CPK-MB

Pathophysiology

Clearance of CK from Blood or Plasma 

CK and its isoenzymes are inactivated in the lymph through proteolysis.[6] Abnormal liver function or renal function does not affect the clearance of CK in a significant manner, and CK is not excreted in the urine.[7] Hypothyroidism retards the clearance of CK.[8]

CK-MB levels can also be elevated in circulation in the absence of acute myocardial infarction.[9] This elevation is due to increased amounts of B subunit production in injured skeletal muscle, as it does during fetal development; ontogeny recapitulates phylogeny.[10]

Rhabdomyolysis, intense exercise, and trauma result in transient elevation of CK and CK-MB; CK-MB is present in skeletal muscles as well, albeit in lesser concentrations.[11] Chronic skeletal muscle disorders such as autoimmune myopathies and inflammatory myopathy can result in persistently high CK-MB levels in the plasma due to ongoing injury and repair.[12][13] Damage to the myocardium releases CK-MB, and since the myocardium contains the largest percentage of CK-MB, patients with rapidly rising and falling CK-MB exceeding the reference range of normal should be considered as having acute myocardial infarction until proven otherwise.[14]

Specimen Requirements and Procedure

A fresh serum that is free from hemolysis is the ideal specimen for analyzing the CK isoenzyme pattern. Among the 3 commonly observed isoenzymes, CK-BB activity is the least stable. Adding a thiol such as 2-mercaptoethanol to the serum improves its stability.[15] CK-MB activity is not significantly reduced when the separated serum is stored for up to 48 hours at 4 °C or 1 month at −20 °C. As the mass measurement is not subject to the loss of enzyme activity, CK-MB protein concentration in serum is stable for weeks when stored under refrigeration and for several years when stored at −20 °C.[16]

Testing Procedures

Analysis of CK Isoenzymes

Electrophoresis and various immunological methods are commonly used to analyze CK isoenzyme.[17]

Electrophoretic methods: Electrophoresis is particularly useful for separating all CK isoenzymes. The isoenzyme bands are visualized by incubating a support medium, such as agarose or cellulose acetate, with a concentrated CK assay mixture using the reverse reaction. This reaction generates NADPH, which is detected by its bluish-white fluorescence under long-wave ultraviolet light (360 nm). NADPH may be quantified by fluorescence densitometry, which can detect bands of 2 to 5 U/L. At pH 8.6, the mobility of CK isoenzymes toward the anode is BB > MB > MM, with the MM remaining cathodic to the point of application.[18] The discriminating power of electrophoresis also allows the detection of abnormal CK bands, such as macro-CK.

However, electrophoresis has several limitations:

• Lengthy turnaround time
• A labor-intensive procedure
• Limited adaptability to clinical chemistry analyzers in emergencies
• The necessity for interpretative skills [19]

Immunochemical methods: Immunochemical techniques, particularly immunoinhibition, are used for direct measurement of CK-MB. In the process, an anti-CK-M subunit antiserum is used to inhibit both the M subunits of CK-MM and the single M subunit of CK-MB, allowing the determination of the enzyme activity of the B subunit of CK-MB, the B subunits of CK-BB, and macro-CKs. This technique assumes the absence of CK-BB and macro-CKs from the tested serum to determine CK-MB.[20] Because the CK-B subunit accounts for half of the CK-MB activity, the change in absorbance should be doubled to obtain CK-MB activity, leading to decreased analytical sensitivity. In addition, atypical macro-CK may result in falsely elevated CK-MB results. Due to its low sensitivity and specificity, the immunoinhibition technique has been largely replaced by mass assays.[21]

Mass Immunoassays: Unlike immunoinhibition, which measures the CK-MB isoenzyme by determination of its catalytic activity, mass immunoassays measure CK-MB protein concentrations.[22] Several mass assays using various labels are now commercially available and used for routine determination of CK-MB. Measurements use the sandwich technique, in which one antibody specifically recognizes only the MB dimer. The sandwich technique ensures that only CK-MB is estimated because neither CK-MM nor CK-BB reacts with both antibodies. Mass assays are more sensitive compared to activity-based methods, with a limit of detection for CK-MB usually <1 μg/L.[23] Other advantages include sample stability; noninterference with hemolysis, drugs, or other catalytic activity inhibitors; full automation; and fast turnaround time.[24]

Interfering Factors

Measurement of CK-MB, Interfering Factors, and Negating Techniques 

CK-MB was initially separated through gel electrophoresis, and densitometry was used to quantify the activity of CK-MB isoenzyme in blood.[25] Adenylate kinase released by red blood cells results in a false elevation of CK activity. Currently, laboratories add reagents to inhibit adenylate kinase activity. Multiple commonly occurring compounds that naturally fluoresce can co-migrate with CK-BB and CK-MB during electrophoresis, including bilirubin, aspirin, antidepressants, and benzodiazepines at high concentrations.[13]

CK-MB elevation was used as one of the criteria for diagnosing acute myocardial infarction. In the late 1980s and 1990s, as the use of CK-MB increased in frequency, it became evident that despite its high sensitivity in detecting acute myocardial infarction, its specificity was low. Better methods to measure CK-MB have been developed to improve its specificity. CK-MB mass measurements using immunoenzymometric assays containing monoclonal antibodies binding to M and B subunits individually were proven to be highly specific and more sensitive than CK-MB activity measurement.[26] Even assays using monoclonal antibodies have been found to have elevations in CK-MB mass due to the cross-reactivity of alkaline phosphatase in plasma with stabilizing agents found in commercial reagents.[27]

Results, Reporting, and Critical Findings

Guidelines for CK-MB reporting and interpretation include the following:

• Reporting of gender-specific results is recommended by the American College of Cardiology (ACC), American Heart Association (AHA), and European Society of Cardiology (ESC)
• CK-MB is typically at undetectable levels in the blood.
• CK-MB levels are reported as positive if they are above the 99th percentile of normal values, 5 to 25 IU/L.
• CK-MB must always be reported along with the CK-MB relative index to add sensitivity and specificity to the test.
• Positive values must be informed to the test-ordering personnel within 1 hour of the positive result.

Clinical Significance

Diagnosis of Acute Myocardial Infarction

Before the advent of cardiac troponins, the CK-MB isoenzyme was the primary biochemical marker for diagnosing acute myocardial infarction.[28] CK-MB first appears 4 to 6 hours after the onset of symptoms, peaks at 24 hours, and returns to normal within 48 to 72 hours. Due to this short timeframe, its diagnostic value for acute myocardial infarction is limited both in the early stages (within the first few hours) and in the late stages (after 72 hours). However, its release kinetics can assist in diagnosing reinfarction if levels rise after initially declining following acute myocardial infarction.[10] The most common diagnostic criterion involved 2 serial elevations above the diagnostic cutoff level or a single result more than twice the upper limit of normal.[29] 

Non-Acute Myocardial Infarction Causes of CK-MB Elevation

Elevated CK-MB can result from skeletal muscle or myocardial cell death due to various causes. Other causes of CK-MB elevation in plasma include the following: 

• False elevations: False elevations in CK-MB occur in the presence of atypical CK isoforms, macrokinases, and adenylate kinase; however, these false elevations can be eliminated by adding reagents to testing kits.[29] 
• Cardiac etiology: Myocarditis and cardiac surgery can damage heart muscle, resulting in the elevation of CK-MB.
• Peripheral sources: Rhabdomyolysis, myositis, inflammatory myopathies, trauma, and medications, such as daptomycin, statins, and antiretrovirals, can also elevate CK-MB levels.

To differentiate the elevation of CK-MB for cardiac etiology versus skeletal muscle source, the CK-MB relative index can be calculated using the formula:

• CK-MB relative index = CK-MB (ng/mL) × 100/CK (IU/L) 

A CK-MB relative index < 3% is consistent with the skeletal muscle source, whereas a relative index > 5% is consistent with the cardiac source of CK-MB.[30] However, prior studies in patients with trauma and chronic skeletal muscle abnormalities have demonstrated the failure of the CK-MB relative index in differentiating skeletal muscle sources of CK-MB from myocardial cell death.[31]

Hence, in patients with clear evidence of no trauma, chronic skeletal muscle abnormalities, and a high index of suspicion for acute myocardial infarction, the use of CK-MB relative index can increase the specificity of CK-MB testing. Miscellaneous causes include hypothyroidism, renal failure, alcohol intoxication, pregnancy, and certain types of malignancies. 

Current Biomarker Use 

According to the World Health Organization (WHO) criteria for diagnosing acute myocardial infarction, various cardiac biomarkers were utilized in the diagnosis process. Among these, CK-MB was considered the most sensitive and specific marker for diagnosing acute myocardial infarction, detecting reperfusion, and estimating the extent of myocardial infarction, particularly in the 1990s. During this period, troponin emerged as a potentially more specific biomarker for myocardial infarction compared to CK-MB.[28]

Troponin is a protein complex composed of 3 units—troponin T, troponin I, and troponin C—present in the actin filament of the skeletal and myocardial muscle cells. Troponin T and troponin I have multiple isoforms, with one specific to cardiac muscle. This isoform is not found in adult skeletal muscle, which enables the development of assays to measure its levels in plasma.[32]

Troponin is present in the myocardium as a 3-unit complex in the contractile apparatus attached to the actin filament of the tropomyosin complex; however, similar to CK-MB, there is unbound or free troponin in the cytosol of myocardial cells, which is known as the cytosolic pool. In the event of myocardial damage, unbound troponin is first released.[33][34] This unbound troponin is about 6% of the total troponin in the myocardium. The rest of the troponin bound to the actin is released slowly with structural damage, resulting in the prolonged duration of elevated troponin levels in the plasma.[35] Troponin elevation >99th percentile is used as the cutoff value for diagnosing acute myocardial infarction.[36] Troponin concentration begins to rise 4 to 6 hours after the onset of symptoms, peaks by about 18 to 24 hours, and remains in detectable levels for 72 to 96 hours.[37]

Troponin is more specific to cardiac muscle compared to CKMB, and current assays for troponin are more sensitive and specific than those used for CK-MB measurement.[29] Due to the expression of CK-MB in skeletal muscle and evidence demonstrating the limitations of the CK-MB relative index, along with its elevation in various non-acute myocardial infarction conditions, troponin has been established as the biomarker of choice for detecting myocardial damage of any etiology.[30]

Use of CK-MB Despite Troponin Being the Biomarker of Choice 

Troponin remains in circulation for a longer duration compared to CK-MB. In conditions where reinfarction is suspected, CK-MB may be useful in classifying a new event due to its shorter duration of elevation at detectable levels in plasma.[38] However, with the advent of troponin and the current aggressive interventional strategies for acute myocardial infarction, and due to a lack of literature comparing CK-MB against troponin for diagnosing reinfarction, the use of CK-MB has declined.[39]

CK-MB Isoforms

The CK-MB isoenzyme exists as 2 isoforms—CK-MB1 and CK-MB2. Laboratory measurement of CK-MB represents the simple sum of the isoforms CK-MB1 and CK-MB2.[9][18] CK-MB2 is the tissue form released from the myocardium after myocardial infarction, and it is converted peripherally in serum to the CK-MB1 isoform rapidly after symptom onset. The tissue CK-MB1 isoform predominates; thus, the CK-MB2/CK-MB1 ratio is typically less than 1. A positive result occurs when CK-MB2 levels are elevated and the CK-MB2/CK-MB1 ratio exceeds 1.7.[29]

CK-MB2 can be detected in serum within 2 to 4 hours after onset and peaks at 6 to 9 hours, making it an early marker for acute myocardial infarction. Two large studies evaluating its use revealed a sensitivity of 92% at 6 hours after symptom onset, compared to 66% for CK-MB and 79% for myoglobin.[14] The major disadvantage of this assay is that it is relatively labor-intensive for the laboratory.[18] 

Troponin remains in circulation for a longer duration compared to CK-MB. In conditions where reinfarction is suspected, CK-MB may be useful in classifying a new event due to its shorter duration of elevation at detectable levels in plasma.[38] However, with the advent of troponin and the current aggressive interventional approach to acute myocardial infarction, and due to a lack of literature comparing CK-MB against troponin in the diagnosis of reinfarction, the use of CK-MB has declined.[39]

Quality Control and Lab Safety

For non-waived tests, laboratory regulations require, at the minimum, the analysis of at least 2 levels of control materials once every 24 hours. Laboratories may perform quality control (QC) testing more frequently if necessary to ensure accurate results. QC samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance.[40] To minimize QC when performing tests for which manufacturers’ recommendations are less than those required by the regulatory agency (such as once per month), the laboratories can develop an individualized QC plan that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.[41] Westgard multi-rules are used to evaluate the QC runs. In case of any rule violation, proper corrective and preventive action should be taken before patient testing is performed.[42]

The laboratory is required to participate in an external QC or proficiency testing (PT) program, as mandated by the Centers for Medicare and Medicaid Services (CMS) under the Clinical Laboratory Improvement Amendments (CLIA) regulations.[43] These programs ensure the accuracy and reliability of test results by comparing performance with other laboratories conducting similar assays. Required participation and scored results are monitored by CMS and voluntary accreditation organizations.[44] The PT plan should be included as an aspect of the quality assessment plan and the overall quality program of the laboratory.

All specimens, control materials, and calibrator materials should be treated as potentially infectious. Standard precautions must be taken when handling laboratory reagents, including wearing gloves, lab coats, and safety glasses. All waste materials should be disposed of in accordance with local guidelines. All plastic tips, sample cups, and gloves that come into contact with blood should be placed in a biohazard waste container.[45] All disposable glassware should be discarded into sharps waste containers. All work surfaces should be protected with disposable absorbent bench top paper, which is discarded into biohazard waste containers weekly, or whenever blood contamination occurs. All work surfaces should be wiped weekly.[46]

Enhancing Healthcare Team Outcomes

Given the significant number of studies and guidelines from the American College of Cardiology recommending the use of troponin instead of CK-MB for diagnosing and ruling out acute coronary syndromes, decreasing the use of CK-MB in hospital and outpatient settings requires a collaborative approach from an interprofessional healthcare team, including nurses, laboratory technologists, pharmacists, and clinicians from various specialties, especially cardiologists and cardiothoracic surgeons. Cardiologists and surgeons rely on troponin's higher sensitivity and specificity to guide patient care, whereas nurses and laboratory staff ensure the proper implementation of testing protocols.

Specialty-trained nurses are crucial in ordering, collecting, and interpreting troponin tests. They help facilitate the smooth integration of troponin testing into clinical workflows, ensuring timely and accurate sample processing. In addition, nurses play a key role in educating patients about the test's purpose and implications and communicating critical results to the healthcare team. Their involvement in these processes ensures that troponin testing is used effectively to guide diagnosis and treatment decisions.