The Evolution of HLA Testing in Clinical Trial Design

Each individual possesses a unique set of HLA, half inherited from each parent.1 HLA typing remainsessential before organ or hematopoietic cell transplantation (HCT) to assess compatibility between donorand recipient cells. Typing may also be used to identify markers for specific diseases. While this testing isprimarily used for investigating immune compatibility in the clinical patient care setting, in research andtrial applications, HLA testing is increasingly used as a strategic tool that can shape study design, patientselection, and data interpretation.

Initially, HLA testing relied on serologic methods. While foundational, these approaches were limited in resolution and sensitivity, leaving ambiguity in antigen identification and interpretation. The transition to molecular typing marked a turning point, dramatically improving allele-level discrimination and reducing uncertainty.

NGS accelerated this evolution further. High-resolution sequencing generates vastly richer datasets, revealing the extraordinary polymorphism of HLA genes and enabling a level of precision that was The Evolution of HLA Testing in Clinical Trial Design With unique clinical, laboratory, and regulatory considerations, the question is no longer whetherHLA testing matters in clinical trials, but how intentionally it is being integrated, and how prepared trial teams are for the complexity it introduces. previously unattainable. At the same time, practical innovations in sample collection and validation such as the move from fresh blood samples to cheek swabs have made testing more scalable and accessible, supporting broader participation and global reach.

The cumulative effect has been transformative: more data, easier logistics, and a foundation for precision medicine. But increased resolution has revealed a new challenge: interpretation.

The field has become adept at identifying HLA variants via emerging technology, but there continues to be a critical shift in focus. Classical HLA class I genes are polymorphic at nearly every amino acid position in the peptide binding region, and human populations actively maintain this diversity. Simply cataloging variants is no longer sufficient. Increasing attention is now focused on identifying the meaning of each polymorphism. The emerging question becomes functional: which differences matter biologically and clinically?

Advances in antibody testing have been equally consequential. Virtual crossmatching and solid-phaseantibody assays are now highly predictive and central to transplant decision-making, yet antibody testingremains one of the most challenging areas to standardize and automate due to human interpretationof machine signals. In some cases, results vary based on reagents, assay conditions, and interpretationthresholds. As a result, expert human judgment remains indispensable.While artificial intelligence holds promise for streamlining interpretation, meaningful automation will requiresystems capable of reflecting immunologic context—not just pattern recognition. Therefore, antibodyinterpretation sits at the intersection of data and expertise, reinforcing the need for skilled laboratories,expert guidance, and thoughtful integration into clinical trial workflows.

As understanding deepens, the field is moving beyond allele-level comparisons toward epitope and epletanalysis. This shift highlights growing recognition that molecular differences are not equally immunogenic,and that chemically and structurally informed comparisons offer great predictive value. As datasets growand correlations with outcomes strengthen, these approaches are expected to play a larger role—not only inclinical decision-making, but in how trials define eligibility, stratify risk, and interpret immune responses.

Today, HLA testing is embedded across clinical trials in ways that extend far beyond donor–recipientmatching. It is used for immune risk stratification, immunogenicity assessment, antibody monitoring, andsafety signal interpretation, particularly in immune-modulating and HLA-directed therapies.One of the most consequential shifts has been the evolution of HLA testing into a formal inclusion–exclusionbiomarker in drug development. When therapies are HLA-directed, aligning biomarker strategy with thetherapeutic or drug’s mechanism of action best supports precision medicine diagnoses. As a result, testingmust be integrated early to ensure appropriate patient selection, preserve efficacy signals, and reduceadverse outcomes.

Regulatory considerations for HLA testing differ primarily based on how the test is used within a study.In transplant studies where HLA testing supports donor-recipient matching or immune risk assessment,HLA testing is considered standard of care. Accordingly, assuming the assay itself is not the subject ofinvestigation, the FDA allows the use of appropriately validated LDTs conducted in accredited laboratories.By contrast, in HLA-directed therapy studies, HLA testing is typically functioning as a companion orcomplementary diagnostic where the test is used to support assessments of safety and efficacy of thecorresponding therapeutic. In this scenario, the HLA assay is considered an investigational device and issubject to the FDA’s investigational device exemption (IDE) requirements, which include the need to filean IDE application with the FDA and receive authorization prior to initiating use of the HLA test in the drugstudy. However, if the study sponsor can demonstrate the investigational study of the HLA test meets thecriteria of a non-significant risk study, then the sponsor is not required to file an application with the FDA butmust comply with the remainder of the IDE requirements. This is also known as an abbreviated IDE.

Regulatory expectations differ markedly by region. In the United States, under FDA framework, there isflexibility with how HLA testing is governed, depending on the intended use of the test. In transplant studieswhere HLA testing is typically used in line with standard of care and is not itself the subject of the study,the FDA permits the use of validated LDTs on the condition the test and the laboratory performing the testcomply with CMS regulations. When HLA testing functions as a companion or complimentary diagnosticin an HLA-directed therapy trial, the test meets the definition of an IVD and is therefore subject to FDAregulations rather than CMS regulations.

In the European Union under In Vitro Diagnostic Regulation (IVDR), there exists increased complexity andoperational burden. In the context of a clinical study, including transplant studies and HLA-directed therapystudies, the sponsor is expected to use a CE-marked HLA test for the intended use required for the study, ifone already exists. Alternatively, the sponsor can use an “In-House” HLA test performed by Health InstituteWhen therapies are HLA-directed, aligningbiomarker strategy with the therapeutic or drug’smechanism of action best supports precisionmedicine diagnoses. As a result, testing must beintegrated early to ensure appropriate patientselection, preserve efficacy signals, and reduceadverse outcomes.residing in the EU, assuming the Health Institute and HLA test comply with the corresponding section ofthe IVDR reserved for these types of tests. If neither a CE-marked assay nor an In-House test is availablefor the required intended use, then the study sponsor will need to develop their own test and file aperformance study application (similar to an IDE application) with the National Competent Authority inthe member state where the study is being conducted. It is worth noting that the IVDR does not have anequivalent to FDA’s non-significant study exemption. Rather, the IVDR requires performance studies forall Class C and Class D IVDs which includes HLA tests for donor matching purposes and HLA tests forcompanion diagnostic purposes.

Looking ahead, HLA testing is moving beyond its historical role as a static laboratory measurement andbecoming a core enabler of precision therapeutics and clinical trial design. This evolution is shaped as muchby regulatory reality as by technology. In Europe, IVDR has introduced friction—particularly for HLA-directedtherapies—where performance study requirements may delay trials and limit patient access. In response,sponsors are exploring hybrid approaches, enrolling patients through available health institution exemptionswhile regulatory approvals proceed in parallel. This highlights a broader need for regulatory frameworks thatalign more closely with the intent of modern precision medicine.From a testing and technology standpoint, HLA genotyping is fundamentally a form of DNA sequencing.This raises an opportunity to define test classes or standing approvals for categories of HLA-directedtherapies, rather than requiring repeated validations for similar diagnostic strategies across individual trials.Antibody testing remains a challenge, as immune profiling reflects a biological continuum, and currentreliance on semi-quantitative thresholds can oversimplify complex immune realities. Moving away from rigidbinary thresholds toward outcome-linked and standardized frameworks may better reflect immune risk andsupport more informed clinical decision-making.

HLA testing has moved beyond its historical role as a technical prerequisite and is now a strategicconsideration that influences clinical trial design, regulatory planning, and therapeutic success. Advances inmolecular testing and informatics have expanded what HLA data can reveal, while growing use in patientselection and risk stratification has increased both its impact and its complexity. Designing with HLA inmind requires early, intentional integration across scientific, operational, and regulatory domains, ensuringthat testing strategies align with study objectives and therapeutic mechanisms. As clinical trials continueto evolve toward more precise, immune-informed approaches, thoughtful HLA strategy will be essential toreducing risk, preserving interpretability, and enabling meaningful clinical outcomes.

1. Berger A. HLA typing. BMJ. 2001 Jan 27;322(7280):218. https://pmc.ncbi.nlm.nih.gov/articles/PMC1119473/