Laboratory Diagnosis of Cardiac Disease

# OBJECTIVES

By the end of this session the reader should be able to:

• To understand the role of the laboratory in the diagnosis of acute myocardial infarction
• To understand the time frame in which various protein markers of cardiac damage should be monitored
• To understand the differences in the various cardiac markers (such as the use of serum cardiac troponins I and T, serum creatine kinase MB, serum myoglobin) in terms of sensitivity and specificity
• Describe the time course for elevation of serum myoglobin, creatine kinase MB, and the cardiac troponins following acute myocardial infarction
• Describe at least five other (i.e., non-acute myocardial infarction) conditions in which serum creatine kinase MB may be elevated
• Calculate the creatine kinase index, discuss its use, and interpret the results
• To understand the role of the laboratory in the diagnosis and monitoring of congestive heart failure

# KEY TERMS

Acute myocardial infarction (AMI) - blockage of circulation to heart tissue causing necrosis; frequently associated with pallor, perspiration, substernal chest pain radiating down the arm, nausea, dyspnea, dizziness and ECG changes

Acute coronary syndrome (ACS) - a wide range of acute heart conditions including ST-segment elevation myocardial infarction, non-ST-segment myocardial infarction, and unstable angina

Cardiac markers - proteins used to monitor damage to cardiac tissue, typically cTnI, cTnT, CKMB, and myoglobin

Cardiac troponin T (cTnT) - protein released from myocardium due to tissue damage (cardiac specific)

Cardiac troponin I (cTnI) - protein released from myocardium due to tissue damage (cardiac specific)

Congestive heart failure - cardiac dysfunction syndrome caused by insufficient cardiac output to meet perfusion needs of the patient, characterized by breathlessness, abnormal sodium and water retention, and often edema

Creatine kinase (CK) - enzyme released from variety of muscle and other tissues indicating non specific tissue damage

Creatine kinase MB (CKMB) - CK enzyme found in highest concentration in heart, also in other tissue

Dypsnea - difficult or labored breathing

Isoenzyme - a group of different proteins (different sequence) that have the same function

Myoglobin - protein released from multiple sources indicating non specific tissue damage

Natriuretic peptides - the two main biomarkers used in the diagnosis of heart failure are
B-type natriuretic peptide (BNP) and its amino terminal-related fragment NT-proBNP; myocardial stretch causes elevations of these peptides which are diagnostic and prognostic in the setting of heart failure

# BACKGROUND/SIGNIFICANCE

It is estimated that about 10 million people present to the emergency department in the US each year with complaints of chest pain. Of these about 6 million people are admitted for workup of AMI and 1.5 million people rule in as having an AMI. Unfortunately about 11,000 of the remaining patients are discharged with an undiagnosed AMI [1, 2] .

# ACUTE MYOCARDIAL INFARCTION

Following ischemic injury to cells, cell membrane integrity is damaged allowing intracellular constituents to leak into the blood stream. Important parameters that determine utility of a cellular component as a marker for ischemic cardiac damage include:

• Intracellular location; cytosolic components generally reach bloodstream faster than structural components
• Size; smaller components tend to reach bloodstream faster than larger components
• Tissue specificity; markers that are specific for cardiac tissue provide increased specificity of the assay

A variety of proteins are used to detect myocardial damage. The choice of which cardiac markers to analyze, depends on the time elapsed since the suspected beginning of the infarction, which is usually taken as the time of the onset of chest pain or discomfort. Diagnostic problems arise because many of these substances circulate in the blood, albeit at lower levels, in health.

Currently the following proteins are monitored when evaluating patients for myocardial infarction:

Table 1. Cardiac Markers
Marker Reference Range
Cardiac troponin I (cTnI) <0.07 ng/mL *
Cardiac troponin T (cTnT) < 0.1 ng/mL *
Creatine kinase MB isoenzyme (CKMB) <10 ng/mL
Myoglobin <170 ng/mL (>25% increase over 90 min. suggests AMI)

*As assays become more sensitive and specific the reference range for troponins continues to decrease. Values greater than 99th percentile of the upper reference limit (greater than 99th percent of the normal population) should be considered abnormal. [3]

Figure 1: Release of Cardiac Markers following AMI (courtesy of A.H. Wu)

Table 2: Protein Elevations in Serum after MI
Serum Protein Time After Infarction: 1st Elevation (hours) Time After Infarction: Peak Elevation (hours) Duration of Elevation (days)
CK 4-6 18-36 3(~2-3xnormal only)
Myoglobin 2-3 6-9 1
Troponin I & T 4-6 24-36 depends on extent of damage

Definition of acute, evolving or recent MI [4]

1. Rise and fall of troponin or CKMB with at least one of the following:
• Ischemic symptoms
• Q waves
• ECG changes (increase or decrease of ST segment)
• Coronary artery intervention (e.g., coronary angioplasty)
2. Pathological findings of AMI

## Troponin

The diagnosis of AMI relies heavily on the analysis of troponins. Of the markers currently available, cTnI and cTnT offer the highest degree of cardiac specificity.

Troponin is a regulatory protein complex located on the thin filament of the contractile apparatus and consists of 3 protein subunits: troponin T, troponin I and troponin C which act to regulate muscle contraction. Troponin T binds to tropomyosin, Troponin I inhibits myosin ATPase, and Troponin C binds to calcium.

Unlike other cardiac markers that are used to detect cardiac damage, cTnI and cTnT have different isoenzymes from those found in skeletal muscle, and thus they are specific for cardiac injury. In addition to detecting an AMI, elevations of either cTnT or cTnI have prognostic value. Marginally elevated concentrations of cTnT and/or cTnI are associated with future adverse cardiac events.

It is important to realize that currently there are many different cTnI assays and reference ranges vary widely depending on who manufactures the assay. At the VA hospital cTnI values between 0.07 and 0.20 ng/mL suggest myocardial ischemia or early infarction serial testing is indicated. Values greater than 0.2 ng/mL are consistent with MI, indicating the patient should undergo serial testing. Reference range: < 0.07 ng/mL.

Since there is only one assay available for cTnT, the reference ranges have less variability between laboratories. Serum cardiac troponin T > 0.1 ng/mL suggests myocardial damage, an MI, or high risk for future myocardial events. Many end-stage renal failure patients on hemodialysis (60%) have elevated cTnT and/or CTnI, which does not represent an AMI, but is associated with future adverse cardiac events. The percentage of end stage renal failure patients on dialysis that are cTnI positive is less than that observed with cTnT.

Figure 2: Structure of Troponin Complex

Elevations of cardiac troponins are diagnostic of heart tissue injury but do not always indicate myocardial infarction. In the setting of ischemic symptoms a rise and fall indicated myocardial infarction, but elevations can be caused by other factors such as heart failure, drug toxicity, myocarditis, etc.).

## Creatine Kinase (CK) Isoenzymes in Myocardial Infarction

CK (molecular weight 86 kilodaltons (KD)) is an enzyme responsible for the conversion of creatine into phosphocreatine, the energy source for muscle contraction. Since CK is found in all muscle tissue, elevations of the total activity of this enzyme are not specific for cardiac damage. However, CK is a dimer and there are three different forms of the enzyme (isoenzymes) with varied tissue distribution. In skeletal muscle the predominate form of CK is CKMM. In heart muscle CKMM is also the predominate form, but CKMB makes up about 20% of the CK activity, whereas in skeletal muscle CKMB is generally about 1% of the CK activity. The third form of CK, which is not used as a cardiac marker, is CKBB.

Figure 3: Relationship between Creatine, Creatine Kinase, Phosphocreatine, and Creatinine

Since cardiac tissue has the highest percentage of CKMB isoenzyme (22% CKMB and 77% CKMM), this isoenzyme is monitored in the diagnosis of AMI. The key finding in myocardial infarction is an elevation of CKMB to greater than 10 ng/mL, approximately 6-48 h after the onset of chest pain.

It is important to remember that CKMB is not cardiac specific, as about 1% of the CK found in skeletal muscle tissue is CKMB. The tissue distribution of CK is shown on the figure on the next page (Figure 4). The lack of specificity of CKMB for cardiac tissue has led the use of the CK index (“relative index").

(1)
\begin{align} CK Index = \frac{CKMB\\mass\\(ng/mL)}{Total\\Ck\\activity (U/L)} X 100 \end{align}

CKMB < 10 ng/mL and an index < 2.5 is considered normal
CKMB > 10 ng/mL and an index > 2.5 suggests cardiac damage
CKMB > 10 ng/mL and an index < 2.5 suggests skeletal muscle damage

Other reasons for elevations of CKMB are:

 Myocarditis Shock Coronary insuffiency Polymyositis Cardiac surgery Other myopathy Muscle trauma Surgery Dermatomyositis Muscular dystrophies Severe angina Drug-induced rhabdomyoloysis Malignant hyperthermia Idopathic (rare)

In these cases the use of the CK index helps distinguish between cardiac and non-cardiac causes of elevated CKMB.

It is advisable that during the first 24 hours at least three samples be drawn for in order to pinpoint the peak of the elevation of CKMB. Demonstration of the peak (rise and fall) within 48 hours increases the specificity of the test since it occurs in MI but not in muscle disease in which values plateau. An elevation of CKMB above 10 ng/mL with an elevated CK index indicates myocardial infarction with very high specificity.

Figure 4: Relative Activities of the CK Isoenzymes in Selected Human Tissues (adapted from Tsung, 1976)

## Myoglobin

Myoglobin is a low-molecular weight protein (about 18 KD) found in all skeletal and cardiac muscle and is involved in oxygen binding. Due to its small size, it appears in serum rapidly after release from injured muscle and is cleared into the urine. Since myoglobin is found in most tissues, it has the least specificity of any of the cardiac markers. False positive results for diagnosis of myocardial infarction may be encountered with skeletal muscle injury and renal failure.

Serum myoglobin > 70 ng/mL is an early, but not specific indicator of myocardial infarction and thus should never be used by itself. For patients who arrive within several hours of onset of chest pain, myoglobin is analyzed on admission and 90 minutes later. If the concentration has increased by at least 25%, this is consistent with an early MI. This finding should be confirmed with more specific techniques (cTnI, cTnT, or CKMB).

Table 3: Table of Sensitivity and Specificity of Various Markers of Cardiac Injury (from Wu, A.H., Diagnostic Enzymology in Clinical Laboratory Medicine, 1994)
Specificity (%)
Marker 2-8 h 8-24 h 24-72 h 72 h Specificity (%)
Myoglobin 95 75 0 0 70
CKMB 60 95 98 50 95
Troponin 75 95 98 98 >95

Drug Therapy for AMI
Thrombolytics - Streptokinase, Anistreplase, Alteplase, Reteplase
Compounds with antiplatlet activity and Anticoagulants - aspirin, heparin, glycoprotein IIb/IIIa inhibitors
Beta-blockers - propranolol
Vasodilators - nitrates
Analgesics - morphine or meperidine if not relieved by nitroglycerin
Other therapies - oxygen, antiarrthymics, stool softeners, percutaneous transluminal angioplasty

# CONGESTIVE HEART FAILURE (CHF)

An estimated 4.8 million Americans have CHF with an additional 550,000 new cases each year. Of the CHF patients, about 300,000 patients will die each year [5] . Symptoms of CHF are often nonspecific (dyspnea) and consequently this syndrome is sometimes difficult to diagnose. Analysis of B-type natriuretic peptides (BNP) have been approved as diagnostic markers for CHF. BNP is synthesized as a pro-molecule consisting of 108 amino acids, pro-BNP (1-108). With left ventricular dysfunction (volume overload and myocardial stretch) pro-BNP (1-108) is released from the left ventricle. During this process pro-BNP (1-108) is cleaved into the active hormone BNP (77-108) and an inactive N-terminal fragment known as NT-pro-BNP (1-76). Assays with antibodies that recognize to the carboxy-terminal of the molecule, BNP (77-108), or the amino-terminal, NT-pro-BNP are used to diagnose and monitor treatment of CHF. Both NT-pro-BNP and BNP concentrations have been shown to correlate with the degree of cardiac failure, but these assays have different reference ranges. Thus it is important to know which type of analysis is being performed on your patients.

Table 4
Marker Reference Range
BNP (77-108) <100 pg/mL
NT-pro-BNP (1-76) if less than 75 years old <125 pg/mL
NT-pro-BNP (1-76) if greater than 75 years old 450 pg/mL

# CASE STUDIES

## Case 1

(Can. J. Emerg. Med. 8:289-294, 2006)

A 29-year-old woman was prescribed penicillin V for pharyngitis by her primary care provider 2 days before ED presentation. On that same day she took 1 dose in the afternoon and a 2nd dose between 6 and 8 p.m. that night. She presented to the ED at approximately 1:45 am complaining of lip swelling and shortness of breath. Lip swelling commenced approximately 10 hours after the initial dose. She denied chest pain or pressure, dyspnea on exertion, and cough or sputum production.

Physical exam revealed an anxious young woman in moderate respiratory distress. Initial vital signs were: temperature 36.7°C, blood pressure 113/64 mmHg, pulse 73 beats/min, respiratory rate 28, O2 sat 94% on room air.

Immediate medical treatment included diphenhydramine 50 mg IV, methylprednisolone 125 mg IV, and cimetidine 300 mg IV. As she continued to be tachypneic and distressed, she was given 1:10 000 epinephrine, 0.1 mg (1 cc) IV. The dose was confirmed, as it was a preloaded 10-cc syringe.

She began to complain of severe pressure-type central chest pain. Results of an ECG revealed ST elevations in several leads.
Laboratory results (Reference Ranges):

time Troponin I (<0.04 ng/mL) CK (20-212 IU/L) CKMB (0-4.5 ng/mL)
3:45 a.m. <0.04 66 0.1
8:40 a.m. 1.99 85 3.3
3:32 p.m. 1.63 106 3.3
1. Calculate the relative index for the three time points.
2. Based on these laboratory findings did the patient rule in for an MI?
3. How do you explain the normal values of CK and CKMB?
4. Is this a common ADR for epinephrine?

## Case 2

A 57-year-old man presented to the emergency department with a 1-hour history of shortness of breath, coughing, and “chest tightness.” He states that his symptoms appeared suddenly while he was watching television on his couch. He describes a constant “squeezing” sensation in his chest limited to the parasternal region associated with a paroxysmal nonproductive cough. He describes his shortness of breath as “difficulty getting air,” denying any pain on inspiration or expiration. He denies any similar episode in the past. His past medical history is significant for diabetes mellitus, hypertension, and severe peripheral vascular disease. In the past 6 years his right leg was amputated just above the knee and the left leg below the knee. He denies any syncopal episode, thigh, or abdominal swelling. He sleeps on one pillow at night and usually does not wake up to urinate. He used to drink alcohol heavily but has not had a drink since 1978. He denies any tobacco or illicit drug use.

Vital Signs: T 97 degrees Fahrenheit, P 125 BPM, RR 28/min, BP 179/102 mmHg

Physical Exam:

Neck No jugular venous distension (JVD) Sinus tachycardia, regular rhythm; grade I/VI nonradiating systolic ejection murmur at the left sternal border Fine rales in the lower half of each lung field; no rhonchi or friction rubs No tenderness to palpation No ascites or hepatosplenomegaly; no tenderness

CXR: Bilateral fluffy infiltrates consistent with pulmonary edema

EKG: Sinus tachycardia; no acute changes from previous EKG; no S-T segment depression or elevation (on admission)

Laboratory Tests: Admission 30 min 60 min 90 min 3 hrs 8 hrs 12 hrs
CKMB (ng/mL) 2.7 4.3 5.3 6.5 9.8 11.4 8.8
Troponin I (ng/mL) <0.5 <0.5 <0.5 0.8 3.6 8.2 5.9
Myoglobin (ng/mL) 49 98 165 184 129 86 56
1. How do you interpret these data? Are they diagnostic?
2. Given the above results, graph their relative crescendos, peaks, and durations of elevation.
3. What do the lab tests tell you about the chronology of events?
4. Why might the patient not have presented with typical chest pain?

## Case 3

(modified from J. Emerg. Med. 18(1):55-59, 2000)

A 34-year-old woman presented with squeezing substernal chest pain radiating across her chest and down both arms, diaphoresis, palpitations, light-headedness, and shortness of breath for 3 h. She did not smoke cigarettes or use recreational drugs, and had no history of hypertension, hypercholesterolemia, diabetes mellitus, or family history of cardiac disease. EKG changes showed 0.5 to 1.0 mm ST changes.

Vital signs: blood pressure 159/97 mmHg, heart rate 52 beats/min, respiratory rate 16 breaths/min, room air oxygen saturation 100%, and oral temperature 37.0°C. Cardiac examination revealed a regular rate and rhythm with I/VI systolic flow murmur. Lung, extremity, skin, peripheral pulse, and neurologic examinations were normal.

Lab Test
Time (hours) 6 12 24 30
CK U/L (normal <200 U/L) 176 195 175 84
CKMB (normal <10ng/mL) 8 14 10
1. Calculate relative index for the 6 hour, 12 hour, 24 hour time points.
2. Based on these findings discuss whether or not this patient ruled in for MI.
3. What other lab tests would have been useful to diagnose cardiac damage?