Use of a Rapid Assay of Subforms of Creatine Kinase MB to Diagnose or Rule Out Acute Myocardial Infarction

Study Design

Patients evaluated in the emergency room of Ben Taub General Hospital in Houston for chest pain occurring at rest during the preceding 24 hours and lasting at least 30 minutes were eligible for enrollment. All patients were required to sign consent forms of the Baylor College of Medicine Institutional Review Board before enrolling in the study. Patients suspected of having myocardial infarction were monitored for cardiac rhythm, if necessary, in the emergency room. Enrollment was consecutive, with the exception that patients who needed intravenous vasopressor therapy or mechanical ventilation or had been resuscitated after cardiac arrest were excluded.

Blood samples were obtained every 30 to 60 minutes until at least 6 hours had elapsed since the onset of chest pain; among patients subsequently admitted to the hospital, blood samples were obtained every 4 hours for up to 48 hours. The samples were collected in vacuum tubes pretreated with preservatives, as previously reported¹¹. The assay for plasma MB₂ and MB₁ activity was performed with rapid high-voltage electrophoresis on an automated analyzer (Rep, Helena Laboratories, Beaumont, Tex.)¹¹; the performance time was 25 minutes. CK-MB subforms to be used as standards were initially purified from human myocardium¹⁵ and generated with the use of lysine carboxypeptidase, but more recently, they have been produced in recombinant form¹⁶. Blood samples were analyzed for total CK-MB activity by the hospital laboratory with the quantitative glass-bead assay,^17,18 which has an upper limit of normal of 14 U per liter.

At the discretion of the physicians in the emergency room, patients were admitted to the coronary care unit or a non-coronary care medical unit or were discharged home. Those with abnormal electrocardiograms and a history of chest discomfort consistent with myocardial ischemia were generally admitted to the coronary care unit. Patients with pain suggestive of myocardial ischemia, a normal electrocardiogram, and no history of documented coronary disease were admitted to an intermediate-care unit for continuous electrocardiographic monitoring. Patients with pain thought to be noncardiac in origin were admitted to a general medical unit or sent home at the discretion of the emergency room physicians. The diagnostic evaluation and care of all patients were conducted by physicians who were unaware of the results of the assays for CK-MB subforms, and the subform assays were performed by personnel who were unaware of the clinical outcomes in the patients and other clinical data, including the results of the routine assays of total CK-MB. Among patients admitted to the hospital, the final clinical diagnosis was determined by the team of the coronary care unit or, in the case of patients admitted to the general medical unit, by a cardiology consultant. The type of infarction (Q wave or non-Q wave) was determined from the electrocardiogram^19,20.

For each patient, the results of the assay for CK-MB subforms and the conventional assay of total CK-MB were analyzed separately and independently by at least two of the investigators without knowledge of the clinical findings. The diagnosis of myocardial infarction was confirmed or excluded solely on the basis of abnormal or normal total CK-MB activity, respectively, according to criteria that have been outlined previously^18,21. The criteria for myocardial infarction were as follows: a CK-MB level ≥ 14 U per liter in two or more sequential blood samples obtained 6 to 12 hours apart; a CK-MB level ≥ 14 U per liter in a single sample, if the value represented a threefold increase above the previous value; and a single CK-MB value ≥ 14 U per liter if only one sample was analyzed. In all the patients with myocardial infarction, except the three who died early, serial blood samples were obtained over a period of 24 to 48 hours. The diagnostic criteria for myocardial infarction, based on the subform levels, were an MB₂ level ≥ 1.0 U per liter and a ratio of MB₂ to MB₁ ≥ 1.5, occurring within six hours after the onset of symptoms¹². The diagnosis of myocardial infarction by subform analysis was confirmed if one or more samples obtained during the six-hour interval met the subform criteria and was excluded if none of the samples obtained during that interval met the subform criteria.

Statistical Analysis

The statistical significance of differences in the time from the onset of symptoms to arrival in the emergency room between patients with myocardial infarction and patients without myocardial infarction was determined by the (two-sample) Wilcoxon test²². The significance of differences in the proportions of male patients in the group with myocardial infarction and in the group without infarction was determined by chi-square analysis²². Exact confidence intervals for binomial proportion were calculated for sensitivity and specificity²². Confidence intervals for the relative risk of myocardial infarction were determined from the log transformation of relative risk²³. The receiver operating characteristics were calculated as previously described²². All P values are based on two-sided comparisons.

During the 14-month study period, 5741 blood samples were obtained from 1110 consecutive patients evaluated in the emergency room because of chest pain that had occurred within 24 hours before their arrival. The diagnosis of myocardial infarction was confirmed in 121 of the patients. A total of 531 patients (47.8 percent) were admitted to the coronary care unit; 118 received a subsequent diagnosis of myocardial infarction confirmed by serial elevation of plasma CK-MB levels; in the other 413 patients, myocardial infarction was ruled out. Of the 129 patients (11.6 percent) admitted to an inpatient unit other than the coronary care unit, 3 subsequently received a diagnosis of myocardial infarction. A total of 450 patients (40.5 percent) were discharged home from the emergency room. Thus, 121 patients received a subsequent diagnosis of myocardial infarction (65 with Q-wave infarction and 56 with non-Q-wave infarction), of whom 118 were admitted to the coronary care unit, accounting for 22.2 percent of all admissions to the coronary care unit during the study period.

Table 1. Table 1. Myocardial Infarction, Death, or Serious Complications in Patients with Chest Pain, According to Sex, Age, and the Interval from the Onset of Pain to Arrival in the Emergency Room.

Fifty-nine of the 65 patients with Q-wave infarction presented with typical ST-segment elevation; 4 of the 56 patients with non-Q-wave infarction presented with ST-segment elevation. The other patients with non-Q-wave infarction presented with either ST-segment depression or T-wave inversion. The sex and mean age of the patients are shown in Table 1. Patients with myocardial infarction presented significantly earlier after the onset of symptoms than did the patients without myocardial infarction (P<0.001) (Table 1).

Table 2. Table 2. Sensitivity and Specificity of the Assay of CK-MB Subforms.

Among the patients with myocardial infarction, the overall sensitivity of the CK-MB subform assay to detect infarction within six hours after the onset of symptoms or in the first available blood samples was 96.6 percent (114 of 118 patients; 99 percent confidence interval, 90 to 99 percent) (Table 2), as compared with a sensitivity of 48 percent for the conventional CK-MB assay. In this group of patients, the mean (±SD) CK-MB₂ level at six hours was 5.1 ±3.2 U per liter, and the mean ratio of CK-MB₂ to CK-MB₁ was 4.4. For 10 of the patients with myocardial infarction, the time of the onset of symptoms was unclear, but all 10 had elevated plasma CK-MB subforms in their first blood samples. Sixteen patients with myocardial infarction came to the emergency room more than six hours after the onset of symptoms, and each of them had a positive test for plasma CK-MB subforms in the first blood sample obtained. Ninety-five of the patients with myocardial infarction came to the emergency room within six hours after the onset of symptoms. Three of these 95 patients died before six hours had elapsed; their subform activity was normal, but the CK testing was incomplete. In three other patients, plasma MB₂ activity was not elevated until 6.5, 7.0, and 8.3 hours after the onset of symptoms. False negative results were obtained in one patient in whom chest pain developed and a new ST-segment elevation occurred more than 12 hours after admission, with the subsequent development of a Q-wave myocardial infarction. Thus, a total of 92 patients with myocardial infarction admitted within six hours after the onset of symptoms had complete CK testing throughout the interval, and the subform assay was positive in 88 of these patients (95.7 percent) within this six-hour interval.

Figure 1. Figure 1. Receiver-Operating-Characteristic Plots for Total CK-MB and for Its Subforms Four Hours (Upper Panel) and Six Hours (Lower Panel) after the Onset of Pain.

The arrows indicate the diagnostic cutoff values determined in advance on the basis of previous studies (a CK-MB level of 1.0 U per liter, a CK-MB:CK-MB ratio of 1.5, and a total CK-MB level of 14 U per liter). At both four and six hours, the assay of CK-MB subforms was more reliable in detecting infarction than the conventional assay of total CK-MB.

Figure 2. Figure 2. Comparison of the Sensitivity of the Assays of Total CK-MB (Light Bars) and Its Subforms (Dark Bars) Two, Four, and Six Hours after the Onset of Symptoms.

The diagnosis of myocardial infarction (MI) in the study patients was confirmed by the total plasma CK-MB activity during the subsequent 48 hours.

We determined the sensitivity and specificity using a ratio of CK-MB₂ to CK-MB₁ ≥ 1.5 with a CK-MB₂ level ≥ 1 U per liter. Although this ratio was selected on the basis of previous data,¹² receiver operating characteristics were analyzed for a ratio of CK-MB₂ to CK-MB₁ ranging from 0.75 to 3.0 and an absolute CK-MB₂ value ranging from 0.6 to 3.0 U per liter. The sensitivity was plotted against 1 minus the specificity at each diagnostic cutoff point. A similar plot was calculated for total CK-MB ranging from 5.0 to 17.0 U per liter. The receiver operating characteristics were analyzed for results four and six hours after the onset of symptoms, and at each interval the area under the curve was calculated. The assay for CK-MB subforms was consistently more sensitive than the assay for total CK-MB, with equal or greater specificity over a wide range of diagnostic cutoff points (Figure 1). The sensitivity was 95.7 percent and the specificity 93.9 percent with a ratio of CK-MB₂ to CK-MB₁ of 1.5 at six hours, which represents the best diagnostic cutoff value at that time. In contrast, when the same cutoff value was used four hours after the onset of symptoms, the sensitivity was only 56 percent, with a specificity of 93 percent. A ratio of 0.75 with a CK-MB₂ level of 1.0 U per liter resulted in a sensitivity of 69 percent and a specificity of 91 percent. Figure 2 compares the sensitivity of the conventional CK-MB assay with that of the subform assay two hours after the onset of symptoms in the patients with myocardial infarction (0 percent [0 of 24] and 8.3 percent [2 of 24], respectively), four hours after the onset of symptoms (22.7 percent [17 of 75] and 56.0 percent [42 of 75], respectively), and six hours after the onset (47.8 percent [44 of 92] and 95.7 percent [88 of 92], respectively).

Among the patients with myocardial infarction, the average interval from the arrival in the emergency room until plasma CK-MB subform activity was elevated was 1.22 ±1.17 hours. Of the 88 patients with abnormal CK-MB subforms within six hours after the onset of symptoms, 50 had abnormal subform activity in the first blood sample, which was obtained a mean of 3.25 ±1.70 hours after the onset of symptoms. In the other 38 patients, CK-MB subforms were normal in the first plasma sample (2.37 ±1.07 hours after the onset of symptoms) and became elevated a mean of 4.22 ±1.13 hours after the onset of symptoms. Of the 44 patients who died or had a serious complication, 32 had myocardial infarction. Most of the patients who died (13 of 18) had presented within six hours after the onset of symptoms (Table 1).

Myocardial infarction was ruled out by the conventional assay of plasma CK-MB in 539 patients admitted to the hospital. The test of plasma CK-MB subforms was abnormal in only 33 of these patients (specificity, 93.9 percent; 99 percent confidence interval, 91 to 96 percent); 26 (78.8 percent) had unstable angina; of the other 7 patients (21.2 percent), 2 had rhabdomyolysis, 2 had hypothyroidism, and in 3 the diagnosis was unknown.

The assay of CK-MB subforms was negative in 432 of the 450 patients (96 percent) sent home from the emergency room (18 patients left the emergency room before six hours had elapsed since the onset of symptoms). The activity of plasma CK-MB subforms was abnormal in 17 of the 450 patients sent home (specificity, 96.2 percent; 99 percent confidence interval, 93 to 98 percent). Four patients were readmitted to the Ben Taub General Hospital and two were admitted to another hospital within 36 hours after their initial discharge; all six of these patients had elevated total CK-MB activity and were given the diagnosis of non-Q-wave infarction. Seven patients had no other symptoms and appeared to do well. Four patients could not be reached. Thus, the estimated false positive result of 3.8 percent is probably an overestimate, but in view of the time lapse, we could not be certain that the two sets of CK-MB values represented the same clinical event, so no changes were made in the estimate for false positive results. It is also possible that the sensitivity of the assay in patients who were discharged home was overestimated, since we had no follow-up data on patients with normal plasma CK-MB subforms who were discharged home.

For the total study population, the risk of myocardial infarction within six hours after the onset of symptoms was 164 times greater (99 percent confidence interval, 45 to 598) if the subform test was positive than if it was negative and 99 times greater (99 percent confidence interval, 27 to 359) if the patient was admitted to the hospital. The risk of death among hospitalized patients with normal plasma CK-MB subforms six or more hours after the onset of symptoms was 0.004 (99 percent confidence interval, 0.0002 to 0.018, unadjusted for the length of the hospital stay).

With an admission policy whereby patients with elevated plasma subform activity were admitted to the coronary care unit and those with normal plasma subform activity for six hours after the onset of symptoms were admitted to a non-coronary care unit, 114 of the 118 patients (96.6 percent) with myocardial infarction would have been admitted to the coronary care unit, and the total number of admissions to that unit would have been reduced from 531 to 164, a 69.1 percent reduction. Seventy percent of the patients admitted to the coronary care unit under this policy would have had myocardial infarction, as compared with 22.2 percent with the use of standard clinical criteria.

We found that the sensitivity and specificity of a rapid assay of CK-MB subforms to diagnose myocardial infarction within six hours after the onset of symptoms were 95.7 and 93.9 percent, respectively, as compared with 48.2 and 94.0 percent, respectively, for the conventional CK-MB assay. The emergency room physicians, who were unaware of the results of the subform assay, discharged 40.5 percent of the patients home, admitted 47.8 percent to the coronary care unit, and admitted 11.6 percent to the non-coronary care unit. The frequency of myocardial infarction was 10.9 percent (121 patients), which is similar to that reported in the emergency rooms of both university and community hospitals^2-6. Among the 450 patients discharged home, only 3.8 percent had abnormal plasma subform activity, and of the 539 patients admitted to the hospital without myocardial infarction, only 6.1 percent had abnormal plasma subform activity, of whom 79 percent had a diagnosis of unstable angina at discharge. Of the 17 patients sent home with abnormal subform activity, 6 were subsequently found to have myocardial infarction. It is possible that several of the patients with unstable angina who were admitted to the hospital had small areas of myocardial necrosis that were below the threshold of detection of the conventional assay²⁴. However, the possibility that very small quantities of CK were released because of transient ischemia cannot be excluded^25,26.

In this study the sensitivity (95.7 percent) and specificity (93.9 percent) of the subform assay indicate that it accurately identifies patients with myocardial infarction within the first six hours after the onset of symptoms. Based on high-voltage (1450 V) rather than conventional (150 V) electrophoresis, this assay is simple and highly reproducible, and it separates the subforms in six minutes, as compared with the conventional assay, which takes one hour¹¹. The subform assay can be performed in any community hospital at a cost similar to that of standard isoenzyme electrophoresis and requires altogether only 25 minutes. An increase in the ratio of plasma CK-MM₃ to plasma CK-MM₁ isoforms or in the level of plasma myoglobin has also been documented in the early hours of myocardial infarction^27-29. However, both markers are present in skeletal muscle, and their elevation in the blood is not as specific as CK-MB for detecting myocardial injury^30,31. The assay of CK-MB subforms would also be expected to be falsely positive in patients with muscular dystrophy³² or severe skeletal-muscle damage,³³ such as rhabdomyolysis, as we observed in two patients in our study. Although one group of investigators has reported three subforms of CK-MB,³⁴ we have detected only two subforms in human plasma. The early detection of myocardial infarction by sensitive nonradioactive immunoassays has also been reported,³⁵ although others have not found these assays to be useful for the diagnosis of myocardial infarction in the emergency room³⁶.

If one were to use a system of triage based on the results of the assay for CK-MB subforms obtained within six hours after the onset of symptoms and to assume that all patients with negative results, even those with unstable angina, could be adequately cared for in a regular hospital unit, the number of patients admitted to the coronary care unit would be reduced by nearly 70 percent. Since it costs an estimated $4 billion per year to care for patients in the coronary care unit who do not have myocardial infarction, admission to a regular hospital unit would reduce the cost substantially. The cost of the subform assay is the same as that of the conventional CK-MB assay, but since testing is discontinued at six hours, this portion of the cost, albeit minor, would be reduced. Several studies have shown that the incidence of death or serious complications before discharge is low in patients in whom myocardial infarction has been ruled out,^7,8,37 including those with unstable angina. Exclusion of the possibility of myocardial infarction has not been feasible in practice, since the clinical history and electrocardiographic findings lack specificity⁹ and conventional markers require a delay of up to 12 hours after the onset of symptoms for a reliable diagnosis of myocardial infarction^9,10,12,38. Another approach has been the establishment of a special unit for the purpose of ruling out myocardial infarction,³⁹ necessitating additional dedicated beds. Such a unit, used in combination with the assay of CK-MB subforms, would probably be efficient and cost effective.

The protocol proposed here requires no additional facilities or staff. One concern is the time patients may have to remain in the emergency room; however, this was not a problem among our patients with myocardial infarction, most of whom received the diagnosis within two hours after their arrival in the emergency room (within one hour in 57 percent). It should be emphasized that patients who have ST-segment elevation and are otherwise candidates for thrombolytic therapy should receive such therapy immediately. Among patients without myocardial infarction, a negative subform assay is not reliable unless the blood sample is obtained at least six hours after the onset of symptoms. In this study patients without myocardial infarction arrived at the emergency room much later than those with myocardial infarction; consequently, among the patients without infarction, the diagnosis was ruled out within 2 hours after their arrival (mean, 1.18 hours).