For prenatal NGS labs, new accreditation requirements

“The checklist doesn’t mandate that labs incorporate prior risk into their report, but if they collect the information, they should realize that not all positives and negatives are created equal.” Dr. Palomaki believes that a number of informative conclusions could potentially be drawn from these collected data that could improve the final risk estimate.

Often, he notes, physicians are provided with test results and have to assemble the interpretation themselves. If a woman has a nuchal translucency measurement and maternal serum screening showing she is at high risk, for instance, and then has the NGS test, the physician may wonder what the negative result on the molecular test means in the context of the prior test results.

“Interpretation of both positive and negative tests is not so straightforward. That’s one of the reasons why the professional organizations have suggested that these issues be more fully explored before we move to testing in the general pregnancy population.”

Two phase II checklist items on quality control (MOL.34923 and MOL.34924) are among the additions and require that labs monitor test performance limits and QC parameters, and include positive and negative controls in each analytical run. Longitudinal monitoring of the assays, another checklist requirement (MOL.34925), will help with a process called epidemiologic monitoring, Dr. Palomaki says.

“What that means is that most of the pregnancies, and tests, will be normal even in the high risk setting. This can be used to actually help monitor your assay.” One company uses a laboratory performance index known as a z-score; two others use something similar. “This score can be monitored as an indicator of assay quality, and when it does not meet expectations, the reference data may not be appropriate. In any event, the laboratory should develop a monitoring scheme, if possible, and have an associated action plan when epidemiological monitoring indicates a potential problem.”

He is hopeful that collecting those data will help improve the tests’ performance. “There are numerous reports from serum screening labs showing what happened to their assays month by month because they monitor results carefully. When one month’s results appeared to be out of control, they went back and found, for example, the problem occurred because they had switched to new reagents.

“Because of this, we now suggest that laboratories preview their serum screening reagents when they are received and assess them using a method comparison before using them clinically. Testing has improved because of this practice. We’ve learned these things because people were willing to share their findings, including their remedies. Whether that will be true for NGS laboratories, I don’t know.”

Monitoring of targeted disorders is another important quality assurance measure that the checklist now requires (MOL.34926). At least quarterly, laboratories should calculate and review the percentages of women with positive results for each targeted disorder such as Down syndrome or Turner syndrome, test failure rates due to such factors as low fetal fraction, and rates of “inconclusive” test results.

Longitudinal mon­itoring is a challenging area for clinical laboratories, Dr. Voelkerding points out. “The importance of NGS testing for fetal aneuploidy is that if there is a positive result, such as presence of trisomy 21, this should trigger a discussion between the pregnant mother/couple and the care provider with respect to confirmatory testing.”

The mother may elect the use of a confirmatory followup method, he says. “In this case, amniocentesis coupled with cytogenetic analysis would be considered standard of care.”

But some believe that the confirmatory method may eventually not be needed. “Will the NGS test become the standard of care so that one can rely on the NGS result and not need an amniocentesis? The only way to determine this is to do longitudinal studies wherein NGS results are compared to those obtained by amniocentesis and cytogenetic analysis.” Important to note, Dr. Voelkerding says, is that cytogenetic analysis may identify other chromosomal abnormalities not currently detected by NGS.

Longitudinal studies are essential going forward, he says. “While laboratories offering NGS-based testing for fetal aneuploidy have performed validation studies, longitudinal studies would provide a larger database to determine the sensitivity and specificity of this testing in clinical practice.”

Finally, the new checklist contains a requirement (MOL.34927) that laboratories’ reports include qualitative and/or quantitative test results for each target chromosome (for example, z-score, fetal fraction, likelihood ratio), reference ranges or cutoff values as appropriate, and a summary set of risks/categorical interpretations.

Variation in these specific test results can make a big difference in a test’s information content, Dr. Palomaki says. “I and others have been suggesting that labs report not only the test interpretation but also the test results. Two of the labs now routinely include fetal fraction on patient reports, one result of testing. But I don’t know of any labs that include results such as the z-score or normalized chromosome value.” However, current practice has been considered satisfactory for CLIA and CAP inspections, he says. “But the checklist suggests that if actual test results are available, they should be reported along with the final interpretation.” This would be similar to serum screening where the raw assay values and the results in multiples of the median are mandated, in addition to the Down syndrome risk.

He gives an example of why including these results is important. “If the fetal fraction were four percent, most of these tests will be reliable, but at 10 to 20 percent, the performance will be better. At 20 percent or higher, even fewer errors would be expected to occur.” This has implications for reporting results, as knowing the fetal fraction for each test may help practitioners better understand the individual interpretations. “For example, as they gain experience, they may clearly see more ‘no calls’ made among women with lower fetal fractions that tend to have higher maternal weights.”

The checklist requirement doesn’t stipulate that the companies have to change their reporting, he says, because it uses the language “as appropriate.” But he hopes the companies will begin issuing more informative and detailed results of the testing, not just assessment of risk.

It’s significant that the College is looking at this type of prenatal testing that is being introduced rapidly into clinical practice, Dr. Palomaki believes. “For the first time, there are specific checklist requirements for the labs offering this type of NGS prenatal screening. This type of testing is a very sensitive area for patients, yet is one of the most common applications for NGS right now. CAP is looking out for physicians and patient care by quickly introducing checklist requirements that are relevant to prenatal screening.”

The NGS-related checklist requirements will continue to evolve, Dr. Voelkerding says. “We initially responded to this new area for labs with a series of general checklist requirements and we are also progressively adding yearly revisions, including diagnostic topic-specific requirements, in response to feedback from the field, to continually improve the checklist as new applications are being developed.”

NGS proficiency testing is next, he adds. “We are now working in parallel toward launching proficiency testing challenges for the diagnostic community for assays based on NGS. That will include PT challenges for genetics/inherited disorders and oncology,” and for fetal aneuploidy, NGS PT is under discussion.

Anne Paxton is a writer in Seattle.