HLA Genotyping Assay Validation Checklist: Reference Materials, Controls, and Analytical Performance

HLA genotyping assays require careful validation because the HLA system contains a large number of highly polymorphic alleles. A reliable result depends not only on primers, probes, sequencing chemistry, or analysis software, but also on the suitability of reference materials, the design of controls, and the way analytical performance is measured.

A validation plan should demonstrate that the assay can correctly identify the intended HLA loci and allele resolution under defined operating conditions. It should also show how the method performs when DNA input, operators, reagent lots, instruments, or testing days change.

This checklist provides a practical framework for validating an HLA genotyping assay. It focuses on reference materials, positive and negative controls, accuracy, precision, analytical sensitivity, analytical specificity, reportable range, invalid results, and ongoing quality monitoring.

Before using this checklist, HLA reference standards for assay validation and quality control can provide additional background on the role of characterized materials in HLA workflow evaluation.

Key Takeaways

• Define the intended use, target loci, allele resolution, specimen type, and reporting format before validation begins.

• Select reference materials that match the assay’s target loci, genotype resolution, sample format, and testing workflow.

• Use positive, negative, extraction, amplification, and contamination controls where appropriate.

• Evaluate accuracy, repeatability, reproducibility, analytical sensitivity, specificity, robustness, and invalid-result rates.

• Establish acceptance criteria before testing rather than adjusting them after results are available.

• Record raw data, deviations, software versions, interpretation rules, and final approval.

• Continue using suitable controls after validation to monitor routine assay performance.

What Should HLA Assay Validation Demonstrate?

HLA assay validation should provide documented evidence that the method is fit for its intended purpose. The goal is not to prove that an assay can identify every HLA allele that exists. Because HLA diversity is extensive, the laboratory should define a realistic analytical scope and support that scope with appropriate evidence.

The validation should answer several practical questions:

• Can the assay correctly identify the target loci?

• Can it distinguish the required allele groups or allele-level designations?

• Does it produce consistent results when testing is repeated?

• Does it remain reliable with different operators, days, reagent lots, or instruments?

• What is the minimum acceptable DNA input?

• How are ambiguous or no-call results handled?

• What results are considered valid, invalid, or outside the reportable range?

• What controls must pass before a run is accepted?

Validation should cover the entire workflow that will be used in practice. If the routine process includes DNA extraction, amplification, sequencing, data analysis, and reporting, the validation should not evaluate only the final sequencing step.

Step 1: Define the Intended Use and Validation Scope

The first part of the checklist is to define exactly what the assay is designed to do.

Intended Application

The intended use may include:

• Single-locus HLA research

• Multi-locus genotyping

• Donor-recipient matching research

• Pharmacogenomic assay development

• HLA kit evaluation

• Method comparison

• Routine laboratory quality control

• Research involving HLA population variation

The intended use determines the amount of evidence required. A focused assay designed to identify one HLA-B allele does not require the same validation scope as a multi-locus assay intended to report several class I and class II loci.

Target Loci

Document every locus included in the assay, such as:

• HLA-A

• HLA-B

• HLA-C

• HLA-DRB1

• HLA-DQB1

• HLA-DPB1

• HLA-DQA1

• HLA-DPA1

• HLA-DRB3, HLA-DRB4, and HLA-DRB5

Do not describe an assay as “multi-locus” without listing the actual loci covered. The reference material must contain assigned results for the same loci that the assay is expected to report.

Required Typing Resolution

Define whether the assay reports:

• Antigen-level results

• Allele-group results

• Two-field allele designations

• Three-field allele designations

• Four-field allele designations

• A higher-resolution sequence-defined result

• An ambiguous allele group

The laboratory should also specify whether the assay reports one genotype, two possible alleles, or a list of unresolved allele combinations.

Specimen and Material Type

The validation plan should identify the sample type used in the routine workflow, such as genomic DNA or a human-cell-based material. Important factors include:

• DNA concentration

• DNA purity

• DNA integrity

• Extraction method

• Storage conditions

• Freeze-thaw history

• Sample volume

• Possible inhibitors

A reference standard added directly to a PCR reaction may evaluate amplification, sequencing, and interpretation, but it does not necessarily evaluate the DNA extraction process.

Step 2: Select Appropriate HLA Reference Materials

Reference materials provide assigned genotypes against which assay results can be compared. Their value depends on how well they match the analytical scope of the assay.

CB-Gene provides an HLA reference standard product category covering single-locus and multi-locus HLA genotyping materials.

Match the Reference Material to the Target Locus

For a focused assay, the reference material should contain the specific target locus and allele designation required for evaluation.

CB-Gene’s single-locus HLA standards mainly cover HLA-B typing. The product information includes HLA-B27:04, HLA-B27:05, HLA-B57:01, HLA-B15:02, HLA-B58:01, HLA-B13:01, and HLA-B*27:07 reference standards.

The HLA single-locus genotyping standard is provided as genomic DNA and is intended for research use. Its listed verification method is PCR-SBT/Sanger sequencing.

For a broad matching workflow, a multi-locus reference material may be more appropriate. CB-Gene’s HLA matching multi-locus standards cover a broad group of HLA loci, including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DPA1, HLA-DPB1, HLA-DQA1, and HLA-DQB1.

Confirm the Assigned Genotype and Resolution

Before testing, record:

• Assigned allele designation

• Target loci

• Resolution level

• Verification method

• Material format

• Catalog number or lot number

• Certificate or technical data

• Storage conditions

• Expiration information

The assigned value must be detailed enough to support the intended comparison. If an assay reports a two-field result, a standard with only an allele-group designation may not be sufficient. Similarly, a high-resolution reference result cannot prove assay performance in sequence regions that the assay does not examine.

Consider Method Compatibility

The reference material should be compatible with the selected workflow. Relevant questions include:

• Is the material genomic DNA?

• Can it be used in PCR-SBT or Sanger workflows?

• Is it suitable for targeted NGS?

• Is it suitable for long-read sequencing?

• Does it enter the workflow before or after DNA extraction?

• Does the material replicate the expected sample background?

• Is the assigned genotype supported by an appropriate verification method?

CB-Gene states that its HLA reference products have been evaluated using Sanger sequencing, PCR-SBT, or third-generation sequencing, with genotype confirmation through sequence alignment against HLA reference sequences. This information should be compared with the method being validated.

Reference Material Selection Checklist

Step 3: Build a Complete Control Strategy

Controls should be placed at the stages where failures may occur. A single positive control at the end of the workflow cannot identify every possible source of error.

Positive Genotyping Control

A positive control should contain a known HLA genotype that the assay is designed to detect. It can be used to verify:

• DNA amplification

• Primer or probe performance

• Sequencing quality

• Allele assignment

• Software interpretation

• Report formatting

For a single-locus assay, one or more allele-specific standards may be sufficient for initial evaluation. For a multi-locus assay, a control covering several target loci may provide better evidence of integrated workflow performance.

Negative or Non-Target Control

A negative or non-target control can help assess whether the assay produces an inappropriate positive result for a defined target. Its suitability depends on the assay design.

For example, a targeted PCR-SSP assay may require samples that lack the target sequence. A sequencing-based assay may instead rely more heavily on contamination controls and allele-discrimination materials.

The term “negative control” should therefore be clearly defined in the protocol. It should not be assumed that one generic negative sample can evaluate every aspect of HLA genotyping specificity.

No-Template Control

A no-template control contains no added DNA template. It is used to monitor contamination introduced during reaction preparation or amplification.

If the no-template control produces an unexpected amplification signal or sequence result, the run should be investigated before any patient or research sample results are released.

Extraction Blank

An extraction blank passes through the DNA extraction process without a biological sample. It helps identify contamination introduced during:

• Sample preparation

• Extraction reagents

• Extraction equipment

• Work surfaces

• Transfer steps

If the HLA standard is added only after extraction, it cannot evaluate contamination or recovery during the extraction stage.

Internal Amplification or Process Control

Where supported by the assay design, an internal control can help identify:

• PCR inhibition

• Reaction failure

• Insufficient DNA

• Incorrect reagent preparation

• Poor amplification efficiency

The control should have defined acceptance criteria. A control that is present but not interpreted consistently does not provide meaningful quality evidence.

Control Acceptance Rules

The validation protocol should state:

• Which controls must pass

• What signal or sequence quality is acceptable

• How failed controls affect the run

• Whether a failed control requires repeat testing

• Who is authorized to approve or reject a run

• How control failures are documented

Step 4: Evaluate Analytical Performance

The analytical performance section should be designed around the actual output of the HLA assay.

Accuracy

Accuracy is the agreement between the assay result and the assigned or accepted reference result.

Accuracy evaluation may include:

• Testing characterized HLA reference materials

• Comparing results with an established method

• Comparing results at the same resolution

• Confirming discordant results using an appropriate method

• Evaluating representative allele groups

• Reviewing the final report rather than only raw sequence data

The comparison should distinguish between a true genotype disagreement and a difference caused by nomenclature formatting, database version, or reporting resolution.

For example, two results may appear different because one uses a legacy allele format while the other uses a colon-based format. Such differences should be normalized before classifying a result as discordant.

Repeatability

Repeatability evaluates performance under the same operating conditions. Consider repeating the assay using:

• The same operator

• The same instrument

• The same reagent lot

• The same laboratory

• The same testing day or short testing interval

Repeatability should assess both correct allele assignment and the quality metrics used to determine whether a result is valid.

Reproducibility

Reproducibility evaluates performance when normal operating conditions change. Depending on the laboratory, this may include:

• Different operators

• Different testing days

• Different reagent lots

• Different instruments

• Different DNA preparation batches

• Different analysis sessions

The goal is to determine whether the assay produces consistent results beyond a single ideal run.

Analytical Sensitivity

For a qualitative HLA genotyping assay, analytical sensitivity usually relates to the minimum amount and quality of DNA that can produce a valid and correctly assigned result.

The validation should evaluate:

• Minimum DNA input

• DNA concentration range

• DNA purity range

• Degraded DNA, if relevant

• Low-input samples

• Heterozygous allele detection

• Allele dropout risk

• Invalid or no-call rates at lower input levels

The laboratory should define a minimum acceptable input rather than assuming that any measurable DNA concentration is suitable.

If the assay is designed for a specific low-abundance or mixed-sample application, the validation should separately define how minor allele detection is evaluated. The acceptance criteria should reflect the actual intended use.

Analytical Specificity

Analytical specificity describes the assay’s ability to distinguish the intended HLA genotype from related or non-target sequences.

The evaluation should consider:

• Closely related allele groups

• Alleles with similar primer-binding regions

• Potential cross-reactive sequences

• Non-specific amplification

• Incorrect allele assignment caused by incomplete coverage

• Interference from non-target DNA

• Contamination between samples

Because the HLA system is highly polymorphic, specificity should not be demonstrated with only one positive and one negative sample. The test panel should include representative challenging genotypes whenever they are relevant to the assay’s target.

Reportable Range and Resolution

The reportable range should define:

• Loci that can be reported

• Allele groups that can be distinguished

• Maximum reportable resolution

• Sequence regions covered

• Acceptable ambiguous results

• Conditions that require a no-call

• Results that require supplementary testing

An assay should not report a more detailed allele designation than its validated evidence supports. High read depth or a strong amplification signal does not compensate for missing target regions or unresolved phase information.

Invalid and No-Call Rate

The validation should establish how often the assay produces:

• Invalid results

• No-call results

• Ambiguous results

• Failed amplification

• Failed sequencing

• Insufficient coverage

• Unresolved allele assignments

The laboratory should define separate acceptance criteria for technical failure and biological ambiguity. A no-call caused by inadequate sequence coverage is different from an ambiguous result caused by two alleles sharing the same tested sequence.

Robustness

Robustness evaluates whether small, realistic changes in operating conditions affect the result. Potential variables include:

• DNA concentration

• Pipetting variation

• Annealing temperature

• Reaction time

• Reagent lot

• Storage duration

• Number of freeze-thaw cycles

• Sequencing run quality

• Analysis threshold

• Software version

Only relevant variables should be challenged. The purpose is to identify conditions that may occur during routine operation and determine whether they could change the reported genotype.

HLA Genotyping Analytical Performance Matrix

Step 5: Design the Validation Run

A validation run should be planned before samples are tested. The protocol should define:

• Number and type of reference materials

• Number of replicates

• Control placement

• Operators involved

• Testing days

• Reagent lots

• Instruments

• Sample randomization

• Data analysis procedure

• Acceptance criteria

• Rules for repeat testing

Replicates should be sufficient to evaluate the performance claim being made. A small number of repeated tests may demonstrate basic feasibility, but it may not support claims about reproducibility across operators, instruments, or reagent lots.

The validation should also include representative failure conditions where appropriate. For example, low DNA input, insufficient amplification, or poor sequence quality may be deliberately evaluated to confirm how the assay handles invalid results.

Step 6: Define Result-Comparison Rules

Before reviewing the data, define how results will be classified.

Exact Agreement

The observed result matches the assigned result at the validated reporting resolution.

Acceptable Equivalent

The observed result uses a different formatting convention but represents the same allele designation. This may include legacy and colon-based nomenclature formats.

Partial Agreement

The observed result agrees at a lower resolution but does not support the full assigned designation. This should not be counted as full agreement when the assay claims higher-resolution reporting.

Discordant Result

The observed result conflicts with the assigned genotype after nomenclature and resolution differences have been excluded.

Invalid or No-Call

The assay does not generate a reportable result because of failed controls, insufficient DNA, poor amplification, inadequate sequence quality, or unresolved analysis.

All comparison rules should be documented in the validation plan. The laboratory should avoid changing the classification criteria after seeing the results.

Step 7: Document the Validation and Approve the Assay

A complete validation file should include:

• Validation protocol

• Intended-use statement

• Assay version

• Primer, probe, kit, and reagent information

• Instrument information

• Software and database versions

• Reference material certificates

• Control configuration

• Raw instrument data

• Sequence or amplification files

• Analysis reports

• Result-comparison tables

• Deviations and investigations

• Failed-run records

• Corrective actions

• Final performance summary

• Approval signatures and dates

The final report should clearly state whether the assay meets its predefined acceptance criteria. If a limitation remains, it should be described in the reportable-range or interpretation section.

Validation is not complete when the laboratory obtains a set of correct results. It is complete when the method, controls, limitations, analysis rules, and operating conditions are documented and approved.

Step 8: Establish Routine Quality Control After Validation

After validation, the laboratory should continue monitoring the assay.

Routine QC may include:

• Positive control testing

• No-template control testing

• Extraction blank testing

• Control trend review

• Reagent-lot verification

• Periodic reference-material testing

• Review of invalid and no-call rates

• Software and database version control

• Investigation of unexpected allele patterns

• Documentation of corrective actions

A control should be used at a frequency appropriate to the assay risk and testing volume. The laboratory should define when a new reagent lot, instrument, software version, primer design, or interpretation algorithm requires partial or complete revalidation.

Potential revalidation triggers include:

• Change in target loci

• Change in reporting resolution

• New primer or probe design

• New sequencing chemistry

• New instrument platform

• Major software update

• New HLA database version

• Change in extraction method

• Significant control failure

• Repeated increase in no-call results

How CB-Gene HLA Standards Can Support Validation

CB-Gene’s HLA materials can be selected according to the scope of the assay being developed.

For focused HLA-B or single-locus workflows, the HLA single-locus genotyping standard provides characterized genomic DNA materials with listed allele assignments and PCR-SBT/Sanger verification information.

For broader HLA matching workflows, the HLA matching multi-locus genotyping standard includes multiple HLA loci and is listed as being verified by third-generation sequencing. The product information also describes sequence-based genotype confirmation using HLA database alignment.

These materials can support accuracy studies, method comparison, operator training, repeatability assessment, and routine quality control. The laboratory should still confirm that the selected material matches its own target loci, resolution, sample format, and intended use.

Common HLA Assay Validation Mistakes

Using Only One Positive Control

One positive sample may show that the assay can detect one genotype, but it does not demonstrate broad allele discrimination or multi-locus performance.

Choosing a Standard at the Wrong Resolution

A control assigned only at allele-group level cannot fully validate a two-field or three-field reporting claim.

Treating a DNA Control as an Extraction Control

A purified genomic DNA standard added after extraction cannot demonstrate extraction recovery or extraction-stage contamination control.

Assuming High Sequence Coverage Guarantees Accuracy

High coverage does not resolve variants outside the target region. It also does not automatically solve phase ambiguity or incorrect reference database interpretation.

Ignoring No-Call Results

Invalid and ambiguous results are part of assay performance. They should be measured, investigated, and included in the validation report.

Changing Acceptance Criteria After Testing

Acceptance criteria should be defined before the validation run. Adjusting them after reviewing results can make the validation difficult to defend.

Failing to Control Software and Database Versions

HLA allele assignment depends on analysis rules and reference data. Software and database versions should be recorded and controlled throughout validation and routine testing.

Practical HLA Genotyping Assay Validation Checklist

Scope

• ☐ Intended use is documented.

• ☐ Target loci are listed.

• ☐ Required allele resolution is defined.

• ☐ Specimen and DNA input requirements are documented.

• ☐ Reportable and non-reportable results are defined.

Reference Materials

• ☐ Reference genotype is assigned.

• ☐ Reference material covers the target loci.

• ☐ Verification method is documented.

• ☐ Material format is compatible with the workflow.

• ☐ Storage and stability information are available.

• ☐ Catalog number and lot number are recorded.

Controls

• ☐ Positive genotyping control is included.

• ☐ No-template control is defined.

• ☐ Extraction blank is included when extraction is part of the workflow.

• ☐ Internal amplification or process control is evaluated.

• ☐ Control failure rules are documented.

Analytical Performance

• ☐ Accuracy is evaluated.

• ☐ Repeatability is evaluated.

• ☐ Reproducibility is evaluated.

• ☐ Analytical sensitivity or minimum DNA input is defined.

• ☐ Analytical specificity is evaluated.

• ☐ Reportable range is documented.

• ☐ Invalid and no-call rates are measured.

• ☐ Robustness is assessed.

Documentation

• ☐ Raw data are retained.

• ☐ Software and database versions are recorded.

• ☐ Deviations are investigated.

• ☐ Acceptance criteria are applied consistently.

• ☐ Final validation conclusions are approved.

• ☐ Revalidation triggers are defined.

Conclusion

A reliable HLA genotyping assay requires more than a successful amplification or sequencing run. The laboratory must demonstrate that the complete workflow can produce accurate, consistent, and interpretable results within a clearly defined analytical scope.

The most important validation steps are to define the required loci and resolution, select suitable reference materials, establish control rules, evaluate analytical performance, and document all results against predefined acceptance criteria. Single-locus standards may be appropriate for focused HLA-B assays, while multi-locus materials can support broader HLA matching workflows.

After validation, routine controls, trend monitoring, version control, and change-management procedures help maintain performance over time. When reference materials and controls are integrated into a structured validation plan, laboratories can identify limitations earlier and make HLA genotyping results more consistent, comparable, and defensible.

FAQs

What Is the Most Important Control in HLA Genotyping Validation?

A characterized positive reference material is essential because it provides an assigned genotype for comparison. However, no-template, extraction, and process controls may also be required to identify contamination or workflow failures.

Can One HLA Reference Standard Validate a Multi-Locus Assay?

Not always. The standard should cover the loci and resolution that the assay claims to report. A single-locus standard may support focused validation but may not provide sufficient evidence for a broad multi-locus workflow.

How Is Analytical Sensitivity Evaluated for HLA Genotyping?

It is commonly evaluated by testing different DNA input levels and quality conditions. The laboratory should determine the lowest input that still produces valid amplification, sequencing, allele assignment, and reporting.

What Should Happen When the Control Fails?

The run should be placed on hold, the cause should be investigated, and affected samples should not be reported until the problem is resolved. Repeat testing and corrective actions should be documented.

Does High-Resolution Reference Material Automatically Validate High-Resolution Reporting?

No. The assay must cover the sequence regions and analytical features needed to distinguish the reported allele designation. A detailed reference result cannot compensate for incomplete assay coverage.

When Should an HLA Assay Be Revalidated?

Revalidation may be required after changes to primers, target loci, sequencing chemistry, instruments, extraction procedures, software, allele databases, reporting resolution, or other conditions that may affect the reported genotype.