--- title: "Six Mistakes That Can Impact Your HPV Assay Validation" description: "Discover six common HPV assay validation mistakes, from sample selection and cut-off optimisation to population differences and platform changes." url: https://qbdgroup.com/en/blog/hpv-assay-validation-mistakes type: "Blog post" language: en published: 2026-10-14 author: "Sara Peeters & Davy Vanden Broeck" category: "Clinical" publisher: "QbD Group" citation: "QbD Group, \"Six Mistakes That Can Impact Your HPV Assay Validation\", https://qbdgroup.com/en/blog/hpv-assay-validation-mistakes" --- # Six Mistakes That Can Impact Your HPV Assay Validation > Discover six common HPV assay validation mistakes, from sample selection and cut-off optimisation to population differences and platform changes. A validation study can be scientifically sound and still fall short of its objectives because of decisions made too early, or too late. For HPV assays, the choice of laboratory, sample set, population and study design can have a lasting impact on the credibility of the resulting evidence. These are six challenges highlighted by Prof. Davy Vanden Broeck of AML, Belgium's HPV National Reference Laboratory, and why they deserve attention before a validation study begins.

In This Blog Post:

## 1. Choosing a Laboratory Without the Right Expertise Not every laboratory is equally well placed to perform an independent HPV assay evaluation. Some studies are conducted by the manufacturer or by laboratories without specific HPV reference-testing experience. For an independent evaluation, the laboratory's expertise, quality systems and familiarity with the relevant methods are important. The team needs to understand not only how to run the assay, but also how to interpret the results in the context of cervical cancer screening. Where possible, consider a national or WHO HPV reference laboratory. Involve the laboratory early enough to review feasibility, sample availability and the proposed evaluation approach before the protocol is finalised. ## 2. Using a Poorly Characterised Sample Set A collection of known HPV-positive samples may look attractive for validation. However, it may not reflect the population in which the assay will actually be used. If the source population, selection process and disease status are not clearly defined, it becomes harder to interpret the results or judge whether they are representative of the intended screening population. A strong sample strategy therefore starts with clear documentation: where the specimens came from, how they were selected, which clinical information is available and how disease status was established. Whenever feasible, use defined and appropriately approved sample sets that are suitable for the validation question. The aim is not simply to assemble enough specimens, but to assemble the right ones. ## 3. Overlooking Geographical and Population Differences The Meijer criteria were developed using European screening populations. That scientific foundation remains valuable, but it does not mean results automatically generalise to every screening context. Under-screened populations may differ in screening history, HPV prevalence and disease spectrum. These factors can affect observed clinical performance and how confidently results can be applied to the intended population. There is another distinction worth keeping in mind: a hospital population in a low- or middle-income country (LMIC) is not automatically representative of a population invited for routine screening. Where organised screening programmes are limited or absent, collaboration with local reference laboratories and suitable recruitment networks can help identify participants and specimens relevant to the intended use. This is particularly important when designing an evidence strategy intended to support both European IVDR requirements and WHO Prequalification. ## 4. Optimising the Cut-Off During Validation For an HPV assay, the chosen cut-off affects the balance between clinical sensitivity and specificity. Pushing sensitivity higher may reduce specificity, with consequences for the number of people referred for follow-up. The problem arises when the cut-off is adjusted after reviewing the validation results. At that point, the dataset intended to provide independent evidence may effectively become part of the optimisation process. A clearer approach is to optimise the cut-off using a separate preliminary sample set, then assess the selected cut-off in an independent validation set. Document the rationale for the chosen threshold and establish the evaluation approach before the confirmatory assessment begins. That separation helps protect the credibility of the results. ## 5. Assuming Performance Transfers Across Platforms An HPV assay validated on one instrument is not automatically clinically validated on another, even when the underlying chemistry remains unchanged. Differences in instruments, workflows or other platform-related characteristics may affect performance. The size of that impact depends on the specific change and needs to be assessed rather than assumed away. A scientifically justified bridging study may be appropriate. The key is to define the bridging strategy before starting the evaluation, including what existing evidence can be relied upon and which potential differences still need to be addressed. This can prevent teams from discovering late in the process that evidence generated on one configuration does not adequately support another. ## 6. Investing in an Outdated Assay Design Validation strategy is not only about demonstrating how well an assay performs today. It is also about considering whether its design remains appropriate for the intended clinical and market context. As the author points out, an HPV test without genotyping capability, including HPV 16 and 18 differentiation, may be at a disadvantage in parts of the current market. That does not mean genotyping is a universal requirement for every HPV assay. The relevant question is whether the test's capabilities match its intended purpose, target screening pathway and applicable evidence expectations. It is worth addressing that question early, before committing substantial resources to a validation programme for a product whose intended positioning may need to change. ## The Common Thread: Plan Your HPV Validation Before You Start These six mistakes have something in common. Many are difficult or expensive to correct once the study is underway, but can be addressed through earlier planning. Choosing experienced partners, defining appropriate sample sets, considering population differences, fixing the cut-off before validation and planning platform bridging all help build a more coherent clinical evidence strategy. For manufacturers pursuing both IVDR and WHO Prequalification, that planning should also include an early comparison of the two pathways. An integrated strategy may help avoid unnecessary duplication, while recognising that each pathway can require additional evidence for its intended settings of use. The objective is not simply to complete a validation study. It is to generate evidence that answers the right questions for the assay, its users and the markets it is intended to serve. --- Source: https://qbdgroup.com/en/blog/hpv-assay-validation-mistakes — © QbD Group. Quote freely with attribution and a link back.