Research articleIncidence and impact of allele-specific anti-HLA antibodies and high-resolution HLA genotyping on assessing immunologic compatibility
Introduction
The importance of antibodies against allogeneic Human Leukocyte Antigen (HLA) molecules as a primary barrier to allogeneic solid-organ transplantation is well established. Antibodies against mismatched donor HLA present at the time of transplantation can mediate hyperacute rejection while donor-specific antibodies (DSA) present after transplantation can drive antibody-mediated rejection [1], [2], [3]. Detection and definition of anti-HLA antibodies in transplant patients has undergone significant refinement since the initial discovery of their pathogenic potential in the 1960s. In particular, introduction of multiplexed solid-phase single-antigen bead (SAB) assays in the 2000s [4], [5] revolutionized the ability to define the specific reactivity of alloantibodies. SAB assays are designed to provide representative coverage for the 165 commonly recognized serologic antigens [6], [7]. This is achieved by the inclusion of one or more different HLA molecules from each of the serologic antigen groups. For several serologically-defined antigens, SAB assays contain HLA molecules encoded by multiple alleles; for instance, an assay may include beads with molecules encoded by the A*02:01, A*02:03, and A*02:06 alleles to represent the A2 serologic antigen. SAB assays are most directly interpreted based on the average reactivity against all beads containing molecules within a serologic antigen group. Differential reactivity among beads is sometimes interpreted as possible false positive or false negative reactions. However, similar to the well-defined concept of serologic-split antigens, HLA allele-specific reactivity is directed at epitopes differentially present among alleles within a broad serologic group [8], [9], [10], [11].
Emerging evidence for allele-specific antibodies has prompted debate of the importance of these antibodies in evaluation of donor-recipient immunologic compatibility. In the pre-transplant setting, this evaluation increasingly relies on virtual crossmatch analysis (VCXM) [12], [13], [14]. VCXM is performed by examination of donor HLA genotype for the presence of antigens against which the recipient has alloantibodies. This process is facilitated by registry systems such as UNET and the National Kidney Registry that utilize patient anti-HLA antibody information and donor HLA genotyping to direct organ offers to recipients who are most likely to be immunologically compatible. The potential importance of anti-HLA antibodies with reactivity outside of standard serologic antigens has been recognized by updates to these systems enabling reporting of antibodies against specific HLA alleles, including DQA1 and DPB1 which are not well-represented by the classically defined serologic antigens. However, standards for donor genotyping currently require only typing to the level necessary to determine serologic split antigen equivalents, potentially limiting the ability to accurately assess immunologic compatibility for patients with HLA allele-specific alloantibodies. This has spurred debate as to the necessity and appropriateness of high-resolution allele-level genotyping of donors [11], [15], [16], [17].
While demonstration of potential clinical impact in individual cases has been reported [9], [11], the prevalence of allele-specific antibodies is not well-defined. Understanding the prevalence of allele-specific antibodies in the context of existing ambiguity in donor genotyping is necessary for evaluating the costs and benefits of potentially updating donor HLA typing requirements. To this end, we performed a retrospective analysis of 2 years of HLA antibody testing for patients listed for transplant at our center combined with examining the ambiguity and accuracy of standard of care low-resolution donor HLA typing to evaluate the clinical impact of allele-specific antibodies. We also examined the prevalence of allele-specific DSA in post-transplant monitoring during the same period to evaluate the benefit of high-resolution donor genotyping information in this context.
Section snippets
Human subjects
All testing was performed as standard of care. Collection and analysis of de-identified data is considered exempt from institutional review board approval.
Anti-HLA antibody testing
Anti-HLA antibody testing was performed between 5/1/2018 and 4/30/2020 at the University of California San Diego Immunogenetics and Transplantation Laboratory as standard of care. Testing included pre-transplant testing of 4547 samples from 1939 patients listed for heart, lung, or kidney transplantation or post-transplant testing and
Allele-specific alloantibodies are frequent among patients awaiting transplantation
To evaluate the potential impact of allele-specific antibodies, we first assessed the frequency of allele-specific antibodies among patients awaiting solid organ transplantation. Allele-specific antibodies were defined as selective reactivity in SAB testing against HLA-A, B, C, DRB1, DRB3/4/5, DQB1, or DPB1 alleles within a defined serologic antigen. Selective reactivity was defined as positive reactivity against 1 or more beads within a serologic antigen group ≥3000 MFI with 1 or more beads
Discussion
The data presented here demonstrate that HLA allele-specific antibodies are relatively common among HLA-sensitized patients and should be considered as an important element of evaluating a patient’s anti-HLA alloantibody profile. These data are a single-center 2-year retrospective review of standard clinical testing of patients listed for heart, lung, and kidney transplantation, and are representative of a typical pre-transplant patient population. Approximately 15% of total pre-transplant
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: UCSD Immunogenetics and Transplantation Laboratory has received educational funds from Thermo-Fisher and CareDx. G.P.M. has received speaker honorarium and travel support from Thermo-Fisher.
Acknowledgement
The authors would like to thank Bing Yang (UCSD) and Arnold Jasman (UC Irvine) for technical assistance related to this project.
Authorship
D.Z., J.A., and P.R. performed data collection and analysis, and participated in writing the manuscript. G.P.M. designed the study, performed data collection and analysis, and participated in writing the manuscript.
Funding
This study was not funded by any sponsor.
References (25)
- et al.
Flow cytometric detection of HLA antibodies using a spectrum of microbeads
Hum. Immunol.
(1999) - et al.
Root cause analysis of limitations of virtual crossmatch for kidney allocation to highly-sensitized patients
Hum. Immunol.
(2017) - et al.
Mapping molecular HLA typing data to UNOS antigen equivalents
Hum. Immunol.
(2018) - et al.
Assessing the utilization of high-resolution 2-field HLA typing in solid organ transplantation
Am. J. Transplant.
(2019) - et al.
Renal transplantation with final allocation based on the virtual crossmatch
Am. J. Transplant.
(2016) - et al.
(F)Utility of the physical crossmatch for living donor evaluations in the age of the virtual crossmatch
Hum. Immunol.
(2018) - et al.
High-resolution HLA typing for sensitized patients: advances in medicine and science require us to challenge existing paradigms
Am. J. Transplant.
(2015) - et al.
A blueprint for electronic utilization of ambiguous molecular HLA typing data in organ allocation systems and virtual crossmatch
Hum. Immunol.
(2020) - et al.
Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant
Am. J. Transplant.
(2012) - et al.
Persistent strong anti-HLA antibody at high titer is complement binding and associated with increased risk of antibody-mediated rejection in heart transplant recipients
J. Heart Lung Transplant.
(2013)
Can solid phase assays be better utilized to measure efficacy of antibody removal therapies?
Hum. Immunol.
Impact of persistent and cleared preformed HLA DSA on kidney transplant outcomes
Hum. Immunol.
Cited by (11)
Impact of allele-specific anti–human leukocyte antigen class I antibodies on organ allocation
2023, American Journal of TransplantationThe Highly Sensitized Recipient: Pretransplant and Posttransplant Considerations
2023, Clinics in Chest MedicineVirtual crossmatch for deceased donor kidney transplantation in the United States: A survey of histocompatibility lab directors and transplant surgeons
2023, Human ImmunologyCitation Excerpt :RT-PCR/SSO/SSP methods only provide low to intermediate level resolution which are reported as antigen equivalents. However, many anti-HLA antibodies are allele specific, the prevalence of which can be ∼ 15 % among waitlisted patients. [34] High-resolution recipient HLA typing does help differentiate between self vs non-self HLA antibodies. [35]
Current HLA testing recommendations to support HCT
2022, Human ImmunologyCitation Excerpt :High-resolution typing of HLA-DRB3/4/5, - DQA1, -DQB1, and -DPA1 is highly recommended for a better match and to assist with DSA risk assessment. Adequate gene coverage to yield 2-field assignments at HLA-A, -B, -C, -DRB1, -DRB3/4/5, - DQA1, -DQB1, -DPA1 and -DPB1 loci may be needed in some cases when allele-specific HLA antibodies are identified in the recipient [44]. Typically, a sequencing-based method with coverage of exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles can achieve HLA P/G level assignments.
Next-generation sequencing and clinical histocompatibility testing
2021, Human ImmunologyCitation Excerpt :This limitation can have clinical consequences as lack of recognition of donor allele-specific antibodies can result in antibody mediated rejection (AMR) and poor transplant outcomes. NGS based typing methods offer the highest resolution available for donor typing which should be utilized to identify potential DSA pre-transplant[66]. In past studies utilizing other methods of HLA typing, mismatching at the HLA-A, -B, -C, -DRB1, -DQB1, and –DPB1 loci in kidney transplant patients has been shown to be immunogenic and associated with increased risk of de novo DSA[67,68].