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A historical background in cardiac markers.

Advances in the use of cardiac markers to evaluate suspected heart attacks has resulted in steady progress over five decades. During that time some tests such as total lactate dehydrogenase (LD) and aspartate amino transaminase (AST) have become discarded for that purpose, while new tests such as creatine kinase (CK), CK-MB isoenzyme, CK-MB isoforms 2 and 1, myoglobin, troponin I and troponin T have been developed.

Innovative technology such as mini-column separation methods (in the '70s and early '80s) for quantitative measurement of CK-MB and automated electrophoretic separation of CK-MB into two isoforms (in the '90s) have played vital roles in the development of our current automated enzyme immunoassays, which utilize highly sensitive and specific monoclonal antibodies to capture and detect CK-MB, myoglobin, troponin T, and troponin I.

Recent cardiac markers of current interest and some of the reported claims to their unique clinical utility are listed below:

Myoglobin: Very sensitive to muscle necrosis and appears in circulation earlier than CK-MB. Enhanced sensitivity compared to CK-MB, especially in early-stage (2-6 hours) post-infarct.

CK-MB: Excellent for evaluation of patients with chest pain and possible myocardial infarction, provided a CK-MB peaking pattern over time is observed. Sensitivity is good enough to detect early-stage infarction (2-6 hours) provided results from serial collections are evaluated.

CK-MB isoforms: Excellent for evaluation of myocardial infarction especially in the early stage (2-6 hours) of cardiac damage; enhanced sensitivity to total CK-MB isoenzyme.

Troponin T or troponin I: Improved cardiac specificity and longer periods of evaluation in circulation after myocardial infarction (up to two weeks). Troponin assays supersede LD-1 or LD-1/LD-2 measurements.

Rapid response qualitative troponin T, myoglobin, or CK-MB tests: Whole blood screening tests with turnaround time less than 20 minutes. These tests provide opportunity for faster triaging of suspected myocardial infarction patients.

All of these tests are now in kit form and naturally, representatives of the kit companies tend to highlight the good points and overlook the bad points about their respective test kits. As a consequence, selecting the cardiac test that provides your hospital the best cost-effective and clinically useful indicator of myocardial infarction can be difficult and confusing.

This article will attempt to evaluate the various possibilities and recommend an approach to cardiac markers than can assist you in making the proper choices for your hospital. Note: Comments are based on results obtained in my research and development laboratory that is fortunate enough to contain the following instrument systems: Cardio-REP; CK-MB isoforms (Helena, Beaumont, Texas), ES-300; troponin-T (Boehringer Mannheim, Indianapolis, Ind.), IMx; CL-MB (Abbott, Abbott Park, Ill.), Axsym; CK-MB (Abbott), Opus; Myoglobin, troponin-I and CK-MB (Behring, Somerville, N.J.), Stratus; CK-MB, Myoglobin and troponin I (Baxter, McGraw Park Ill.) and Cardiac T Rapid Assay; troponin-T (Boehringer Mannheim).

Evaluations of cardiac markers

Myoglobin. Myoglobin is an oxygen-binding protein found in both cardiac and skeletal muscle. Its most important diagnostic feature is its early release kinetics after muscle damage (rise and fall within 6 hours after onset of myocardial infarction). Thus, it would appear to be ideal for effective triage and earlier treatment. We have found, however, that myoglobin does not perform consistently in this manner. There is not only the problem of low cardiac specificity (false-positive results in cases of trauma, renal failure, and shock) but also in cases of MI, samples collected after onset of chest pain are within a rather wide normal range (6-85 ng/mL).

One study(1) appears to have identified the problem, and this work supports the fact that sample collection time is critical, and impractical hourly collection times are needed to consistently detect abnormal myoglobin within 6 hours of chest pain. The apparent reason for this is due to the rapid release kinetics of myoglobin and a "staccato phenomenon," where myoglobin appears in multiple short bursts, often lasting only 1 to 2 hours.

A sample case is shown in Table 1 to illustrate this point. Here, myoglobin was still well within the reference range at the 2-hour collection time. At 7 hours myoglobin was about 1.7 times higher in concentration but still below the upper reference range of 85 ng/mL, whereas CK-MB exhibited an abnormal result equal to approximately two times the normal reference range of up to 5 ng/mL.

CK-MB isoenzyme

With the use of more sensitive and automated total CK assays in the 1960s, it soon became apparent that CK was not as specific an indicator for acute myocardial infarction as initially thought. Numerous reports began to appear of CK elevations resulting from noncardiac conditions such as chronic alcoholism, cardioversion, cerebrovascular disease, hypothyroidism, intramuscular injections, and surgical trauma. Thus, in the early '70s, attempts to enhance the diagnostic specificity of CK assays were focused on the isolation and identification of a cardiac-specific CK isoenzyme.

Initially the early methods for determination of cardiac CK-MB were performed by either electrophoretic or chromatographic procedures. Results from these early studies, especially from the column chromatographic reports from Montefiore University Hospital in Pittsburgh, Pa., provided quantitative CK-MB activity measurements at both high and low CK-MB levels.(2,3) This work and other reports set the stage for the discovery and production of CK-MB monoclonal antibodies.(4) This accomplishment was the key factor in the development of our current, fully automated, rapid, and cost-efficient CK-MB assays.

Now, many laboratories were demonstrating the clinical utility of CK-MB previously observed with earlier and more labor-intensive techniques (column and electrophoresis) of the late '70s and '80s. In fact, data from the new immunoassays analyzers has made it easier to obtain serial (0, 3-, 6-, 12-, 24-hour) measurements of CK-MB to identify more quickly the increasing CK-MB levels and peaking CK-MB.

Another highlight of CK-MB is its ability to detect reinfarction since it normalizes quickly (in 36 to 48 hours) after the first episode of chest pain. A sample case is shown in Figure 1.

Unfortunately, CK-MB is not the perfect cardiac marker; there are certain clinical situations where CK-MB results are very difficult to interpret.

1. Patients with chest pain and delayed arrival to hospital (after 36 hours). CK-MB is a poor market in this situation since peak values are missed and normal CK-MB levels are usually observed.

2. Patients with extreme skeletal muscle injury (perioperative or trauma). Here, total CK is frequently greater than 4,000 U/L and since trace amounts (up to 1%) of CK-MB are found in normal skeletal muscle, CK-MB values as high as 40 U/L are observed due to skeletal muscle necrosis. This frequency leads to confusion unless an MB index (CK-MB ng/mL over total CK) is calculated. Unfortunately, even with the index calculation and the use of an upper index limit of 2.5, there still remains some difficulty in differentiating between a non-cardiac and cardiac injury condition, especially when a relatively small myocardial infarction has occurred simultaneously with massive skeletal necrosis.
Table 1
Myoglobin and CK-MB in the 16 hours after an AMI


 Time of sample(*) Myoglobin CK-MB
Sample no. (hr.) (ng/mL) (ng/mL)


1 2 31 1.3
2 7 53 9.4
3 16 20 18.9


* After onset of chest pain in confirmed MI patient.


CK-MB isoforms

It is not clear that CK-MB exists in two other forms called isoforms 1 and 2. They have been shown to be a result of postsynthetic modification of the primary structure of CK-MB. CK-MB 2 originates from cardiac tissue and is named according to its relative anodic migration after electrophoresis. CK-MB 1 is a product of CK-MB 2, which results from the enzymatic action of a serum enzyme named carboxypeptidase. The proteolytic action of serum carboxypeptidase removes terminal positively charged amino acids (lysine) to produce a more negatively charged CK-MB with greater anodal mobility.

Studies(5,6) have found CK-MB isoform ratios of 1.5 or greater to be an excellent indicator for the early-stage myocardial infarction. Automated high-voltage electrophoresis is the current method of choice of CK-MB isoform separation.

There are, however, several problems with the use of CK-MB ratios obtained by electrophoretic separation and staining techniques:

1. Technical skill is required to produce precise results. This is especially the case when low total CK-MB levels are observed. This is an important point because early detection usually involves the separation of CK-MB isoforms from samples with CK-MB levels of 2 to 5 U/L.

In addition, 24-hour coverage is necessary and many different technologists are required to perform this technique. It would be interesting to see precision results over a period of time from a hospital laboratory currently using the electrophoretic system 24 hours a day. When compared to the use of automated immunoassay procedures for total CK-MB, there is no question which system would be favored by the technologies.
Table 2
Troponin T, CK, and CK-MB values following physical activity


Sample Time of sample Total CK CK-MB Troponin T
no. (hr.) (U/L) ng/mL Index (ng/mL)


1 12 693 21.1 3.0 0.01
2 36 418 13.5 3.3 0.00


2. Frequent false-positive results. Although claims that the isoform ratio test never yields false-negative results(7) appear to be acceptable, many false-positive ratios (1.7 or greater) have been observed in my laboratory. Patients with urinary tract infections, cholecystitis, pulmonary edema, congestive heart failure, urosepsis, and seizure have exhibited ratios (n=15; range 1.7 to 3.1). Other reports(8) also have demonstrated elevated isoform ratios in many types of muscle disease.

3. Limited number of samples in one run. This is another major problem, especially in large laboratories performing high volumes of CK-MBs daily. Since only five patients with one control can be performed at any one time (30 minutes), long delays in the reporting of results for CCU and ER patients would result unless multiple Cardio REPs were available and electrophoretic runs were performed frequently at 2- or 3-hour intervals.

Troponin T and troponin I

As rapid, automated immunoassays for CK-MB permitted increased use of this sensitive test for laboratory diagnosis of myocardial infarction, its limitations in certain clinical situations such as trauma and late-admission ER patients became even more apparent. Thus, attempts to develop a more specific cardiac marker, which would be present in cardiac tissue alone, began to accelerate.

Recent studies(9) have demonstrated that one of the structural components of muscle cells, the troponin complex - an important regulatory element - appears in the circulation of post-infarct patients with characteristics that provide for a more ideal cardiac marker. Numerous clinical studies of troponin T and troponin I(10, 11) have demonstrated these new cardiac markers to be as accurate and reliable a test for myocardial injury as CK-MB. Based on these reports, there are several clinical conditions regarding whether the use of the troponin assays would appear to offer several advantages over CK-MB. Some of the conditions are listed below:

1. Greater cardiac specificity. Troponin T (second-generation assay) and troponin I both possess greater cardiac specificity than does CK-MB. In addition, troponin normally is not present in serum until cardiac cell necrosis occurs, thus negligible levels of troponin usually are observed as a reference range.

Enhanced cardiac specificity allows for improved diagnosis of cardiac injury, especially in a clinical situation where severe skeletal muscle damage has occurred.

An excellent example of the clinical use of troponin T in a case where skeletal muscle necrosis was involved is shown in Table 2. Here CK-MB and CK-MB index values of 3.0 appeared at first to indicate myocardial infarction in a patient who has been involved in unaccustomed strenuous yard and garage work the day before. However, troponin T values subsequently were found to be within normal limits (less than 0.1 ng/mL), which ruled out MI. After additional CK-MB and total CK determinations, it became apparent that this individual consistently demonstrated slightly elevated CK-MB and index values.

2. Troponins remain elevated for longer periods. Both troponin T and troponin I offer a diagnostic window of five to nine days with troponin I, and up to 14 days with troponin T. Thus, sensitivity remains high long after CK-MB levels have returned to normal. This long-term delay is an advantage when dealing with patients who delay seeking medical care of myocardial infarction because of misleading symptoms.

CK-MB sensitivity for early-stage detection appears superior to the troponins based on several observed results.(12) The apparent reason for troponin's late apperance is related to its relative insolubility as a contractile protein. In contrast, CK-MB is very soluble and can penetrate the cell membrane easily.

3. Troponin T offers prognostic value in unstable angina. Recent studies(13) have shown the potential for elevated values to be predictive of poor outcomes in patients given the clinical diagnostic of unstable angina.

However, even with the above advantages provided by troponin compared to CK-MB, troponins still have several disadvantages:

* Troponins fail to detect recurrence of myocardial infarction. One advantage of troponins - their ability to detect cardiac damage up to several days - also turns out to be a major disadvantage when attempting to determine if a second or third cardiac event has occurred soon after the first episode. This situation is common and cardiologists are more confident in the ability of CK-MB to detect recurrences.

* Troponins are not as sensitive as CK-MB in early stages of infarction. As shown in Table 3, greater delta changes (incremental increases) are observed with CK-MB compared to troponin T during early-stage periods (0 to 2 hours) post-admission. This patient case is typical of many.(12) Sometimes, CK-MB above-normal values are detected 4-6 hours before troponin T elevations.

Rapid troponin T

Development of a whole blood assay for troponin T recently has been cleared by the FDA for use at the bedside in the Emergency Department or CCU to rapidly identify myocardial damage in patients with chest pain or other cardiac-related symptoms. This rapid semiqualitative assay was developed as an alternative to the slowness of the existing troponin T quantitative assays on the ES-300; the first-generation assay takes 90 minutes, the second-generation assay takes 45 minutes, and a third-generation assay - which requires a new random access analyzer and takes only 8 minutes - is in production.

A comparison between the rapid assay and the second-generation quantitative assay performed with the ES-300 demonstrated excellent correlation. Seven of seven positive samples determined on the ES-300 (range, 0.23 to 5.3 ng/mL) and 21 of 23 negative samples (range, 0.00 to 0.17) were observed. Only two false negatives were detected with the rapid assays; ES-300 determinations demonstrate one sample of 0.24 ng/mL and another at 0.58 ng/mL.

A national, multi-center trial of cardiac markers coordinated by the University of Cincinnati currently is in progress. Patients with suspected cardiac chest pain are eligible for inclusion in this study. Some 2,500 patients will be evaluated.

Initial results on the first 234 patients revealed 33 with confirmed myocardial infarction. Serial blood samples were obtained at 0, 3, and 6 hours. The rapid assay was found to have similar sensitivity when compared to the ES-300 troponin T quantitative assay at all three time points.(14)

Based on the above results, my laboratory currently is offering both the rapid troponin T assay to the ER on a routine basis and to other non-critical hospital sites by special order. The ES-300 troponin t quantitative assay is utilized to confirm rapid T assay results and to assist in the interpretation of confusing CK-MB results.

Another rapid response whole blood cardiac test strip (Spectral, Cardiac STATus, Dade International, Deerfield, Ill.) also has been introduced recently in the U.S. This device provides simultaneous, qualitative measurement of CK-MB and myoglobin in 15 minutes. My laboratory has not evaluated this product, but I would question the use of myoglobin based on the need for hourly blood sampling to detect the abnormal myoglobin peak.

[TABULAR DATA FOR TABLE 3 OMITTED]
Table 4
Recommendations for the use of cardiac markers


Patient status Choice of lab test


Acute MI CK-MB remains the test of choice for acute
(chest pain) MI. Early serial sampling every 2 to 4 hours
(early-stage) over 9 to 12 hours after arrival in the
0 to 12 hours Emergency hours Room provides adequate
 sensitivity and assists in the appropriate
 triage and management of patients.


Reinfarction CK-MB remains the test of choice because its
 normalizes sooner than troponin T (3 to 4
 days) after the first MI episode.


Acute MI Troponin T is the test of choice because
(chest pain) levels have been known to be elevated up to
(late-stage) 10 days after the onset of chest pain. Thus,
2 to 10 days troponin T is an ideal late market (better
 than lactate dehydrogenase isoenzymes) for
 diagnosing infarction in patients who are
 admitted to the hospital with
uncharacteristic
 symptoms and normal range CK-MBs.


Suspected MI In patients with extreme skeletal muscle
(perioperative injury and markedly elevated total CK (more
of trauma) than 2,000 U/L), troponin T assay appears to
 be the test of choice because troponin T is
 not influenced by massive skeletal muscle
 injury compared with CK-MB. If CK-MB levels
 are to be used in this situation, percent
 index or percent MB calculation is desirable.
 Expected ranges:


 * [less than]1:normal (MI unlikely)
 * [greater than]1 but [less than] 2.5:
 indeterminate (gray zone)
 * [greater than]2.5: probable MI


Minor myocardial In patients exhibiting unstable angina, both
injury CK-MB and troponin T are required and their
 elevation, especially troponin T, may
indicate
 accelerating myocyte damage suggesting
 impending cellular necrosis.


Thrombolytic Monitoring of serial CK-MB assists in the
non-
therapy invasive assessment of myocardial reperfusion
 after treatment. Successful reperfusion
causes
 an earlier and greater CK-MB peak.


Invasive treatments Both CK-MB and troponin T are recommended to
 evaluate side-branch (PTCA) occlusion. CK-MB
 appears sensitive to simple leakage, whereas
 troponin T reflects more severe damage to
 myocytes.


Supplied courtesy of Allegheny General Hospital


Evaluation of multiple cardiac markers as panels

Based on the documented problems regarding the use of individual cardiac markers alone, herewith is a recommendation for a multiple-marker or panel approach to achieve the ultimate goal of a clinically useful and cost-effective cardiac test.

Various cardiac combinations or panels have been suggested, such as myoglobin and CK-MB, CK-MB and LD-1/2, and CK-MB and troponin T. Previously, I was involved with the CK-MB/LD-1 panel[15] and found it to be an excellent panel during the '70s and '80s, but since the introduction of the troponins in the '90s, my laboratory has found the substitution of troponin T for LD-1 or LD-1/2 to produce enhanced specificity superior to the classic panel of CK-MB and LD isoenzymes.

With regard to myoglobin as a potential panel test, it could be surmised that any panel involving myoglobin is going to experience major problems due to rapid clearance and the staccato phenomenon described when discussing myoglobin as a single marker.

Currently at Allegheny General Hospital, a panel consisting of CK-MB and troponin T is utilized. Recommendations on how to use CK-MB and troponin T in combinations, or CK-MB or troponin T alone in different clinical situations, is shown in Table 4.

Note: Troponin T is not performed serially for the detection of early-stage myocardial infarction since CK-MB has been shown in my laboratory and by Alan H.B. Wu, Ph.D., director of clinical chemistry, Hartford (Conn.) Hospital, et al,[12] to be a better indicator of early cardiac damage. In other clinical situations such as trauma or with post-operative patients, troponin T or troponin I are the tests of choice, not CK-MB.

Instrumentation required to accomplish the above involve the use of a random access analyzer 24 hours a day to perform the CK-MB. We found CK-MB performed by the Axsym to be very precise and accurate, especially in the low-level CK-MB range of 1 ng/mL-5 ng/mL. This is an important range level, especially when attempting to determine early myocardial infarction 0-6 hours post-chest pain by examining incremental increases in CK-MB close to reference range CK-MB levels (up to 5 ng/mL).

In combination with CK-MB, we routinely use the rapid troponin T assay for ER patients after transport of the sample to the laboratory by a pneumatic tube system. To support the qualitative rapid assay and to provide troponin T for patients in non-critical hospital sites, the ES-300 quantitative assay with second-generation reagents is available.

Recently, we designed a study(16) to evaluate the use of CK-MB and troponin T in five patient groups: myocardial infarction, ventricular arrhythmias, percutaneous coronary transluminal angioplasty, unstable angina, and non-cardiac disease. Here, the use of CK-MB and troponin T in combination appeared to offer additional clinical information when compared to the use of CK-MB or troponin T alone. For example, patients experiencing serious problems with prolonged arrhythmias exhibited elevations in both CK-MB and troponin T in contrast to patients with relatively mild arrhythmias such as atrial fibrillation, when only CK-MB was elevated.

In addition, my laboratory and another(17) have demonstrated in patients undergoing angioplasty that elevations in both CK-MB and troponin T during this procedure appear to indicate an episode of severe myocardial damage or infarct. In cases where only CK-MB is elevated, simple cell leakage due to the angioplasty procedure itself appears responsible.

Several experiments comparing troponin T (second generation) and troponin I have revealed little difference between the two troponins when used to detect the possibility of myocardial infarction in patients with chest pain. Similarly, when troponin levels were evaluated in patients suffering severe renal failure, no major differences between the two assays have been observed.

In this author's opinion, CK-MB and troponin T assays in combination as a panel or used individually (See Table 4) currently are the answer to a rapid, cost efficient and effective test for the evaluation of cardiac status.

References

1. Kagen L., Scheidt S. Butt A. Serum myoglobin in myocardial infarction: The staccato phenomenon. Am J. Med. January 1977; 62: 86-92.

2. Mercer DW. Separation of tissue and serum creatine kinase isoenzymes by ion-exchange column chromatography. Clin Chem. 1974; 20: 36-40.

3. Mercer DW, Varat MA. Detection of cardiac-specific creatine kinase isoenzymes in sera with normal of slightly increased total creatine kinase activity. Clin Chem. 1975; 21: 1088-1092.

4. Vaidya HC, Maynard Y, Dietzler DN, Ladenson JH. Direct measurement of creatine kinase-MB activity in serum after extraction with a monoclonal antibody specific to the MB isoenzyme. Clin Chem. 1986: 32(4): 657-663.

5. Puleo PR, Guadagno PA, Roberts R. Perryman B. Sensitive, rapid assay of subforms creatine kinase MB in plasma. Clin Chem. 1989: 35: 1452-1455.

6. Puleo PR, Guadagno PA, Roberts R, Scheel MV. Early diagnosis of acute myocardial infarction based on assay for subforms of creatine kinase-MB. Circulation. 1990; 82: 759-764.

7. Puleo PR, Meyer D, Wathan C, et al. Use of a rapid assay of subforms of creatine kinase MB to diagnose or rule out acute myocardial infarction. N Eng J Med. September 1, 1994; 331(9): 561-566.

8. Wu AHB, Wang XM, Gornet TG, Ordonez-Llanos J. Creatine kinase MB isoforms in patients with skeletal muscle injury: Ramifications for early detection of acute myocardial infarction. Clin Chem. 1992; 38(12): 2396-2400.

9. Katus HA, Scheffold T, Remppis A, Zehlein J. Proteins of the troponin complex. Lab Med. May 1992; 23(5): 311-317.

10. Gerhardt W, Katus H, Ravkilde J, et al. Troponin T in suspected ischemic myocardial injury compared with mass and catalytic concentrations of S-creatine kinase isoenzyme MB. Clin Chem. 1991; 37(8): 1405-1411.

11. Adams JE, Schechtman KB, Landt Y, Ladenson JH, Jaffe AS. Comparable detection of acute myocardial infarction by creatine kinase MB isoenzyme and cardiac troponin I. Clin Chem. 1994: 40(7): 1291-1295.

12. Wu AHB, Valdes R, Apple F, et al. Cardiac troponin T immunoassay for diagnosis of acute myocardial infarction. Clin Chem. 1994; 40(6): 900-907.

13. Hamm CW, Ravkilde J, Gerhardt W, et al. The prognostic value of serum troponin T in unstable angina. New Eng Jour of Med. July 16, 1992; 327: 146-150.

14. Baxter MS, Gibler WB, Brogan GX, et al. Bedside whole blood rapid assay of cardiac troponin T: A sensitive indicator of acute myocardial infarction. Annals of Emergency Med. May 1996; 13: 411.

15. Mercer DW. Simultaneously separation of serum creatine kinase and lactate dehydrogenase isoenzymes by ion-exchange column chromatography. Clin Chem. 1975; 21: 1102-1106.

16. Mercer DW, Pickeral J, Harbison P. Clinical evaluation of panel testing for myocardial injury using CK-MB and troponin T. Clin Chem. 1995; 41: S233.

17. Talasz H, Genser N, Mair J, et al. Side-branch occlusion during percutaneous transluminal coronary angioplasty. Lancet. 1992; 339: 1380-1382.

DONALD W. MERCER, PH.D., head of chemistry for Allegheny General Hospital in Pittsburgh and professor of pathology at Allegheny General Hospital campus of the Medical College of Pennyslvania and Hahnemann University.
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Author:Mercer, Donald W.
Publication:Medical Laboratory Observer
Date:Jul 1, 1996
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