UCB unit reports. Alternatively, it is provided as a part of QC releasing tests.
Clonogenic Efficiency (CLONE) is a combined parameter for CBU potency assessment to 'holistically' describe the ability of stem cells, usually measured as the percentage of CD34 + cells, to grow into colonies indicating their projected effectiveness in repopulating a recipient's haematopoietic system. It is measured by the ratio between post-thaw CFU and pre-cryopreservation CD34 + cells. It is suggested that this value should be higher than 10%, and any decrease, below this threshold, indicates impairment of the functional ability of CD34 + cells and therefore the unit should be considered at risk of engraftment failure (Querol et al., 2010). A later study showed that CLONE ≥20% predicted faster neutrophil and platelet engraftment and contributed to a decrease in the nonrelapse mortality (Castillo et al., 2015).

ABO matching (see also Section 11.2)
There are no conclusive studies showing a beneficial impact of ABO matching on UCB transplant outcome, but in general, a match is preferred due to fewer risks of adverse events at time of infusion of UCB cells. In 2015, the Eurocord group presented the results of multivariate analysis demonstrating ABO major and minor incompatibility resulting in a significant increase of TRM in patients receiving dUCBT (Rocha et al., 2015). Based on Eurocord data, EBMT recommend where possible to avoid ABO incompatible units in dUCBT (Querol & Rocha, 2019).

UCB unit age
Although some published studies demonstrated a lower correlation between pre-cryopreserved and post-thaw CD34 + cell doses in units banked before 2005 (Purtill et al., 2014), the general consensus is that UCB age is not a factor of inferior potency. Analysis  (Politikos et al., 2020).

UCB processing method
Variations in UCB manufacturing are common. Nowadays, the majority of CBBs use automated processing methods, but manually processed UCB units are still available in the worldwide inventory.
Compared with automated processed units, manually processed UCB units were reported to be associated with significantly lower day 28 neutrophil recovery after single UCBT (Ballen et al., 2015).
A concern of UCB processing is the method of RBC depletion.
Manually processed UCB units are more often RBC replete and con-  (Rocha et al., 2012). Considering NIMAs as permissive mismatches significantly increases the potential number of 'virtual' 5/6 and 6/6 UCB matches for recipients (van der Zanden et al., 2014) including patients with rare HLA alleles and haplotypes, thus increasing chances of finding a suitable graft for Black, Asian and Minority Ethnic patients (Powley et al., 2016). This principle has been applied by the Hellenic Cord Blood Bank (Panagouli et al., 2018).
Evidence, albeit indirect, of maternal cell microchimerism present in UCB units that may mediate a GVL effect in UCBT suggests a match on Inherited Paternal Antigens (IPAs) may also be beneficial (van Rood et al., 2012), but has not been proven by other studies (Politikos et al., 2020).
Advice on UCB unit selection is described in Section 14.

| HL A ALLOANTIBOD IE S
The presence of HLA alloantibodies in recipients directed against HLA mismatches of their donors has been linked with primary GF characterized by the absence of initial donor neutrophil engraft- versus current DSA is unknown; however, a patient who has had historic DSA but is currently negative could be considered to have undergone natural desensitization. A differential clinical impact due to cause of sensitization, that is pregnancy or transfusion or other source, is also unknown.
At present, there is no formal testing of donors for the presence of antibodies reactive with HLA mismatches of the recipient; recipient-specific antibodies (RSA). One single-centre study has described an increased incidence of either aGVHD or cGVHD in patients transplanted with donors who were sensitized to HLA class II, predominantly mismatched HLA-DPB1 alleles (Delbos et al., 2016). It has been reported that donor-derived HLA antibodies can be detected post-transplant in patients transplanted with donors that are positive for HLA antibodies which suggests they could impact on engraftment in HLA-mismatched transplants (Taniguchi et al., 2012).

| Cytomegalovirus
Cytomegalovirus (CMV) infection can cause significant complications post-transplantation. CMV disease affects different organs including lung (pneumonia); liver (hepatitis); gut (gastroenteritis); eye (retinitis) and the brain (encephalitis). Even with improvements in anti-viral prophylactic therapies, CMV seropositivity remains associated with an adverse prognosis and is still a major cause of morbidity and mortality in allogeneic HPCT (reviewed in Ljungman, 2014).

| ABO blood group incompatibility
ABO incompatibility (ABOi) between patient and donor is a common feature of HPCT. The ABOi can be major, minor or bidirectional (

| Donor sex
Using a male donor has been reported in some studies as having a positive effect on long-term survival regardless of the sex of the recipient (Gustafsson-Jernberg et al., 2004;Pond et al., 2006) but not in others (Lee et al., 2007). Regardless, donor sex selection priority is usually given to male donors due to their usually larger size associated with higher HPC counts obtained and also the increase in GVHD reported with female multiparous donors (Kollman et al., 2001). A German study of 2,646 transplants performed in patients with haematological malignancies found that transplants performed with international donors had a worse outcome compared to transplants with national donors and male patients transplanted with female international donors showed an even higher hazard ratio in analysis of OS than other sex-matched groups .
In contrast, a multicentre analysis of the effect of donor characteristics on the outcome of 709 RIC transplants (Passweg et al., 2011) demonstrated no association between donor age, parity and sex matching with transplant outcome, with only HLA matching being predictive for survival.
An investigation into the impact of cord blood donor sex compatibility has demonstrated no impact on survival in adults with haematological malignancies receiving a myeloablative single unit cord blood transplant. However, a higher incidence of chronic GVHD was observed in male recipients of female cord blood donors and a lower incidence of platelet engraftment in female recipients with male cord blood donors (Yuji et al., 2014). These findings require confirmation in further studies.
In unmanipulated haploidentical transplantation, Chang et al. (2016) concluded that a male donor is preferred with usage of anti-thymocyte globulin (ATG) or PTCy, due to the potential for superior survival of the patient. The more recent EBMT consensus recommendations for donor selection in haploidentical haematopoietic cell transplantation (Ciurea et al., 2020) suggest that a male donor should be the preferred choice when selecting a haploidentical donor for a male recipient, with PTCy. However, the EBMT recommendation also highlights conflicting evidence regarding benefits versus detriments of using mothers as donors for their children, regardless of the recipient's sex. These apparently conflicting results suggest that perhaps the donor's relationship (mother), rather than the donor's gender (female) has a stronger influence on transplant outcomes.

| Donor age
Donor age has been a consideration in unrelated donor selection, with younger donors being preferentially selected based on factors including their predicted better medical fitness to donate; being better HLA typed due to being recruited more recently and also on early data that indicated better post-transplant outcome (Kollman et al., 2001). Shaw et al. (2018) have followed up on an earlier CIBMTR study (Kollman et al., 2016)  In their reviews of the effect of donor age on haploidentical donor transplants, both Ciurea et al. (2020) and Chang et al. (2016) concluded that younger donors were preferable in both T-cell depleted (TCD) and unmanipulated haploidentical donor transplants.  (Askar et al., 2017;Carapito et al., 2016;Wulf et al., 2016;Martin et al., 2020;Patel et al., 2020). In addition, the MICA-129 V/V genotype in the donor (but not MICA-129 mismatching) has been indicated to increase the risk of CMV infection and disease posttransplant (Patel et al., 2020).  (González-Galarza et al., 2015), containing up to 16 KIR genes including two to six activating KIR genes, giving them the moniker of being 'activating haplotypes', and are considered to effect greater alloreactivity than KIR A haplotypes (Hsu et al., 2002;Uhrberg et al., 1997).  Ruggeri et al. (1999Ruggeri et al. ( , 2002. Further evidence followed, to support the preferential selection of a donor with an increased number of B/x diplotypes (activating haplotypes) in patients with AML, or other myeloid malignancies, when unrelated donors are the stem cell source (Cooley et al., 2009(Cooley et al., , 2010(Cooley et al., , 2014. These studies were performed in a cohort of patients receiving T-cell replete, myeloablative conditioning with bone marrow being the predominate source of stem cells.

| NK cell receptors in graft selection
The KIR B content scoring model classifies all potential donors as 'neutral' (lowest priority ranking), 'better' or 'best' (highest priority ranking) according to the donor's A or B haplotype content of both the centromeric and telomeric regions, with priority given to donors who possess two KIR B haplotypes in the centromeric region ('Best') (Cooley et al., 2009(Cooley et al., , 2010. In the international donor registry pool, approximately 70% of donors are categorized as neutral, 20% as better and 10% as best (Weisdorf et al., 2019). By selecting donors with activating KIR haplotypes (high B content), there is mounting evidence that recipients experience reduced relapse and improved survival, possibly due to selective elimination of minimal residual disease (MRD) in the recipient (Bao et al., 2016;Cooley et al., 2009Cooley et al., , 2010Cooley et al., , 2014. This effect has also been observed in paediatric haploidentical donor transplantation (Oevermann et al., 2014 This was attributed to better functional NK cell reconstitution post transplantation (Zhao et al., 2019).
Donor KIR characteristics not only impact on the GVL effect of transplants but can also contribute to immunological responses to infectious agents. Reduced incidence of CMV reactivation has been observed in transplants using donors who possess ≥5 activating KIR genes (Sobecks et al., 2011), or B/x diplotype characteristics in the telomeric region (TelB/x) of the KIR gene cluster (Heatley et al., 2018;Sobecks et al., 2011). Furthermore, transplantation using donors possessing KIR2DS2, a centromeric activating KIR gene, was found to be protective against CMV reactivation (Behrendt et al., 2013). Preferential cytotoxic activity against infected malignant cells may manifest clinically as promoting a GVL effect (Oevermann et al., 2014;Ruggeri et al., 2016;Zhao et al., 2007).  (Sobecks et al., 2015). Unfortunately, many existing KIR assessment algorithms for donor selection (such as the B content scoring model) have been found to be ineffectual in C2/C2 individuals (Cooley et al., 2014;Faridi et al., 2016). For those C2/C2 homozygous patients where a choice of fully HLAmatched donors exist, the process of additional KIR gene profiling should be applied to facilitate optimal outcome with fewest posttransplant complications. When considering transplant options, HLA typing must remain the primary priority in donor selection, as HLA-C mismatching results in detrimental transplant outcomes (Hoff et al., 2017).
A second group of recipients in whom there is a recognized risk are those with CMV serostatus-mismatched donors. Evidence is accumulating to suggest that if these transplants are unavoidable, prior knowledge of the KIR background of both donors and recipients may be able to direct CMV prophylaxis, highlight the requirement for closer post-transplant monitoring, and early therapeutic intervention for recipients with KIR B haplotype-negative donors.

| CCR5-Δ32
The successful transplantation of an HIV-infected patient, with AML, using an unrelated donor who was homozygous for a 32 basepair deletion in the CCR5 gene (CCR5 Delta 32/Delta32, (Δ32/Δ32)), resulted in the patient being free of antiretroviral medication and without any evidence of virus (Hűtter et al., 2009). Homozygosity for the CCR5Δ32 mutation prevents entry of HIV into target cells via the encoded CCR5 receptor. This success was repeated with an HIV-positive patient with Hodgkin's Lymphoma (Gupta et al., 2019).
Individuals homozygous for CCR5Δ32 are present in only approximately 1% of caucasian populations, and therefore, the chance of finding an HLA-matched CCR5Δ32/Δ32 donor is very low; however, some bone marrow donor registries have CCR5Δ32 genotypes available and this information can be requested when conducting an unrelated donor search.

| BACKUP OP TI ON S
For all transplants, it is advised to identify a backup option in case there are last minute issues with the preferred donor option. The backup donor could be related or unrelated, but preferably one where the workup could be turned around quickly. Consideration of location of the preferred and backup donor is a necessity should situations affecting global transportation be affected during a crisis, for example pandemic 2020.

| TUMOUR-S PECIFIC MUTATIONS
The improvement and wide-scale application of high resolution HLA typing methods has led to a significant increase in the number of mutations identified when HLA typing patients using DNA extracted from peripheral blood. These mutations can be attributed to a novel HLA allele expressed in all tissues or could be specific to the patient's tumour (Mrazek et al., 2014). If discovered, effort must be made via HLA typing of relatives and HLA typing of DNA extracted from patient tissue not affected by disease (e.g. skin plug) to determine whether the allele is novel or tumour specific. Only novel alleles will be assigned an official HLA allele name Robinson et al., 2015).
The expression of HLA proteins can be reduced within tumours due to deletion or mutations within genes encoding HLA proteins.
Loss of heterozygosity at HLA loci can also occur. Care must be taken when patients are HLA typed from DNA extracted from peripheral blood with a high frequency of tumour cells in circulation.
Homozygosity at HLA loci must be confirmed via family studies or repeat HLA testing when the patient is in remission or by testing DNA extracted from nondiseased cells such as buccal swab or skin plug.

| G R AF T IDENTIFIC ATION ADVISORY S ERVI CE (G IA S)
The provision of a professional GIAS to a transplant centre requires trained staff able to undertake both straight-forward and complex donor selection. GIAS may be delivered from an H&I lab-

| US EFUL WEBS ITE S
Guidance and tools to assist in donor selection and allele frequencies are available at the websites listed in Table 6.