Evaluation of combined biochemical markers in the diagnosis of acute coronary syndrome in Nineveh governorate

Objective: To evaluate the importance of combination of biochemical markers in patients with Acute Coronary Syndrome (ACS) and to determine the optimal biochemical strategy for highly sensitive, early diagnosis of myocardial injury. Patients and methods: This study was carried out in coronary care unit in Ibn- Sena Teaching Hospital in Mosul city from January to November, 2008. Two hundred and forty nine patients with (ACS) presented with chest pain and one hundred and eleven apparently healthy subjects. Four cardiac markers Creatine Kinase (CK) and Creatine Kinase-MB (CK-MB) activities (markers of necrosis), myoglobin (marker of muscle injury), and troponin I (marker of necrosis) were estimated in addition to CK-MB index. The collected data were analyzed by chi square, unpaired t-test and analysis of variance (ANOVA). Receiver Operating Characteristics (ROC) analysis was used to assess the role of selected parameters in the diagnosis of ACS and to determine optimal cut-off values for all biochemical markers. Results: The optimal cut-off value of each of 5 parameters with reasonable validity were used to define single test criteria. The serum troponin I was with highest validity among other parameters. The serum troponin I was used in combination with each of the remaining 4 criteria and the test performance was assessed. All combinations were associated with higher specificity than that of serum troponin I alone. However, the accuracy of serum troponin I alone 94.0% was higher than the accuracy of all other combinations. Conclusion: The study has suggested that combining troponin I, myoglobin and CK-MB index yields satisfactory diagnostic sensitivity and thus provide valuable information for clinicians in managing Acute Coronary Syndrome (ACS). myoglobin, Creatine Kinase (CK), Creatine Kinase-MB (CK-MB), CK-MB index, acute coronary syndrome, biochemical markers.

Biochemical marker identification which is sensitive and specific for myocardial ischemia and can easily and rapidly measured in serum would be clinically valuable 3 . Elevated levels of cardiac markers: Creatine kinase (CK-MB) activities (marker of necrosis), in addition to troponin I (marker of necrosis), and myoglobin (marker of muscle injury), could be useful in early diagnosis of acute coronary syndrome when patients admitted to coronary care unit (CCU) 3 .
Use of the combination of a marker that appears early (myoglobin) and either Creatine Kinase CK-MB isoenzyme (CK-MB) or cardiac troponin I (TnI) may facilitate rapid exclusion of AMI and enable discharge of patients who do not require prolonged observation 4 . Myoglobin is advantageous because it appears 1 to 2 hours after symptom onset, and studies have demonstrated its high sensitivity for detection of AMI within the first few hours after presentation (4) . However, use of myoglobin alone has significant limitations. Myoglobin has low specificity for cardiac necrosis in patients with renal failure or skeletal muscle trauma 5 . Also, given that serum myoglobin rises and falls quickly in AMI, a single measurement at presentation may be normal for patients who present early and who present 24 hours after symptom onset 6 . On the other hand, CK-MB and cTnI appear 3 to 6 hours after symptom onset and remain elevated for S 24 to 36 hours and 7 to 10 days, respectively 7 . However, these markers must be ordered as a panel in the ED because no single marker meets all criteria for an ideal marker of AMI diagnosis for all patients who arrive at the ED at various times after the onset of the symptoms 8 .
The aim of this study was to evaluate the importance of combination of biochemical markers in patients with Acute Coronary Syndrome (ACS) and to determine the optimal biochemical strategy for highly sensitive, early diagnosis of myocardial injury.

Subjects and methods
This study was carried out in Ibn-Sena Teaching Hospital in Mosul from January 2008 to November 2008. It was carried out on 249 patients with Acute Coronary Syndrome (ACS) who were presented with chest pain where 193 (77.5%) patients with a diagnosis of possible Acute Myocardial Infarction (AMI) and 56 (22.5%) patients with Unstable Angina (UA) . A control group that includes 111 apparently healthy subjects who attended the Out-Patient department was chosen for comparison. None of the control subjects had any chronic diseases and they were not taking regular medicine. All patients who admitted to the CCU were with provisional diagnosis of ACS. The patients were looked for risk factors, "Smokers" were defined as patients currently smoking at the time of admission; Hypertension was defined by self-report of a diagnosis and use of an anti-hypertensive medication, or if systolic blood pressure > 140 mmHg or if diastolic blood pressure> 90 mmHg 9 ; "Diabetes" as patients on insulin or taking oral hypoglycemic agent 10 ; " Hypercholesterolemia " as total cholesterol of >5.0 mmol/L on admission 11 . A family history for CHD is considered positive if relatives have experienced an MI prior to the age of 50 in men, and 55 in women 1 .
History of ischemic heart diseases is defined as any group of acute or chronic cardiac disabilities resulting from insufficient supply of oxygenated blood to the heart, or is a group of diseases characterized by reduced blood supply to the heart muscle usually due to coronary artery disease ( atherosclerosis of coronary arteries) 11 .
Male gender and obesity were also considered as risk factors 1 .
All patients had cardiac markers tested within 12 hours post chest pain 12 . Their ECG was assessed and any ischemic or progressive changes were documented 12 . Anthropometric Measurements were also taken. The socioeconomic state was grouped into low, medium and good. The study was carried out with the cooperation of senior cardiologist in Coronary Care Unit (CCU). On admission, patients satisfying two out of the three criteria of WHO were considered as having myocardial infarction 2 . The definitive diagnosis of myocardial infarction required all three criteria to be satisfied. Patients with chest pain and non-Q ECG pathology but with no changes in cardiac enzymes were diagnosed as having unstable angina 13 .
The case notes were analyzed and More than 30% of patients had more than four risk factors ( Table 2).

Using Test Combinations
At time of admission to coronary care unit, the combination of serum troponin I, myoglobin, CK activity, CK-MB activity and CK-MB index was performed. The optimal cut-off values determined by selecting the point on each curve at maximum curvature in ROC curve for each of 5 parameters with reasonable validity ( Figure 1).
As shown in Table 3. serum troponin I was the most valid test when used alone in differentiating MI from unstable angina cases. The optimal cutoff value of each of 4 tests with reasonable validity were used to define single test criteria. After that, combination test criteria were used to improve the specificity of diagnosis by considering a subject as positive if the test positive on both criteria, otherwise the subject will be considered negative.
The serum troponin I was used in combination with each of the remaining 4 criteria and the test performance was assessed. All combinations were associated with higher specificity than that of serum troponin I alone. However, the accuracy of serum troponin I alone 94.0% was higher than the accuracy of all other combinations (Table 4). In this study, a large proportion of unstable angina ( 33 patients from 89 unstable angina patients), which is 13.2% of total (249 ACS patients), would be reclassified as having acute myocardial infarction due to utilizing cardiac markers. All combinations were associated with higher or equal specificity than that of serum troponin I alone (91.1%), while the accuracy of serum troponin I alone (94.0%) was higher than the accuracy of all other combinations.

Cardiac Biochemical Markers Combination
Triple combination of TnI, myoglobin with CK-MB/CK Ratio offered a high improvement over the combination of TnI and myoglobin testing recording (triple panel sensitivity, 91.2%; specificity, 96.45% and highest accuracy 92.4%) for diagnosis of myocardial infarction which means the best combination among others (Table  4).

Discussion
The National Academy of Clinical Biochemistry (NACB) has recommended that the protocol for using cardiac markers in evaluation of patients with possible ACS should include an" early marker such as myoglobin or CK-MB, which is reliably increased in the blood within 6 h after symptoms onset, and a "definitive" marker ( such as TnI or TnT), which is increased in the blood after 6-9 h with a high sensitivity and specificity for myocardial injury and remains with abnormal levels for several days thereafter 19 . The use of combination of marker that appears early [myoglobin] and either CK-MB or cardiac troponin I may facilitate rapid exclusion of MI and enable discharge of patients 20 .
As for diagnostic purposes, an algorithm applying a panel of biochemical markers reflecting different aspects in ACS appears to be a promising option for improving risk stratification 21 .
In contrast multimarker strategies incorporating CK-MB or myoglobin in combination with troponin I results have only been evaluated by few authors [20][21][22][23][24] . However, it makes intuitive sense that combinations of two or more cardiac biomarkers increase the early predictive value of these types of strategies 25 .
In this study, four markers were used in addition to CM-MB index as criteria that favor the diagnosis and differentiation of MI from UA in cases of ACS, when equal to or higher than an optimal cut-off value. When the subject is positive in at least three of these tests he is considered to have diagnosis of AMI. Troponin I sensitivity was the highest one among other parameters with (99.4%) negative predictive value. But however, at these cut-off values, the diagnostic predictive value for combination of two tests was less significant (Table 4).
In the present study, a combination of TnI and CK-MB index can achieve a diagnostic sensitivity of 93.8% for AMI with a 99.3% negative predictive value at time of admission to CCU, which is the highest diagnostic sensitivity among other combination. This is consistent with the study of Engel and Rockson where the sensitivity of combination of TnI and CK-MB index was 90.6% at time of presentation 24 .
However, the combination of myoglobin and TnI can achieve a diagnostic sensitivity of 88.1% for AMI with a 98.6% negative predictive value, at presentation to CCU which similar to the results of others 23,27,28 . Thus, the combination of a highly sensitive marker such as myoglobin with a highly specific marker such as troponin might optimize diagnostic accuracy.
The triple combination of the measured parameters when used to diagnose patients with ACS showed combination of myoglobin,troponin I and CK-MB ratio recorded high sensitivity 91.2% with a specificity of 96.45% and highest accuracy 92.4% and high positive predictive value 99.6%. These results are in line with the study of Hsu et al where a triple marker panel of myoglobin, TnI and CK-MB had a sensitivity of 93% and specificity of 95% and the study of Rathore et al which showed a sensitivity of 85.7% and specificity of 96.5% and positive predictive value 92.3% for the same combination 20,29 .
Most studies have compared only two or three markers at the same time and have not compared them with the initial electrocardiogram.
In conclusion, no single biochemical marker can be used to predict diagnosis in AMI patients, therefore, it is conceivable that combination of biochemical markers may help for this purpose. Combination of Troponin I, myoglobin and CK-MB index can achieve the best diagnostic yield.