Quality control and cardiac marker assays

When a patient presents with chest pain, is it myocardial infarction (MI) or pulmonary embolus (PE)? When a patient presents with shortness of breath, is it heart failure (HF) or chronic obstructive pulmonary disease (COPD)? Critical treatment decisions often hinge on the answers to questions like these, and the results of cardiac marker assays can be crucial to those answers. Troponin and D-dimer assays are used to differentiate MI from PE. An elevated natriuretic peptide (BNP or NT-proBNP) is indicative of HF but not COPD. C-reactive protein (CRP) and homocysteine are independent risk factors for the development of cardiovascular disease. A lot depends on the laboratory results being correct. 

Cardiac troponins

Today, cardiac troponin is the preferred biomarker for myocardial injury and the centerpiece of the third universal definition of myocardial infarction.¹ Troponins are contractile proteins found in cardiac muscle. When cardiac muscle is damaged, troponins are released into the serum. Better control of troponin assays at low levels is critical because, according to Tietz, “all concentrations above the 99th percentile upper reference limit (URL) are designated as irreversible myocardial injury” and in the presence of the clinical setting of ischemia “indicative of acute myocardial infarction.”² 

Assays are available for the cardiac troponins, Troponin I (cTnI) and Troponin T (cTnT). The term “high sensitivity” (hs) refers to the assay’s ability to detect low levels. In 2012, an International Federation of Clinical Chemistry (IFCC) Task Force proposed that cTnI and cTnT assays receive the designation hs if they met two criteria: 

• Total impression (CV) at 99th percentile ″10%; 
• Measurable concentrations below 99th percentile attainable with an assay concentration value above the assay’s limit of detection for at least 50% (ideally >95%) of healthy individuals.³

The 99th percentile URL is provided by the manufacturer of the assay in the package insert and varies by platform. Tables showing the analytical characteristics of troponin assays are available online from the IFCC. A list of published peer-reviewed papers that describe the analytical characteristics of troponin assays is also available on the IFCC website. The cited papers “give a realistic assessment of the performance of manufacturer’s assays in routine clinical laboratories.” ⁴

Recently, the FDA issued a safety communication regarding the identification and verification of falsely elevated results for troponin. It states that “different troponin assays have wide variations in lower detection limits, upper reference limits, diagnostic cut points, assay imprecision (coefficient of variation), and specimen matrices….The presence of a large number of manufacturers of troponin assays in the United States market makes standardization more difficult.” In the communication, the FDA made the following quality recommendations:

• Follow manufacturer’s recommendation for running proper quality control samples. At least one control should be run at the cutoff level (emphasis in original). If the risk stratification and acute myocardial infarction cutoff are different, separate controls should be considered at those levels. 
• Follow the manufacturers’ recommended calibration and/or maintenance schedules. Analyzer malfunction is one of the common assay interfering factors that leads to inaccurate results. Laboratories reporting troponin results should perform thorough and regular system maintenance to ensure peak performance of their analyzers and to reduce the possibility of inaccurate results.⁵

Determining number of control levels

The Clinical and Laboratory Standards Institute (CLSI) guideline on QC, C24-A3, states: “The number of levels and concentration of quality control materials should be sufficient to determine proper method performance over the measuring range of interest.” It continues:

For most analyte-method combinations, a minimum of two levels (concentrations) of control materials is recommended. Where possible, analyte concentrations should be at clinically relevant levels to reflect values encountered in patient specimens….

Control materials may be selected to cover the measuring range. Routine testing of these materials may be helpful in confirming the expected range of the procedure.⁶

While for most analyte-method combinations a minimum of two levels (concentrations) of control materials is recommended by CLSI, consider analyte-method combinations that require multipoint calibration and have a non-linear response. Although two points may provide sufficient quality control for a straight line (single point calibration), at least three points are needed to provide quality control for a curve (multipoint calibration). For example, some troponin assays require five point calibration; others have a predefined master curve that is adapted to the analyzer by using a bi-level calibrator. Often, widely separated values are used for calibration and control. Likewise, the low control value may be far above both the medical decision point and the lower limit of the measuring range. However, tri-level controls from an independent manufacturer can provide quality control for this critical region and validate the adaption of the predefined calibration curve to the analyzer.

Patient risk

Whether your laboratory produces five hundred or five million results a year, providing quality patient care should include a plan to minimize the risk of reporting incorrect results in the event of an out-of-control condition. A patient-focused assessment enables the laboratory to evaluate appropriate QC levels, frequency, and rules for quality control. This risk-based approach looks at potential sources of error, frequency with which they may occur, likelihood of the error being detected, and impact to the patient should an incorrect result be acted upon (severity of harm). CLSI’s EP23-A (Laboratory QC Based on Risk Management) and CMS’ IQCP (Individualized Quality Control Plan) are two means of looking at patient risk when evaluating a QC Plan. 

Other considerations

To accommodate the lack of standardization in Troponin I assays, laboratories should select quality control material at a low value better suited to the 99^th percentile URL of their platform. Likewise, they should select a low value of hs cTnT better suited to its 99^th percentile URL.

CLSI’s C24-A3 also provides considerations for quality control selection: “The control materials specified are separate external specimens to be analyzed repeatedly by the measurement procedure. The quality control materials should be different from the calibrator materials to ensure the QC procedure provides an independent assessment of the measurement procedure’s performance in its entirety, including the procedure for calibration of the measurement.”⁶

It is generally agreed that the most reliable monitoring of instrument performance is provided by independent controls from a third-party (independent) manufacturer. Independent control materials are designed and manufactured free of any instrument, kit, or method bias. In addition to overt failures, independent controls can often detect more subtle changes in calibration status and instrument and reagent performance. International Organization for Standardization (ISO) 15189 recommends the use of independent control material, either instead of or in addition to any control materials supplied by the reagent or instrument manufacturers. 

Any patient result reported without real-time review of quality control data is at risk. The ability to successfully manage and interpret QC results is essential to improving laboratory analytical performance. Software tools include support for run time decision making, trouble shooting, and QC design. Instrument connectivity allows QC data to be quickly and easily imported from instruments, middleware, or LIS, regardless of whether the cardiac assays are performed in the laboratory or at the point of care, allowing labs to manage their QC results and review peer group data using the same workflow as they do for hundreds of other core lab assays. 

Conclusion

The importance of cardiac markers in making timely decisions in critical situations cannot be understated. Clinicians and laboratorians alike need to feel confident that the results are accurate and actionable; cardiac presentations rarely come with the luxury of allowing for repeat testing. Confidence in testing results comes from not only selecting the most appropriate instrument for the clinician’s needs but also in developing a robust quality control regimen. Considerations must include matching the quality control target values for the given method, number of levels to ensure multi-point calibration curves are appropriately assessed, risk of harm to the patient, independence from instrument calibrators, and data management/peer group comparison solutions.