Valerie Neff Newitt
November 2023—In the 2023 edition of the CAP accreditation program checklists is new guidance on analytical measurement range verification and new and revised requirements for mass spectrometry.
All are part of an effort to clarify the checklists based on questions that are commonly asked and observations made during inspections, says William J. Castellani, MD, a member of the CAP Checklists Committee and interregional commissioner for the accreditation program.
The requirements on analytical measurement range verification have been revised to provide additional guidance for chemistry and toxicology, clinical biochemical genetics, immunology, point-of-care testing, and coagulation laboratories. CHM.13550 AMR Verification Materials says verification of the analytical measurement range is performed with matrix-appropriate materials that, at minimum, include the low, mid, and high range of the AMR. (The same requirement is in the checklists for the other types of laboratories.) New content was added for factors to consider when verifying the AMR, such as the expected analytic imprecision near the limits, the clinical impact of errors near the limits, and the availability of test specimens near the limits.
“The closeness of sample concentrations and activities to the upper and lower limits of the AMR are defined at the laboratory director’s discretion,” the requirement says.
For calibration verification, CLIA requires showing there is a consistent response across the measurement range, Dr. Castellani notes. One acceptable way, he says, is if the method is calibrated at the extremes of the measurement range in addition to at least one more point within the range itself. “This satisfies the requirement,” he says. For methods not calibrated in this manner, the checklist has long held that the CLIA requirement does not mandate that the materials used to verify the calibration include specific values at the midpoint and extreme limits of the range.
“Although the laboratory may use the entire manufacturer’s analytical range for reporting patient results, there is no requirement that verifiers are at or within some arbitrary closeness to the upper and lower limit of the manufacturer’s range,” Dr. Castellani says. The laboratory director may apply medical decision-making to determine the extent of the analytical range where the analytical response is tied to values that may affect or change clinical decisions, he says.
“For many analytes, at the extremes of the measurement range, there is no clinically significant difference for any result in that part of the range. High is high, low is low, and any value in the high or low part of the analytical range would have the same clinical meaning.” The entire manufacturer’s range is therefore verified by showing an appropriate response within that part of the range where a specific result may affect clinical decisions, he says.
In those instances, the laboratory can accept and report from the manufacturer’s broader range once it has, in general and by the lab director specifically, determined and verified the range where clinical decisions are affected by the value the method produces. “Within this range of clinically important values, it is typically possible to find material with those values,” Dr. Castellani says.
For some analytes—Dr. Castellani cites beta-hCG as an example—the entire range of values may have clinical significance. For significant clinical states, such as monitoring at-risk pregnancies or gestational tumors, patient samples often need to be diluted to bring them into the analytical range of the method, and there is no way to anticipate where the diluted sample will fall within this range, he says. Since these values are often trended, each value must be reliable wherever the diluted sample falls within the analytical measurement range. Therefore, the laboratory must be confident of its results throughout the range.
“However, for these analytes there is often material, such as patient samples, that can be found at the extremes of these ranges,” he says. “Again, it is up to the laboratory director to determine which analytes require such stringent verification and how close to the upper end of the range the material must be to verify the range for clinical use.” Since these analytes typically require dilution to bring clinically important values into the analytical range of the method, “it is the upper end of the range that is critical,” Dr. Castellani says. “No dilution should ever be necessary to bring an elevated sample into the lower part of the manufacturer’s analytical range.”
Testing the midpoint using a material with a value at the center of the range is not mandated and is “typically useless,” he says. “It’s often much, much higher than any clinically significant diagnostic threshold. It’s more useful to include a sample with a value around such clinical thresholds rather than an arbitrary—and arbitrarily high—midpoint sample.”
CHM.13600 is another AMR verification requirement, which says verification must be performed at least every six months and following defined criteria. It now says AMR verification is not required for calculated test results as long as the individual results contributing to the calculation have AMR verification.
For mass spectrometry, there are new requirements, among them CHM.18640 Validation and Monitoring of MS Data Analysis Tools.
It requires the laboratory to validate data analysis tools used for compound identification and quantification when first installed and after modifications, as applicable, and to verify performance at least annually. (FDT.24950 and CBG.17150 require the same.)
Such tools can be used for various processes such as integrating targeted and untargeted peaks and evaluating acceptability of calibration and control performance. “Data analysis tools take the output of the analyzer and perform higher-level, typically assay-specific processes,” Dr. Castellani explains.
Also new is CHM.18610 Extracted Calibrators, which requires appropriate extracted calibrators to be analyzed or appropriate calibration verification to be conducted with each batch of samples. The requirement says at least one extracted calibrator at the commonly accepted cutoff or reporting threshold for single-point calibration, or multiple calibrators above and below that value for multipoint calibration, must be analyzed with each run.
“This requirement addresses the fact that calibrators that do not go through the extraction process, although useful, do not show that the instrument responds as appropriately as calibrators that have gone through the extraction process,” he says.
CHM.18620 Analytical Performance Monitoring of MS Assays is a new requirement that says the lab must monitor analytical performance of mass spectrometric assays using defined performance criteria and quality metrics and perform corrective action when acceptance criteria are not met.
“This gives clarity to what would be expected to demonstrate that the mass spectrometer is performing appropriately and that the assays are working,” Dr. Castellani says.
For inspectors, especially those less familiar with mass spectrometry, he adds, “it offers guidance on what the laboratory has to do to demonstrate that the method is performing as expected over time.”
The requirement provides examples of performance criteria and quality metrics, among them system suitability testing, thresholds for re-injection of specimens or re-extraction, and adequacy and stability of internal standards response within and across runs.
New this year to the forensic drug testing checklist (as FDT.25150), and revised in other checklists, is the requirement for matrix effect assessment of mass spectrometry assays—validation (CHM.18825 and CBG.17500). An evaluation of matrix effects must be performed during assay development and validation, and the requirement says there must be a record of the assessment.
Matrix effects can affect analyte ionization and performance in both directions: suppression or enhancement. Evaluation protocol examples are provided, such as post column infusion, which is constant infusion of an analyte followed by injection of blank matrix specimen extracts to measure ionization response. A new protocol is included among the examples: internal standard monitoring, which is evaluating the trend in internal standard abundance and signal-to-noise ratios during an analytical run that includes blank and spiked matrix specimen extracts.
“The assays that can be implemented on mass spectrometry are very broad, and they can be extremely different from each other,” Dr. Castellani says. “So a third evaluation protocol option, felt to be equally useful and reasonable for the laboratory to use to look for this interference, was added.”
Another requirement, CHM.18850, requires the laboratory to evaluate mass spectrometry assays for possible ion-suppression or enhancement in patient samples during routine testing. Prior to the 2023 checklist edition, the requirement addressed single-stage liquid chromatography-mass spectrometry. “The reality is that this requirement is valid for any type of mass spectrometry,” Dr. Castellani says, and thus its wording was revised to generalize the requirement. “This is appropriate for all mass spectrometry assays,” he says. The requirement is in other checklists as FDT.25210 and CBG.17600.
CHM.18700 Identification Criteria—Mass Spectrometry, which requires that the criteria for analytes detected by mass spectrometry be defined, was broadened to encompass both single-stage and tandem mass spectrometry, making it possible to eliminate a similar requirement that was applicable to tandem MS only. To streamline the checklist, Dr. Castellani explains, requirements for single-stage and tandem MS that appeared separately in prior checklist editions were merged into single requirements applicable to both, with notes made when needed to address the specifics pertaining to the different devices.
The prior version of CHM.18700 addressed only the single-stage mass spectrometer and was “long and highly prescriptive,” he says, while the tandem MS requirement, CHM.18800 (now deleted), was brief and permitted flexibility in developing identification criteria. “For the merged requirement, a middle course was taken,” Dr. Castellani says, in which the text that describes the overall purpose of the requirement was retained while the more prescriptive text on how to meet the requirement was removed. “The revised requirement is applicable to and sufficient for both single-stage and tandem mass spectrometry,” he says. (In the FDT checklist this requirement is FDT.25130.)
CHM.18600 Mass Spectrometer Tuning also was revised. It requires tuning as defined based on the platform in use, the assay performance requirements, and the specimen types tested. Frequency should be based on the manufacturer’s recommendation. (In other checklists, the requirement is FDT.24630 and CBG.17100.)
Before the revision, Dr. Castellani says, the required frequency had been every day of patient testing. “Simply too often for every situation,” he says. “We felt it is most appropriate for a laboratory, when bringing up a test, to determine how often it needs to tune or retune the mass spectrometer to ensure the assay is reading correctly.” But it must be tuned at least as often as the manufacturer requires, he stresses. “That’s important because the FDA cleared the device dependent on what the manufacturer says is necessary for proper operation. So the lab must follow that or validate by its own studies that it can do something different.”
New and revised requirements in other checklists were reported in the August (https://bit.ly/CT-082023), September (https://bit.ly/CT-0923-visco), and October (https://bit.ly/CT-1023-breakpt) issues.
CAP-accredited labs can access compliance-related resources on www.cap.org (in e-Lab Solutions Suite, log-in required, under Accreditation Resources), including the Oct. 18 Focus on Compliance webinar and other past Focus on Compliance webinars, as well as lab inspection preparation videos. Also online are answers to the most common checklist-related questions, a self- and post-inspection toolbox, and customizable templates and forms for, among other things, competency assessment and quality management.
Valerie Neff Newitt is a writer in Audubon, Pa.