the immuassay handbook parte83

817© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00066-X

Normal Heart FunctionThe heart is a muscular pump that ejects blood into the lung and systemic circulation. For it to function continu-ously, the heart muscle needs an uninterrupted and plenti-ful supply of oxygenated blood. This is supplied from the coronary arteries, which leave the ascending aorta soon after exiting the left ventricle. The main coronary artery divides into right, left, and circumflex arteries, which run over the surface of the upper part of the heart. These arter-ies in turn divide into finer arteries and eventually pene-trate the whole of the myocardium ensuring that all the individual myocardial cells receive adequate oxygen and nutrients. The blood eventually collects into the coronary sinus, which drains into the right atrium before traversing to the pulmonary circulation for further oxygenation.

Much of the oxygen delivered to the myocardium is bound to myoglobin, a heme protein that gives cardiac muscle its deep red color and that is located inside the muscle fibers. The major requirement for energy provi-sion in the myocardial cells is for adenosine triphosphate (ATP), which is used to produce mechanical energy at the level of the contractile myofibrils. Most high-energy phos-phate in the heart is stored not as ATP but as creatine phosphate. The cytosolic enzyme creatine (phospho) kinase (CK) catalyzes the transfer of high-energy phos-phate from creatine phosphate to adenosine diphosphate to give adequate supplies of ATP. The ATP is used for a wide variety of reactions, but the most important is the interaction of myosin and actin in the myofibril thick and thin filaments. During this reaction, the chemical energy of ATP is transduced into mechanical energy, causing thick and thin filaments to slide past each other, making the muscle contract. This process is controlled by the level of intracellular free calcium ions, which act on a series of regulatory proteins (the troponin–tropomyosin complex) that, in turn, allow the thick and thin filaments to interact. To summarize, the cardiac pump function requires a con-tinuous supply of oxygenated blood, ATP production from creatine phosphate, and the interaction of the myofibrillar proteins.

Clinical DisordersAlthough there are a number of different disorders that affect the biochemical, physiological, and mechanical functions of the heart, by far the most common is the spec-trum of changes that comes under the general umbrella of coronary artery disease (CAD).

CORONARY ARTERY DISEASECAD is a continuous spectrum of disorders that for many, begins at an early age, and progresses until death. The major events are atherosclerosis, stable angina, unstable angina, acute myocardial infarction (AMI), and cardiac death. Indi-viduals who survive episodes of myocardial infarction develop congestive heart failure (CHF). As there are other causes of heart failure, this disease is listed separately.

AtherosclerosisThe initial stages of CAD are characterized by atheroscle-rosis, which is the build up and deposition of lipid-filled plaques within the coronary arteries. During this period, the individual is asymptomatic and essentially disease free. The progression of atherosclerosis, and the subsequent risk for CADs, is dependent on numerous risk factors such as abnormally high concentrations of total (>200 mg/dL) and low-density lipoprotein (LDL) cholesterol (>100 mg/dL) and/or high-density lipoprotein (HDL) cholesterol of <40 mg/dL, smoking, hypertension (blood pres-sure > 140/90 mm Hg), diabetes, being a male or a post-menopausal female or having an immediate family with a history of premature (men < 45 years, women < 55 years) heart disease. In addition to cholesterol, LDL, and HDL cholesterol, new biochemical markers have been investi-gated as additional risk factors for CAD. Some of these markers include the apolipoproteins AI and B-100, lipopro-tein (a), homocysteine, high-sensitivity C-reactive protein, coagulation factors such as fibrinogen, factor VII, tissue plasminogen activator antigen, and plasminogen activator inhibitor (PAI-1), nitrous oxide, and oxidized LDL.

Stable Angina Pectoris and Silent IschemiaStable angina pectoris is defined as episodes of chest pain precipitated by physiologic situations of increased oxygen demands to the heart. It occurs most commonly during or immediately after exercise. In patients with atherosclero-sis, angina is caused by the narrowing of coronary arteries to the point where there is insufficient delivery of blood and oxygen to actively respiring myocardial tissue. The affected areas of the heart are said to be ischemic, i.e., they are in danger for permanent myocardial damage, but myo-cardial necrosis does not occur in this condition, therefore, most biomarkers in blood are present within the normal range. However, there may be release of cardiac troponin, providing the basis for risk stratification. The atheroscle-rotic plaques are stable, in that they have a thick fibrous cap and are not in any immediate risk to rupture (Fig. 1a). The chest pain is relieved by rest or by medications such as

Cardiac MarkersDeborah French ([email protected])

Alan H.B. Wu

C H A P T E R

9.12

818 The Immunoassay Handbook

nitroglycerin, which functions to diminish the oxygen demands of the heart.

Patients with silent ischemia have episodes of reduced blood flow that can lead to both myocardial ischemia and cell necrosis. Unlike patients with stable angina and acute coronary syndromes, those with silent ischemia do not present with any symptoms or knowledge that an ischemic event has taken place. Biochemical markers might be increased in the blood of patients suffering an event. How-ever, in the absence of symptoms, the patient would not know to present to a hospital for blood collection. The diagnosis is made on the basis of stress testing, whereby ischemic episodes are induced under controlled conditions and monitored by continuous electrocardiographic record-ings (ECG ST-segment depression of >1 mm) or nuclear imaging techniques (reduced coronary artery blood flow).

Acute Coronary SyndromesPatients who present with chest pain at rest have unstable angina pectoris or AMI, collectively known as acute cor-onary syndromes. In these patients, atherosclerotic plaques have become vulnerable due to the thinning of the fibrous cap (Fig. 1b). Under the shear stress of circulating blood, there is rupture of the plaque, which leads to the exposure of the lipid-filled core, causing an aggregation of platelets and the formation of a thrombus. When the clot is subocclusive, the patient has unstable angina pectoris. This condition is associated with little or no myocardial damage, and the ECG may be normal. Patients with unsta-ble angina are treated with anti-inflammatory drugs (e.g., salicylates), β-adrenergic blockers, antithrombotic drugs (e.g., unfractionated or low molecular weight heparin), and antiplatelet medications (e.g. glycoprotein IIb/IIIa

receptor inhibitors). Additionally, HMG-CoA reductase inhibitors or statins have become the treatment of choice in order to reduce LDL cholesterol in some patients.

When the clot from a ruptured plaque completely blocks coronary artery blood flow, the patient has suffered an AMI. This leads to myocardial cell death and release of intracellular components into the circulation. The World Health Organization criteria for AMI are a triad that includes a clinical presentation of chest pain, ST-segment elevations (>1 mm) on ECG, and increases in serum enzymes. The diagnosis of AMI is given when two of these criteria are fulfilled. In 2000, the definition of myocardial infarction was redefined by a joint committee of the Euro-pean Society (ESC) and American College of Cardiology (ACC), and this was further updated in 2007 by these orga-nizations in conjunction with the American Heart Associa-tion (AHA) and the World Heart Federation (WHF). The current criteria for AMI are increased concentrations of a cardiac marker, preferably troponin, with a least one value above the 99th percentile of the upper reference limit, along with evidence of myocardial ischemia with at least one of the following: chest pain, ECG changes (ST-T changes or new left bundle branch block or development of pathological Q waves), angiographic, or autopsy documentation.

The common biochemical markers of AMI have changed over the years due to the availability of more specific and sensitive biomarkers. Troponin (T or I) has become the preferred biomarker for myocardial necrosis, but if these assays are not available, the best alternative is the CK-MB isoenzyme mass assay. Other proteins that are released into the circulation from damaged myocytes and hence have been utilized in the past as biomarkers include total CK, lactate dehydrogenase isoenzymes, and myoglobin. ECG-documented AMI patients are acutely treated with intravenous thrombolytic agents such as streptokinase and tissue plasminogen activators or revascularization by coro-nary angioplasty. In many cases, the non-Q-wave AMI is indistinguishable from unstable angina, and the therapy is similar.

A significant number of AMI patients will die within minutes after suffering AMI, before emergency treatments can be rendered. These patients die of uncontrolled car-diac arrhythmias. For those who survive, the risk for future myocardial infarctions is high.

HEART FAILUREA clinical syndrome whereby the heart is unable to gener-ate sufficient cardiac output to meet the body’s demands defines heart failure (HF). This is characterized by intra-vascular and interstitial volume overload, including short-ness of breath, dyspnea, rales, and edema, or conditions of inadequate tissue perfusion, including fatigue and exercise intolerance. HF is caused by a primary disorder of the heart muscle (cardiomyopathy), valvular disease, infiltrative pro-cesses, and ischemic heart disease. One measure of left ven-tricular function is the ejection fraction, determined by the echocardiogram. HF occurs when there is overstimulation of the renin–angiotensin–aldosterone axis that leads to vol-ume overload and remodeling (enlargement) of the heart. Aldosterone stimulates vasoconstriction, hypernatremia,

FIGURE 1 Schematic cross-section of a coronary artery. (a) Stable atherosclerotic plaque with a thick fibrous cap. (b) Unstable plaque vulnerable to rupture.

819CHAPTER 9.12 Cardiac Markers

and fluid retention. As a compensatory mechanism, the natriuretic peptides are released in an attempt to reverse these changes. These include A-type natriuretic peptide (ANP) that is found in high concentrations in the atrium as granules, and B-type natriuretic peptide (BNP) and N-terminal pro-B-natriuretic peptide (NT-proBNP) that are upregulated and produced in the ventricles. Novel bio-markers have also emerged including endothelin-1, soluble ST2 receptor, myeloperoxidase, galectin-3 and copeptin, among others, and these are likely to become introduced into clinical use in the near future.

HYPERTENSIONHypertension is defined when the systolic blood pressure exceeds 140 mm Hg and the diastolic blood pressure exceeds 90 mm Hg. It can be the result of a fault in the complex physiological, biochemical, and endocrine con-trol mechanisms. One important factor is the level of activity in the renin–angiotensin system, which generates angiotensin II, a powerful vasoconstrictor that gives rise to elevated blood pressure. Direct or indirect measurements of elevated plasma renin activity can be used to give an indication of hypertension. Other factors are the natri-uretic peptide system (notably atrial and brain natriuretic peptides), which controls fluid and electrolyte balance.

AnalytesCARDIAC TROPONIN (T AND I)

FunctionThe thin filament of contractile muscle contains actin, myosin, and troponin, a complex of three proteins. Cardiac troponin T (cTnT) functions to bind the troponin com-plex to tropomyosin and has a molecular mass of 37 kDa. Cardiac troponin I (cTnI) functions to inhibit calcium-dependent ATPase and has a mass of 24 kDa. Troponin C is so named because it has four binding sites for calcium, and weighs 18 kDa. When bound to calcium, this complex undergoes a stearic rearrangement enabling the thin fila-ment to slide past the thick myosin filament when a signal for muscle contraction is received. As with other myofi-brillar proteins, troponin T and I exist in more than one isoform, most of which have characteristic tissue distribu-tions. The cardiac isoforms of T and I are specific to heart tissue. The cardiac isoform of C is identical to the skeletal muscle form. The majority of cTnT and cTnI are myofi-brillar bound. However, there is a small free cytosolic pool of cTnT (6–8%) and cTnI (2–8%), possibly as a precursor for incorporation into the myofibril.

Reference IntervalThe cutoff concentrations for most cardiac markers, such as CK-MB and myoglobin, are set such that they differen-tiate between non-AMI cardiac diseases such as unstable angina and AMI. These cutoffs are generally higher than the upper limit of normal and are necessary because some healthy subjects can have high concentrations of these

markers due to normal skeletal muscle turnover. For cTnT and cTnI, there is no contribution from skeletal muscle troponin and there is only a small amount of car-diac troponin in healthy individuals. Therefore, low cutoff concentrations are used to detect minor myocardial dam-age. The ESC/ACC/AHA/WHF has recommended use of a cutoff at the 99th percentile of the normal range and has an assay coefficient of variance (CV) of 10% or less. Currently, there are no commercial assays that can meet these criteria. However, evidence-based published data have indicated that assays with a CV of ≤20% at the 99th percentile can be utilized as this degree of imprecision will not lead to misclassification of patients in diagnosis or risk assessment. The majority of the contemporary cTnI assays meet this level of imprecision. New high-sensitivity cTnI (hs-cTnI) assays are in development but at this time are not commercially available. These assays all have ≤10% CV at the 99th percentile. One high-sensitivity cTnT assay is currently available worldwide, but it has not been approved by the Food and Drug Administration in the USA to date, and it has a 13% CV at the 99th percentile. The International Federation of Clinical Chemistry (IFCC) maintains a list of the commercially available and research troponin assays and their analytical characteris-tics on its website. An additional, often overlooked factor is that of biological variation, including differences between gender and age groups (Fig. 2). With the advent of the new high-sensitivity assays, it may one day be pos-sible to include this in the clinical interpretation of tropo-nin results. The current recommendation for a troponin reference interval is still to use the lowest troponin con-centration that can be measured with a CV of at least 10% and serial sampling of the patient is recommended at base-line, 6 h and 12 h to definitively rule out AMI and at 2–3 h to rule in AMI. Figure 3 shows typical precision results for the 95th, 99th, and 10% imprecision levels for cTnI, and Fig. 4 shows the determination of the 99th percentile in a healthy reference cohort for cTnT.

cTnT: 0–0.03 ng/mL (10% CV); cTnI: varies with manufacturer.AMI cutoff: not established for cTnT, varies with manufacturer for cTnI.

FIGURE 2 Influence of age and gender on cTnT results. Individual cohorts are defined by decade of age and gender. Statistical significant differences between female and corresponding male cohorts are indicated by P values noted above the box-and-whisker plots: *P < 0.05 or **P < 0.001. Used with permission from Saenger et al. (2011).


Clinical ApplicationsAfter AMI, necrosis of cardiac myocytes allows intracellu-lar components such as cTnT and cTnI to leak into the circulation. The initial release of proteins is due to the free cytosolic pool, and abnormal concentrations of cTnT are detected in blood within the first 6–12 h after the onset of chest pain. Despite the release of free cytosolic cTnI, there is very little free form found in serum after AMI, because this form is hydrophobic and presumably binds to other proteins. Over the subsequent days, there is degradation of the myofibrillar elements and release of the ternary tropo-nin complex. Much of this protein degrades in blood to the troponin I–C binary complex and free troponin T. The concentrations of cardiac troponin T and I are increased for 7–10 days after AMI, depending on the marker and the reperfusion status. For patients with successful reperfusion (by thrombolytic therapy or spontaneously), cTnT exhib-its a biphasic release pattern (Fig. 5). For patients with unsuccessful reperfusion and for cTnI, a monophasic release pattern is observed. In most cases, cTnI clears from the blood sooner than cTnT.

A major application of assays for cTnT and cTnI is in the risk stratification of patients with unstable angina. Roughly one third of patients with a diagnosis of unstable

angina (UA) will have measurable concentrations of cTnT or cTnI, in the presence of a normal concentration of CK-MB. These patients have suffered minor myocar-dial injury. In prospective clinical trials, UA patients with an abnormal troponin will have a fivefold higher inci-dence of cardiac death or AMI within the following 4 weeks than a matched group of UA patients who have normal troponin. These data indicate that measurement of cTnT or cTnI is useful for risk stratification. These markers may be used to triage patients to receive new antithrombotic and antiplatelet drugs. Those with high concentrations of cardiac troponin will benefit most from these new therapies.

LimitationsBoth cTnT and cTnI have been shown to be highly specific for cardiac injury, but the fourth generation cTnT assay was shown to have cross-reactivity with a protein found in skeletal muscle of patients with primary skeletal muscle dis-ease. There are increases in both cTnT and cTnI in a minority of patients with chronic renal failure, with a higher percentage of abnormal results occurring for cTnT. These results are due to the presence of true myocardial injury, which is common in patients with chronic renal failure. There is no release of cTnT or cTnI in any other noncar-diac diseases. Analytical false positive results may occur in troponin assays mostly due to fibrin interference. Hetero-phile antibodies or human anti-mouse antibodies (HAMA) can also produce falsely positive or negative results.

Assay TechnologyAll troponin assays use a two-site non-isotopic immuno-metric assay format with enzyme, fluorescence, or chemi-luminescence detection. Due to patent restrictions, all cTnT assays are produced by the same manufacturer and are calibrated to the same reference material. Assays for cTnI are available through many manufacturers. These cTnI assays have significant biases between them. How-ever, a cTnI reference standard has become commercially available. Manufacturers will be gradually standardizing

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FIGURE 3 Assay precision vs troponin I concentration curve. The frequency of cTnI concentrations is given on the left y-axis (bars) and the assay precision on the right y-axis (curve). The troponin I concen-trations for the 97.5%, 99%, and 10% CV cutpoints are shown on the x-axis.

FIGURE 4 Determination of the 99th percentile in a healthy reference cohort for cTnT. Used with permission from Saenger et al. (2011).

FIGURE 5 Cardiac marker release patterns vs time after onset of chest pain.


their assays to this material. Qualitative and quantitative point-of-care testing devices are also available using an immunochromatography format. In some cases, multiple analytes are available on the same strip, e.g., cTnI, myo-globin, and CK-MB (e.g., Biosite Diagnostics and First Medical). However, it should be noted that the results of these point-of-care testing devices are not comparable to the results of automated immunoassay analyzers, and serial sampling tests should be carried out on one instrument.

Desirable Assay Performance CharacteristicsAssays should exhibit no cross-reactivity toward skeletal muscle troponin. Cardiac troponin T should have a sensi-tivity to detect the 99th percentile of a healthy population with an assay precision of 10% or less.

Type of SampleSerum or heparinized plasma.

Frequency of UseTroponin has become the standard biomarker for use in acute coronary syndromes.

CREATINE KINASE AND THE MB ISOENZYMECK is a dimeric protein composed of two enzymatically active M (muscle-type) and B (brain-type) subunits. There are three major cytosolic isoenzymes. The CK-MM isoen-zyme is the major form in skeletal and cardiac muscles, whereas CK-BB is found in brain and other organs. CK-MB is found predominantly in cardiac muscle where it accounts for up to 30% of the total CK present, the rest being CK-MM. Small amounts of CK-MB, normally less than 1% of the total, are found in skeletal muscle. There are also two major mitochondrial isoenzymes of CK that can be released into blood after injury. These isoenzymes are unstable as they lose their enzymatic activity rapidly after they appear in blood.

FunctionMitochondrial CK functions to convert ATP generated from the electron transport system to creatine phosphate, which diffuses into the cytoplasm and acts as a reservoir of high-energy phosphate bonds. When energy is required for active muscle contraction, cytosolic CK catalyzes the production of ATP from creatine phosphate stores. CK is also found in other non-contractile tissue such as the distal nephron. The role of CK in other tissues may be to shuttle high-energy phosphate groups for other ATP-dependent functions such as the sodium–potassium membrane pump. Total CK and CK isoenzymes are cleared from the blood by the reticuloendothelial system of the cell.

Reference IntervalTotal CK: males 0–200 U/L, females 0–160 U/L.CK-MB: <5 ng/mL.Relative index: <2.5%.

Clinical ApplicationsTotal CK is not specific to cardiac damage, as it is also increased in patients with skeletal muscle disease or injury. Measurement of the CK-MB isoenzyme improves the specificity of the assay. Both total CK and CK-MB begin to increase in the blood of patients with AMI within the first 4–6 h after the onset of chest pain, peak at 18–24 h, and return to normal with 72 h (Fig. 5). If there are no further episodes of ischemic injury, the activity declines in a mono-exponential fashion.

LimitationsThe major limitation of CK-MB determination as a car-diac-specific marker of heart damage stems from the low levels of CK-MB present in skeletal muscle. In cases where severe skeletal muscle trauma may occur, such as in patients with polymyositis, muscular dystrophy, severe muscle trauma, and after prolonged physical exercise, CK-MB levels can be greatly increased above the absolute cutoff concentration indicative of myocardial infarction. In these cases, it is necessary to calculate a relative index of CK-MB (in ng/mL)/total CK (in U/L). An abnormal concentration of CK-MB with a normal relative index indicates a noncar-diac source of CK-MB. Neither the relative index nor the absolute concentration of CK-MB can be used to deter-mine the presence of myocardial damage when skeletal muscle disease is also present.

Due to the lack of myocardial tissue specificity, CK-MB levels cannot be used to measure minor myocardial injury. To avoid a high number of false positive results, the cutoff concentration used for CK-MB is preset to differentiate AMI from non-AMI disease. Therefore, CK-MB results are often within the normal range in patients with low lev-els of myocardial necrosis, such as in patients with myocar-ditis, HF, and myocardial ischemia. In this respect, there is no clear cutoff, but usually, one might not expect to see the normal time profile of elevated CK-MB levels typical of mild to severe infarction. Single elevated samples should therefore be treated with caution. Typically, a sample would be taken on admission and a minimum of once or twice daily for the subsequent 2–3 days. More frequent sampling not only aids in positive diagnosis but also may provide a semiquantitative measure of the extent of dam-age. Numerous studies have shown that the cumulative release of CK-MB is well correlated with the extent of myocardial necrosis (infarct sizing) when compared to the amount of tissue damaged, when anatomically determined during an autopsy.

Assay TechnologyEither the activity or the mass concentration of CK-MB can be measured. Activity measurements include electro-phoresis and immunoinhibition. However, these assays have largely been replaced by immunoenzymetric mass assays, using a combination of monoclonal or polyclonal antibodies, with one raised against CK-MB, and the other against an individual subunit, usually the B subunit. Most commercial assays make use of the monoclonal antibody licensed from the Department of Laboratory Medicine, Washington University (St. Louis, MO).


Desirable Assay Performance CharacteristicsThe assay for CK-MB should be sensitive to below 1 ng/mL and have a dynamic range of at least 100 ng/mL. The accept-able precision is about 10% at the cutoff concentration.

Type of SampleSerum or heparinized plasma.

Frequency of UseIf a troponin assay is not available, a CK-MB mass assay is the method of choice for diagnosis of AMI. Due to the advent of troponin, many clinical laboratories no longer offer CK-MB testing at all.

MYOGLOBIN

FunctionMyoglobin is an iron-containing protein with a molecular mass of 18 kDa. It resembles hemoglobin but binds one rather than four molecules of oxygen. Its binding charac-teristics are such that it takes up oxygen from hemoglobin in the blood and releases it for use into the mitochondria where oxidative reactions occur.

Reference IntervalMyoglobin: 25–90 ng/mL.

Clinical ApplicationsAfter AMI, when the cardiac myocyte becomes necrotic, myoglobin is released through the damaged cell membrane where it becomes detectable in the circulation. Elevated levels of myoglobin are normally detectable between 2 and 6 h after infarction peaking within 5–18 h. It is generally detectable before CK-MB. Because of its low molecular weight, it is readily filtered by the glomerulus and cleared by the kidneys. Myoglobin levels return to normal within 24 h after injury (see Fig. 5). Because of its rapid return to baseline concentrations, myoglobin can also be used to detect new myocardial injury, such as a reinfarction.

LimitationsMyoglobin is found in all oxidative muscle fibers, includ-ing skeletal muscle, so injury to such tissues will give rise to elevated myoglobin levels. This can occur after skeletal muscle trauma, extreme physical pain, skeletal myopathy, or rhabdomyosarcoma. Myoglobin concentrations are also increased in patients with acute or chronic renal fail-ure due to reduced clearance of the protein. Cardiac-related conditions in which myoglobin elevations may be seen include HF, tachyarrhythmias, ischemia, and some cardiomyopathies.

Assay TechnologyThe original myoglobin assays were based on radioimmu-noassays. Subsequently, immunoturbidimetric assays were developed. These have all been replaced by non-isotopic

immunoassays with enzyme, fluorescence, or chemilumi-nescence signal generation systems. Assays usually use antibodies to human myoglobin raised in rabbits. Both competitive and noncompetitive assay formats are avail-able. The solid phase may be microtiter™ plates or poly-styrene particles in the case of some turbidimetric assays. In all other technical respects, assays for this protein are straightforward.

Desirable Assay Performance CharacteristicsThe myoglobin assay should have a sensitivity of <5 ng/mL and a dynamic range of at least 500 ng/mL. The acceptable imprecision is about 10% at the cutoff concentration.

Types of SampleSerum, plasma, or urine.

Frequency of UseMyoglobin testing has largely been discontinued in clinical laboratories since troponin T or I assays have increased in sensitivity and can now be used to rule in AMI earlier in the patient’s clinical course.

FREE FATTY ACID-BINDING PROTEINS

FunctionFatty acid-binding protein (FABP) functions as a long-chain fatty acid carrier in blood. It plays an important role in lipid metabolism. The heart type isoenzyme is found in the heart and skeletal muscles. It has a molecular weight of 14–15 kDa.

Reference IntervalFABP: <6 ng/mL*

Myoglobin/FABP: <9.0*

Clinical ApplicationThe clinical performance of free FABP is similar to that of myoglobin. Both are low molecular weight proteins that are released within 3–6 h after the onset of chest pain, and both are also increased in skeletal muscle injury or disease. There-fore, measurement of FABP alone offers little clinical advan-tage over myoglobin. However, when myoglobin concentrations are increased, measurement of the ratio of myoglobin to FABP may be useful in determining the source of myoglobin release. This ratio is significantly different when the injury originates from the skeletal muscles, as opposed to myocardial injury. Therefore, the additional mea-surement of FABP increases the specificity of myoglobin.

LimitationsThe addition of FABP to myoglobin doubles the amount of laboratory work and expenses for such testing. Addi-tionally, FABP is also increased in patients with renal insufficiency.

* Provisional cutoffs.


Assay TechnologyAll assays for FABP are based on a two-site immunometric assay format.

Frequency of UseCommercial assays for FABP are available for research use only and are not currently used in clinical laboratories in the USA, but are approved for diagnostic use, and are more widely used in Europe.

APOLIPOPROTEINS AI, AII, AND B

FunctionThe protein part of the lipoproteins is composed of com-ponents termed apolipoproteins. Each type of lipoprotein has a specific and relatively constant composition of apoli-poprotein. Apolipoprotein A (Apo A) is the major compo-nent of the HDLs and is in turn composed of two major subcomponents: Apo AI and Apo AII. Apo AI constitutes about 60–75% of the Apo A in HDL and is necessary for HDL formation. Apo AI is also responsible for the activa-tion of lecithin cholesterol acyl transferase, which cata-lyzes cholesterol esterification before catabolism of cholesterol and liver excretion. HDL formation helps remove cholesterol from blood vessels and as such is anti-atherogenic.

Apo B is the major protein constituent of LDL (about 90–95%) and also makes up about 40% of the very LDLs and chylomicrons. Apo B is a mixed group of proteins, the two major components being Apo B-100 and Apo B-48. Apo B is of major functional importance because it is rec-ognized by the receptors of LDL and therefore plays a crucial role in LDL catabolism, transporting cholesterol to cells for deposition.

Reference IntervalAdult male Apo AI: 94–176 mg/dL.Adult female Apo AI: 101–198 mg/dL.Adult male Apo B: 52–109 mg/dL.Adult female Apo B: 49–103 mg/dL.Adult Apo B/A1 ratio: <0.29 below average risk.0.29–1.30 average risk.>1.30 above average risk.

Clinical ApplicationsPatients with atherosclerosis and associated CAD have consistently lower levels of HDL and consequently Apo AI and AII. This leads to increased blood levels of the LDLs, which in turn results in cholesterol deposition in blood vessels and atherosclerotic plaques. Apo AI levels show a significant correlation with HDL cholesterol although it has been shown that Apo AI levels are a better predictor of CAD than HDL or HDL cholesterol. Additionally, a number of studies have indicated that administering Apo A1 to patients with CAD significantly decreases coronary atherosclerosis, as measured by plaque volume in a matter of weeks. Apo A1 concentrations may also be useful in the diagnosis of inherited or acquired conditions that cause deficiencies, such as Tangier disease.

The indications for Apo B levels in atherosclerosis are directly counter to those for Apo A with increased levels of Apo B being observed consistently in CAD. From this, the ratio of Apo B/Apo AI is being used as a positive predictor of coronary atherosclerosis.

LimitationsThe apolipoproteins have not been studied as extensively as the more traditional total, HDL, and LDL cholesterol for risk assessment in CADs. Because these markers are newer, there is less epidemiologic data available. More-over, studies on the effect of lipid-lowering drugs have largely focused on these traditional lipids and not on the apolipoproteins.

Assay TechnologyMany different assays for detection of Apo A (usually Apo AI) and Apo B concentrations are available from diagnostic companies, most of which are immunoturbidimetric.

Type of SampleFasting serum.

Frequency of UseModerate.

LIPOPROTEIN (A)

FunctionLipoprotein (a) (Lp(a)), a unique lipoprotein molecule, is a spherical lipid particle very similar in structure to LDL, but with an additional apolipoprotein (a) (apo(a)) compo-nent that is covalently bound by disulfide bridges to Apo B-100. In consequence, it differs from LDL in molecular mass, protein:lipid ratio and electrophoretic mobility. Lp(a) has a core rich in cholesterol esters.

Reference IntervalLp(a) >30 mg/dL = borderline risk.Lp(a) >50 mg/dL = high risk.

Clinical ApplicationsSeveral studies have shown a positive correlation between raised plasma Lp(a) levels and the risk of CAD. When the level of Lp(a) is elevated above 30 mg/dL, the risk of athero-sclerosis is doubled. This is thought to be due to the large degree of homology between Lp(a) and plasminogen. Lp(a) may inhibit fibrinolysis by competing for plasminogen receptors on the epothelial surface for binding to fibrin. Lp(a) promotes vascular smooth muscle cell proliferation by inhibiting the activation of growth factor. It is taken up by macrophages and may contribute to the formation of foam cells. Unlike other markers of cardiovascular risk, the con-centration of Lp(a) in plasma is genetically determined, pri-marily by the apolipoprotein (a) genotype, and is independent of diet and exercise. Recent clinical studies have shown, how-ever, that plasma concentrations of Lp(a) can be lowered by


up to 30–40% by niacin therapy in a dose-dependent man-ner and that this therapy concurrently reduces LDL and total cholesterol and triglycerides and increases HDL cho-lesterol. Both scientific and clinical evidence point to causal-ity between elevated Lp(a) concentrations and increased risk of cardiovascular disease (CVD). It is therefore recom-mended that Lp(a) be measured in individuals at intermedi-ate or high risk of CVD, and if therapeutic intervention is given, repeat measurement may be useful. Additionally, a single nucleotide polymorphism in the apo(a) gene encoding an isoleucine to methionine substitution is associated with elevated levels of Lp(a) and therefore CVD, and aspirin treatment in these patients reduced the risk for CVD com-pared to a placebo control group.

LimitationsThe apo (a) structure of Lp(a) exists in polymorphic forms, with a variable number of multiple repeats within the krin-gle 4 protein domain. These apo(a) isoforms have a range of molecular weights from 280 to 800 kDa. Because report-ing units are on a mass basis (milligrams per deciliter) rather than a molar basis, some individuals will have a higher Lp(a) result if they normally produce the higher molecular weight form. Moreover, immunoassays that make use of antibodies directed against the variable por-tions of the apo(a) molecule will have different immunore-activities to these forms. Therefore, it is difficult if not impossible to standardize immunoassay results from dif-ferent manufacturers, unless they all agree to use antibod-ies that are not sensitive to the variable regions of the protein. Additionally, there are wide variations in the Lp(a) concentrations between ethnicities, and as such, there should be race-specific reference intervals. Presently, these do not exist in clinical laboratories.

Assay TechnologyAssays for Lp(a) are mainly of the ELISA or immunoturbi-dimetric type and are commercially available from a vari-ety of diagnostic companies. However, it should be noted that these assays are as yet not standardized.

Type of SampleFasting serum or plasma.

Frequency of UseAssays for Lp(a) have been commercially available for many years, and the frequency of use is growing as it has become clear that elevated Lp(a) is associated with increased risk of CVD.

HOMOCYSTEINE

FunctionHomocysteine is a sulfhydryl-containing amino acid formed by the demethylation of methionine. It consists of a mixture of homocysteine, homocystine, and homocyste-ine–cysteine mixed disulfides. Under normal conditions, it is metabolized to cysteine through the transsulfuration pathway or re-methylated back to methionine through the

transmethylation pathway. In patients with hereditary homocystinuria, a deficiency in a metabolic enzyme such as cystathione β-synthase causes severe elevations of plasma and urinary homocysteine. In individuals who have deficiencies in vitamin B6, B12, and folate, homocysteine accumulates in blood because B6 is a necessary cofactor for the transsulfation pathway, and B12 and folate are neces-sary for the transmethylation pathway.

Reference IntervalTotal homocysteine: ≤10 µmol/L desirable.>10 to <15 µmol/L intermediate (low to high).

15 to <30 µmol/L high.30 µmol/L very high.

Clinical ApplicationsHomocysteinemia has been identified as a potential cardio-vascular risk factor. The original observation was made in untreated homocystinuric children who died of stroke and AMIs before adulthood. Despite normal levels of choles-terol, examination of their coronary arteries revealed exten-sive atherosclerosis, similar in presentation to adults with CAD. Subsequent studies have shown that adult patients with high plasma homocysteine concentrations are at greater risk for CVD than age-matched controls, but others have shown no association. Homocysteinemia has also been asso-ciated with peripheral arterial and venous occlusive diseases.

Initially, it was thought that if there was cardiovascular risk, it could be reduced by vitamin supplementation, but subsequent literature reviews have indicated no benefit or lowering of CVD risk. The current USA recommended daily allowance is 2 mg for B6, 5 µg for B12, and 200 µg for folic acid. To increase the folate concentration in the gen-eral population, grain products are supplemented with folate in the USA.

Assay TechnologyTotal homocysteine can be measured by high-performance liquid chromatography (HPLC) and immunoassay. HPLC eluates can be measured using electrochemical detectors, mass spectrometers, or fluorometrically following derivatization. Commercial immunoassays are also avail-able from most diagnostic companies.

Desirable Assay Performance CharacteristicsStandardized assays should be used, and the analytical per-formance goals are imprecision of <5%, <10% bias, and <18% total error.

Type of SampleFasting serum or plasma.

C-REACTIVE PROTEIN

FunctionC-reactive protein was first described in 1930. It is an acute-phase protein that is released into the circulation during


acute and chronic inflammation. It functions to bind to the polysaccharide component of bacteria, fungi, and parasites. Once bound, CRP promotes opsonization, phagocytosis, and lysis via activation of the classical complement pathway. CRP is a pentamer of identical subunits with a combined molecular weight of 118 kDa. In patients suffering acute inflammation, CRP is released into blood within 24 h to exceptionally high levels (>1000 times the normal range).

Reference IntervalNormal sensitivity assay: <10 mg/L.High-sensitivity assay: <1.0 mg/L low cardiovascular risk. See Fig. 6.1.0–3.0 mg/L average risk.3.1–10.0 mg/L high risk.>10.0 mg/L very high risk.

Clinical ApplicationsThe monitoring of CRP concentrations in blood has been used to provide a marker for acute inflammation for many years. CRP has been shown to be more sensitive and spe-cific than the erythrocyte sedimentation rate. Recently, the role of inflammation has been clarified in the

pathophysiology of acute coronary syndromes. CRP is associated with platelet activation, it also binds to LDL and is found in atherosclerotic plaques, and therefore increases in CRP concentrations have been associated with risk of CAD. Use of CRP as a predictor of cardiac events requires the use of a high-sensitivity assay, as increased risk is shown for CRP concentrations that are within the normal range of conventional (low sensitivity) assays. The predictive value of high-sensitivity CRP assay is independent of total cholesterol and HDL measurements. In the JUPITER trial, it was found that in patients with LDL of <130 mg/dL but hsCRP of ≥2 mg/L, the use of rosuvastatin (a statin) was associated with a 54% reduction in AMI, a 48% reduc-tion in stroke, a 46% reduction in bypass surgery or angio-plasty, a 43% reduction in venous thromboembolism, and a 20% reduction in all cause mortality as compared to the placebo group. The combination of hsCRP and lipids pro-duces more precise risk stratification information than use of lipids and lipoproteins alone.

LimitationsIf CRP is to be used for acute detection of inflammation, an assay with low sensitivity is sufficient for clinical use.

FIGURE 6 Primary cardiovascular risk assessment using a combination of hsCRP and total cholesterol:HDL cholesterol. Cutoffs are broken down into quintiles. Used with permission from the American Association for Clinical Chemistry (Clin. Chem. 47, 28–30 (2001)).


However, for CVD risk, high-sensitivity CRP assays are necessary. The limit of quantitation necessary for this use is ≤0.3 mg/L. Additionally, if the hsCRP assay is to be used, the patient should be metabolically stable and free from infection or any other illness due to the nonspecific nature of this molecule. If the CRP assay is performed by light-scattering immunoassays, the phenomenon of “pro-zoning” or antigen excess can occur. The antigen–anti-body complex is insoluble, and a linear relationship is initially observed between the light-scattering curve and the antigen concentration. At high antigen concentration, however, a soluble complex is formed, resulting in a reduction in the analytical signal back to the baseline. As a result, two different antigen concentrations can produce the same analytical signal. To prevent the reporting of the wrong antigen concentration, the sample must be diluted. A reduction in the antigen concentration upon dilution suggests that the initial sample was within the acceptable linear portion of the curve. A high antigen concentration recovery upon dilution suggests that the sample CRP concentration is too high. Heterogeneous immunometric assays with other signal generation sys-tems do not suffer from the prozone effect but may suffer from the hook effect common to these types of assays. The reporting units for the conventional (low sensitivity) CRP assay have been in milligrams per deciliter. This can produce considerable confusion if the recommended mil-ligrams per liter units are used for the high-sensitivity assay.

Assay TechnologyC-reactive protein can be measured by rate nephelometry and turbidimetry. For high-sensitivity determinations, heterogeneous immunoassays are preferred and are com-mercially available through many diagnostic companies.

Desirable Assay Performance CharacteristicsStandardized assays should be used, and <10% impreci-sion throughout the analytical measurement range is desired.

Types of SamplesSerum and heparinized plasma.

LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2 (LP-PLA2)

FunctionLp-PLA2 is a calcium-dependent serine lipase enzyme expressed by macrophages and other inflammatory cells in atherosclerotic plaques. It is carried in the circulation bound mostly to LDL and propagates inflammation by hydrolyzing oxidized phospholipids to yield pro-inflam-matory products implicated in endothelial dysfunction, plaque inflammation, and formation of a necrotic core in the plaque. There is modest biological variation in Lp-PLA2 concentrations, but it is smaller than that of CRP. Additionally, Lp-PLA2 is not elevated in systemic

inflammation and so its specificity makes it of value in detecting and monitoring CVD risk.

Reference IntervalPatients with moderate CVD risk (two or more risk factors):≤200 ng/mL Lp-PLA2 LDL-C goal < 130 mg/dL.≥200 ng/mL Lp-PLA2 LDL-C goal < 100 mg/dL (reclas-sify patient as high risk).Patients with high CVD (with CHD or CHD risk equivalents):≤200 ng/mL Lp-PLA2 LDL-C goal < 100 mg/dL.≥200 ng/mL Lp-PLA2 LDL-C goal < 70 mg/dL (reclassify patient as very high risk).

Clinical ApplicationsLp-PLA2 concentration can be used in conjunction with clinical evaluation and patient risk assessment to aid in predicting a patient’s risk for coronary heart disease and ischemic stroke associated with atherosclerosis. It is rec-ommended to assess Lp-PLA2 in patients with two or more CVD risk factors. Patients with LDL cholesterol of <130 mg/dL and an elevated Lp-PLA2 are twice as likely to have a coronary event. Additionally, patients with a normal systolic blood pressure and elevated Lp-PLA2 are twice as likely to suffer a stroke, and those with an elevated systolic blood pressure and an elevated Lp-PLA2 are seven times more likely to suffer a stroke than those patients with a normal Lp-PLA2. Therefore, the goal is to treat the patient until the LDL-C is below the recommended concentra-tion and reclassify them as higher risk based on the Lp-PLA2 concentration if required. Additionally, measurement of Lp-PLA2 and hsCRP may be complementary to identify individuals at increased risk for ischemic stroke.

LimitationsThe assay is in a microplate format and is therefore not available to be implemented on chemistry or immunoassay analyzers found in clinical laboratories. This assay must be run in a CLIA-certified high complexity laboratory. Addi-tionally, hemolysis, HAMA, or other heterophilic antibod-ies may falsely elevate or depress the results.

Assay TechnologyThe assay approved for diagnostic use in Europe and the USA for Lp-PLA2 (PLAC®, diaDexus) is a sandwich enzyme immunoassay using two specific monoclonal anti-bodies. The capture antibody is immobilized on the micro-plate, the detection antibody is conjugated to horseradish peroxidase (HRP), and the substrate, tetramethylbenzi-dine, results in a color change, the absorbance of which is directly proportional to the Lp-PLA2 concentration.

Type of SamplesSerum or plasma samples (ethylenediaminetetraacetic acid [EDTA] or heparin), including gel separator tubes.

Patient samples should not be frozen at −20 °C but should be refrigerated, although not for longer than 7 days. If longer storage is required, the samples may be stored at −70 °C. The samples are stable at room temperature for up to 6 h (not including incubation on the microplate).


B-TYPE NATRIURETIC PEPTIDE (BNP) AND NT-PRO-BNP

FunctionThe natriuretic peptides are a collection of hormones that regulate body fluid homeostasis and blood pressure by diuresis, natriuresis, vasorelaxation, and inhibition of the renin–aldosterone axis. Atrial natriuretic peptide is secreted from the atrium and is a 28 amino acid peptide. B-type natriuretic peptide (BNP) is found in both the brain and the ventricles of the heart and is a 32 amino acid peptide. C-type natriuretic peptide is a 22 amino acid peptide and is thought to originate in the endothelial cells. BNP and the inactive metabolite, NT-proBNP are widely used as markers for HF. Unlike lipid markers that participate in the disease process of atherosclerosis or cardiac markers that are released as a consequence of car-diac injury, BNP is released as a compensatory mechanism to the failing heart to improve left ventricular function by reducing cardiovascular load.

Within the myocyte, Fig. 7 shows that BNP and NT-proBNP are derived from preproBNP, a 134 amino acid peptide. This peptide is cleaved to proBNP (108 amino acids) and a signal peptide. Under conditions of ventricu-lar stretch, proBNP is cleaved to BNP, the biologically active hormone (32 amino acids), and the inactive N-terminal fragment (NT-proBNP, 76 amino acids). Both of these peptides circulate in blood.

Reference IntervalThe concentrations for BNP and NT-proBNP from healthy individuals increase with age. Healthy women have higher values than men. Cutoff concentrations for use in HF areBNP: <100 pg/mL no HF.

100–300 pg/mL suggests HF is present.>300 pg/mL mild HF.>600 pg/mL moderate HF.>900 pg/mL severe HF.

NT-proBNP: <50 years old: <300 pg/mL HF unlikely. >450 pg/mL high probability of HF.

>50 years old: <300 pg/mL HF unlikely. >900 pg/mL high probability of HF (in the absence of renal failure).

Clinical ApplicationsThe New York Heart Association (NYHA) and other pro-fessional groups have established four classifications of HF based on subjective criteria of functional capacities. These are listed in Table 1. Clinical studies have shown that there is an incremental rise in blood concentrations of BNP within the increasing severity of NYHA classes. In patients with systolic dysfunction (ventricular ejection defect), the natriuretic peptide concentrations are inversely correlated to the ejection fraction as measured by echocardiographic analysis. There is no correlation with BNP and the LVEF in diastolic HF (i.e., ventricular filling defect). These clinical studies suggest that BNP

may be useful as a diagnostic marker of CHF. BNP might also be useful in monitoring the success of drug therapy given to these patients. For risk stratification, increased BNP is correlated with a higher risk for death and AMI for patients who present with acute coronary syndromes or CHF. In this regard, BNP provides complementary information to hsCRP and cardiac troponin.

LimitationsBNP is not stable beyond 24 h at 4 °C. NT-proBNP is stable for 72 h at 4 °C. Both assays are increased in patients with chronic renal failure due to volume overload and due to AMI. False positive BNP results are more common in females > 75 years of age. The natriuretic peptides have substantial circadian variation that is not related to HF.

Assay TechnologyAssays for BNP and NT-proBNP are based on two-site sandwich immunoassays using monoclonal antibodies and are available on a point-of-care platform using immuno-chromatography and on automated immunochemistry

myocyte

preproBNP (134 aa)

proBNP (108 aa) signal peptide (26 aa)

secretion

NT-proBNP (1-76) BNP (77-108)circulation

FIGURE 7 Derivation and secretion into blood of BNP and NT-proBNP from myocytes.

TABLE 1 New York Heart Association Classification of HFa

Functional assessmentClass I Patients with cardiac disease but without resulting

limitation of physical activity. Ordinary physical activity does not cause undue fatigue, palpitation, dyspnea, or anginal pain,

Class II Cardiac disease resulting in slight limitation of physical activity. They are comfortable at rest. Ordinary physical activity results in fatigue, palpita-tion, dyspnea, or anginal pain,

Class III Marked limitation of physical activity. They are comfortable at rest. Less than ordinary activity causes fatigue, palpitation, dyspnea, or anginal pain.

Class IV Cardiac disease resulting in inability to carry on any physical activity without discomfort. Symptoms of HF or of the anginal syndrome may be present even at rest. If any physical activity is undertaken, discomfort is increased.

Objective assessmentA No objective evidence of CVD,B Objective evidence of minimal CVD,C Objective evidence of moderately severe CVD, andD Objective evidence of severe CVD.

aFunctional capacity and objective assessment. In Dolgin (1994).


analyzers. The IFCC maintains a list of the commercially available BNP and NT-proBNP assays and their analytical characteristics on its website. It should be noted that some commercially available BNP immunoassays may cross-react with endogenous proBNP in HF patients.

Types of SampleBNP: whole blood or plasma collected in EDTA only.NT-proBNP: serum, heparin, or EDTA plasma.

Frequency of UseBNP and NT-proBNP are widely used for diagnosis and management of HF. These markers have been incorpo-rated into the ESC clinical practice guidelines and the National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines (clinical utilization of car-diac biomarker testing in HF).

GALECTIN-3

FunctionGalectin-3 is a carbohydrate-binding lectin that is secreted by macrophages and is best known for its regulatory func-tions in inflammation, immunity, and cancer. Galectin-3 plays a role in HF including tissue repair, cardiac remodel-ing, and fibrogenesis and therefore acts as a mediator of the development and progression of this disease.

Reference Interval≤17.8 ng/mL: low risk of HF.17.8–25.9 ng/mL: intermediate risk of HF, mortality, and/or hospitalization (should be interpreted with caution as these values lie within the reference interval).>25.9 ng/mL: high risk of HF and mortality.

Clinical ApplicationsIncreased concentrations of galectin-3 are associated with increased risk of HF and also the severity of HF—there is an association between increasing NYHA class (Table 1) and increasing galectin-3 concentrations. Galectin-3 mea-surements can be combined with NT-proBNP measure-ments as there is a complementary relationship between these markers. If both markers are low, patients with HF are at low risk for adverse outcomes, and if both markers are high, HF patients are at the highest risk for adverse outcomes. It has been speculated that blocking galectin-3 may slow the progression of, and the morbidity and mor-tality associated with, HF.

LimitationsFalsely elevated galectin-3 levels occur in samples with vis-ible hemolysis, in patients with certain cancers and other conditions associated with organ fibrosis. Specimens with high levels of gamma-globulins (>2.5 g/dL), HAMA, and rheumatoid factor all have the potential to cause falsely high results. The available assay cannot be placed on chemistry or immunoassay analyzers found in clinical lab-oratories, is labor intensive, and takes approximately 3.5 h to obtain the results.

Assay TechnologyAn assay for galectin-3 (BGM Galectin-3™, BG Medi-cine) approved for diagnostic use in Europe and the USA is a heterogeneous, microtiter plate-based ELISA using two monoclonal antibodies directed against galectin-3 and an HRP-labeled anti-galectin detection antibody. After addition of an HRP substrate, a color change occurs that can be quantified by reading the absorbance at 450 nm—the absorbance is proportional to the galectin-3 in the specimens.

Type of SampleSerum and EDTA plasma.

SOLUBLE ST2 (SST2)

FunctionST2 protein is a member of the interleukin (IL) 1-recep-tor family released upon mechanical stimulation of the cardiomyocytes. IL-33 is a ligand of ST2, and this interac-tion plays a role in preventing cardiac hypertrophy and fibrosis following pressure overload. Increased sST2 con-centrations, therefore, identifies patients with a more remodeled ventricle and decompensated hemodynamic profile.

Reference Intervals>35 ng/mL (males and females) is predictive of all cause mortality from HF.

Clinical ApplicationsIncreased concentrations of sST2 have been reported in HF, and it was shown to be a significant predictor of mor-tality or heart transplantation independent of the natri-uretic peptides. sST2 elevations are strongly predictive of mortality at 1 year following diagnosis of HF, independent of NT-proBNP concentrations, although the highest mortality was in patients that had elevations in both bio-markers. sST2 elevations are predictive of a worse progno-sis in patients with HF and myocardial infarction. Moreover, sST2 concentration is not affected by age, prior diagnosis of HF, body mass index, renal function, atrial fibrillation, or etiology of cardiomyopathy (ischemic vs nonischemic), unlike the natriuretic peptides.

LimitationsST2 is elevated in patients with abnormal type-2 T helper cells such as systemic lupus erythematosus and asthma as well as in inflammatory conditions such as septic shock, pneumonia, chronic obstructive pulmonary disease, and trauma (Fig. 8). The available assay cannot be placed on chemistry or immunoassay analyzers found in clinical lab-oratories and are labor intensive.

Assay TechnologyAn sT2 assay is available and approved for diagnostic use in Europe and the USA. This assay is a sandwich immu-noassay (Presage™ ST2, Critical Diagnostics) carried


out on a microtiter plate. The capture antibody is bioti-nylated, and the detection antibody is conjugated to HRP. A color change is initiated by addition of TMB, and this color change is proportional to the sST2 present in the sample.

Type of SampleEDTA plasma.

PLASMA RENIN

FunctionAngiotensin I is formed from angiotensinogen by the action of renin, which is released from the kidney juxtaglo-merular cells. Angiotensin I is further cleaved to angioten-sin II by angiotensin converting enzyme. Angiotensin II is an extremely potent vasoconstrictor but has a very short half-life, being degraded by angiotensinases to inactive fragments. Because angiotensin II is difficult to measure, angiotensin I levels are determined as a measure of renin activity under conditions in which angiotensin breakdown is blocked by inhibiting plasma-converting enzyme and proteolysis by angiotensinases.

Reference IntervalPlasma renin activity (pH 6.0):

renin (supine): 0.2–2.8 ng/mL/h.renin (upright): 1.5–5.7 ng/mL/h.The above values are only indicative for a sodium intake

of 100–150 mEq per 24 h.

Clinical ApplicationsThe determination of plasma renin activity has been widely used to evaluate the renin–angiotensin system in

disease states. In patients with hypertension due to pri-mary hyperaldosteronism, plasma renin activity is reduced. In contrast, in patients with renovascular hypertension, plasma renin activity and aldosterone secretion are both elevated. Measurement of plasma renin activity has there-fore been suggested as an important aid in the differential diagnosis of primary and secondary aldosteronism. A dif-ferentiation between low and high renin hypertensive states may also be helpful in selecting antihypertensive medications (e.g., angiotensin converting enzyme inhibi-tors vs diuretics).

LimitationsBecause the analyte in this case is generated in the assay, the specificity of the antiserum in question is not critical because sample blanks are deducted from the final results. The interference from some sample proteins may limit the assay. In addition, prorenin, the inactive precursor of renin, is cryoactivated to renin when the sample is exposed to 4 °C temperatures for extended time, leading to falsely high results.

Assay TechnologyThe common assay format is a competitive radioimmuno-assay using 125I-labeled angiotensin I mostly carried out in primary antibody-coated tubes. Nichols Institute Diag-nostics developed a two-site immunoradiometric sandwich assay making use of one antibody coupled to biotin and the second radiolabeled renin antibody. Plasma renin activity can also be measured by liquid chromatography–tandem mass spectrometry. Direct renin concentration immuno-assays are also available: Cisbio Bioassays has an assay approved for diagnostic use in Europe and the USA, but most of the others are only sold for research use only.

Type of SamplePlasma, using EDTA as anticoagulant, as heparin inter-feres with angiotensin I production.

References and Further ReadingAnalytical characteristics of commercial and research high sensitivity cardiac tropo-

nin I and T assays per manufacturer: http://www.ifcc.org/PDF/ScientificActivities/Committees/C-SMCD/cTn_Assay_Table_v091209.pdf

Analytical characteristics of commercially MR-proANP, BNP and NT-proBNP assays as per the manufacturer: http://www.ifcc.org/PDF/ScientificActivities/Committees/C-SMCD/NP_Assay_Tablev091209.pdf

Adams, J.E., Bodor, G.S., Davila-Roman, V.G., Delmez, J.A., Apple, F.S., Ladenson, J.H. and Jaffe, A.S. Cardiac troponin I. A marker with high specific-ity for cardiac injury. Circulation 88, 101–106 (1993).

Apple, F.S. Acute myocardial infarction and coronary reperfusion. Serum cardiac markers for the1990s. Am. J. Clin. Pathol. 97, 217–226 (1992).

Apple, F.S. and Wu, A.H.B. Myocardial infarction redefined: role of cardiac tropo-nin testing. [Editorial]. Clin. Cornerstone 47, 377–379 (2001).

Apple, F.S., Parvin, C.A., Buechler, K.F., Christenson, R.H., Wu, A.H.B. and Jaffe, A.S. Validation of the 99th percentile cutoff independent of assay imprecision (CV) for cardiac troponin monitoring for ruling out myocardial infarction. Clin. Chem. 51, 2198–2200 (2005).

Arakawa, N., Nakamura, M., Aoki, H. and Hiramori, K. Plasma brain natriuretic peptide concentrations predict survival after acute myocardial infarction. J. Am. Coll. Cardiol. 27, 1656–1661 (1996).

Azzazy, H.M.E., Pelsers, M.M.A.L. and Christenson, R.H. Unbound free fatty acids and heart-type fatty acid-binding protein: diagnostic assays and clinical applications. Clin. Chem. 52, 19–29 (2006).

FIGURE 8 Distribution of sST2 plasma concentrations in healthy individuals versus diseased patients. Box-and-whisker plots showing the distribution of sST2 in healthy individuals (healthy, n = 22) compared to patients with heart failure (HF, n = 15), to patients with pneumonia (Pneum, n = 15), to patients with chronic obstructive pulmonary disease (COPD, n = 15), to patients with HF and co-morbidity of pneumonia (HF + Pneum, n = 15), to patients with renal disease (Renal, n = 15), and to patients with sepsis (Sepsis, n = 15), respectively. Used with permission from Dieplingere et al. (2009).


Ballantyne, C.M., Hoogeveen, R.C., Bang, H., Coresh, J., Folsom, A.R., Chambless, L.E., Myerson, M., Wu, K.K., Sharrett, A.R. and Boerwinkle, E. Lipoprotein-associated phospholipase A2, high-sensitivity C-reactive protein, and risk for incident ischemic stroke in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study. Arch. Intern. Med. 165, 2479–2484 (2005).

Bessman, S.P. and Carpenter, C.L. The creatine-creatine phosphate energy shuttle. Ann. Rev. Histochem. 54, 831–862 (1985).

Bodor, G.S., Porter, S., Landt, Y. and Ladenson, J.H. Development of monoclonal antibodies for the assay of cardiac troponin I and preliminary results in sus-pected cases of myocardial infarction. Clin. Chem. 38, 2203–2214 (1992).

Cauliez, B., Santos, H., Bauer, F., Basuyau, J.P., Nadolny, A., Galpy, G. and Lavoinne, A. Cross-reacitivty with endogenous proBNP from heart failure patients for three commercial BNP immunoassays. Clin. Chim. Acta 413, 337–338 (2012).

Chan, K.M., Ladenson, J.H., Pierce, G.F. and Jaffe, A.S. Increased creatine kinase MB in the absence of acute myocardial infarction. Clin. Chem. 32, 2044–2051 (1986).

Chapelle, J.P. and Heusghem, C. Semiquantitative estimation of serum myoglobin by a rapid agglutination method: an emergency screening test for acute myo-cardial infarction. Clin. Chim. Acta. 145, 143–150 (1985).

Chasman, D.I., Shiffman, D., Zee, R.Y.L., Louie, J.Z., Luke, M.M., Rowland, C.M., Catanese, J.J., Buring, J.E., Devlin, J.J. and Ridker, P.M. Polymorphism in the apolipoprotein(a) gene, plasma lipoprotein(a), cardiovascular disease, and low-dose aspirin therapy. Atherosclerosis 203, 371–376 (2009).

Christenson, R.H., Duh, S.-H., Wu, A.H.B., Smith, A., Abel, G., deFilippi, C.R., Wang, S., Adourian, A., Adiletto, C. and Gardiner, P. Multi-center determina-tion of galectin-3 assay performance characteristics: anatomy of a novel assay for use in heart failure. Clin. Biochem. 43, 683–690 (2010).

Davidson, M.H., Corson, M.A., Alberts, M.J., Anderson, J.L., Gorelick, P.B., Jones, P.H., Lerman, A., McConnell, J.P. and Weintraub, H.S. Consensus panel recommendation for incorporating lipoprotein-associated phospholipase A2 testing into cardiovascular disease risk assessment guidelines. Am. J. Cardiol. 101, S51–S57 (2008).

Davidson, M.H. Apolipoprotein measurements: is more widespread use clinically indicated? Clin. Cardiol. 32, 482–486 (2009).

de Boer, R.A., Voors, A.A., Muntendam, P., van Gilst, W.H. and van Velduisen, D.J. Galectin-3: a novel mediator of heart failure development and progression. Eur. J. Heart. Fail. 11, 811–817 (2009).

Dieplinger, B., Januzzi, J.L., Steinmair, M., Gabriel, C., Poelz, W., Haltmayer, M. and Mueller, T. Analytical and clinical evaluation of a novel high-sensitivity assay for measurement of solutble ST2 in human plasma – The Presage™ ST2 assay. Clin. Chim. Acta 409, 33–40 (2009).

Dolgin, M. (ed), Functional capacity and objective assessment. In: Nomenclature and criteria for diagnosis of diseases of the heart and great vessels, 9th edn, 253–255 (Little, Brown and Co., New York, 1994).

Feng, Y.G., Chen, C., Fallon, J.T., Lai, T., Chen, L., Knibbs, D.R., Waters, D.D. and Wu, A.H.B. Comparison of cardiac troponin I, creatine kinase-MB and myoglobin for detection of acute ischemic myocardial injury in a swine model. Am. J. Clin. Pathol. 110, 70–77 (1998).

Fuster, V., Badimon, L., Badimon, J.J. and Chesebro, J.H. The pathogenesis of coronary artery disease and the acute coronary syndromes. N. Engl. J. Med. 326, 242–250 (1992).

Genest, J.J., McNamara, J.R., Salem, D.N., Wilson, P.W.F., Schaefer, E.J. and Malinow, M.R. Plasma homocyst(e)ine levels in men with premature coronary artery disease. J. Am. Coll. Cardiol. 16, 1114–1119 (1990).

Hamm, C.W., Ravkilde, J., Gerhardt, W., Jorgensen, P., Peheim, E., Ljungdahl, L., Goldmann, B. and Katus, H.A. The prognostic value of serum troponin T in unstable angina. N. Engl. J. Med. 327, 146–150 (1992).

Hedges, J.R., Young, G.P., Henkel, G.F., Gibler, W.B., Green, T.R. and Swanson, J.R. Early CK-MB elevations predict ischemic events in stable chest pain patients. Acad. Emerg. Med. 1, 9–16 (1994).

Jaffe, A.S., Vasile, V.C., Milone, M., Saenger, A.K., Olson, K.N. and Apple, F.S. Diseased skeletal muscle: a non-cardiac source for increased circulating con-centrations of cardiac troponin T. J. Am. Coll. Cardiol. 58, 1819–1824 (2011).

Joint European Society of Cardiology/American College of Cardiology Committee Myocardial infarction redefined—a consensus document of the joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. J. Am. Coll. Cardiol. 36, 959–969 (2000).

Kelly, D., Squire, I.B., Khan, S.Q., Quinn, P., Struck, J., Morgenthaler, N.B., Davies, J.E. and Ng, L.L. C-terminal provasopressin (copeptin) is associated with left ventricular dysfunction, remodelling and clinical heart failure in sur-vivors of myocardial infarction. J. Card. Fail. 14, 739–745 (2008).

Konstam, M.A., Dracup, K., Baker, D.W., et al. Heart Failure: Evaluation and Care of Patients with Left-ventricular Systolic Dysfunction (U.S. Dept. Health Human Services, Rockville, MD, 1994).

Ky, B., French, B., McCloskey, K., Rame, J.E., McIntosh, E., Shahi, P., Dries, D.L., Tang, W.H., Wu, A.H., Fang, J.C., Boxer, R., Sweitzer, N.K., Levy, W.C., Goldberg, L.R., Jessup, M. and Cappola, T.P. High-sensitivity ST2 for prediction of adverse outcomes in chronic heart failure. Circ. Heart Fail. 4, 180–187 (2011).

Mair, J., Morndell, D., Genser, N., Lechleitner, P., Dienstl, F. and Puschendorf, B. Equivalent early sensitivities of myoglobin, creatine kinase MB mass, creatine kinase isoform ratios and cardiac troponins I and T for acute myocardial infarc-tion. Clin. Chem. 41, 1266–1277 (1995).

Miles, L.A., Fless, G.M., Levin, E.G., Scanu, A.M. and Plow, E.F. A potential basis for the thrombotic risks associated with lipoprotein(a). Nature 339, 301–303 (1989).

McCullough, P.A., Duc, P., Omland, T., et al. For the BNP Multinational study Investigators. B-type natriuretic peptide and renal function in the diagnosis of heart failure: an analysis from the breathing not Properly Multinational study. Am. J. Kidney. Dis. 41, 571–579 (2003).

Mueller, T., Dieplinger, B., Gegenhuber, A., Poelz, W., Pacher, R. and Haltmayer, M. Increased plasma concentrations of soluble ST2 are predictive for 1-year mortality in patients with acute destabilized heart failure. Clin. Chem. 54, 752–756 (2008).

Muller-Bardorff, M., Hallermayer, K., Schroder, A., Ebert, C., Borgya, A., Gerhardt, W., Remppis, A., Zehelein, J. and Katus, H.A. Improved troponin T ELISA specific for cardiac troponin T isoform: assay development and analyti-cal and clinical validation. Clin. Chem. 43, 458–466 (1997).

Myers, G.L., Christenson, R.H.M., Cushman, M., Ballantyne, C.M., Cooper, G.R., Pfeiffer, C.M., Grundy, S.M., Labarthe, D.R., Levy, D., Rifai, N. and Wilson, P.W.F. National Academy of Biochemistry laboratory Medicine prac-tice guidelines: emerging biomarkers for primary prevention of cardiovascular disease. Clin. Chem. 55, 378–384 (2009).

National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III guidelines: http://www.nhlbi.nih.gov/guidelines/cholesterol/atglance.pdf

Nordestgaard, B.G., Chapman, M.J., Ray, K., Boren, J., Andreotti, F., Watts, G.F., Ginsberg, H., Amarenco, P., Catapano, A., Descamps, O.S., Fisher, E., Kovanen, P.T., Kuivenhoven, J.A., Lesnik, P., Masana, L., Reiner, Z., Taskinen, M.-R., Tokgozoglu, L., Tybjaerg-Hansen, A. and for the European Atherosclerosis Society Consensus Panel Lipoprotein(a) as a cardiovascular risk factor: current status. Eur. Heart J. 31, 2844–2853 (2010).

Pearson, T.A., Mensah, G.A., Alexander, R.W., Anderson, J.L., Cannon, III, R.O., Criqui, M., Fadl, Y.Y., Fortmann, S.P., Hong, Y., Myers, G.L., Rifai, N., Smith, Jr., S.C., Taubert, K., Tracy, R.P. and Vinicor, F. AHA/CDC scientific Statement: markers of inflammation and cardiovascular disease. Circulation 107, 499–511 (2003).

Reinhart, R.A., Gani, K., Arndt, M.R. and Broste, S.K. Apolipoproteins A-I and B as predictors of angiographically defined coronary heart disease. Arch. Intern. Med. 150, 1629–1633 (1990).

Redfield, M.M., Rodeheffer, R.J., Jacobsen, S.J., Mahoney, D.W., Bailey, K.R. and Burnett, Jr., J.C. Plasma brain natriuretic peptide concentration: impact of age and gender. J. Am. Coll. Cardiol. 40, 976–982 (2002).

Reichlin, T., Socrates, T., Egli, P., Potocki, M., Breidthardt, T., Anenja, N., Meissner, J., Noveanu, M., Reiter, M., Twerenbold, R., Schuab, N., Buser, A. and Mueller, C. Use of myeloperoxidase for risk stratification in acute heart failure. Clin. Chem. 56, 944–951 (2010).

Remme, W.J. and Swedberg, K. Guidelines for the diagnosis and treatment of chronic heart failure. Task Force for the diagnosis and treatment of chronic heart failure, European Society of Cardiology. Eur. Heart J. 22, 1527–1560 (2001).

Ridker, P.M., Cushman, M., Stampfer, M.J., Tracy, R.P. and Hennekens, C.H. Inflammation, aspirin and the risk of cardiovascular disease in apparently healthy men. N. Engl. J. Med. 336, 973–979 (1997).

Ridker, P.M., Danielson, E., Fonseca, F.A.H., Genest, J., Gotto, Jr., A.M., Kastelein, J.J.P., Koenig, W., Libby, P., Lorenzatti, A.J., MacFadyen, J.G., Nordestgaard, B.G., Shepherd, J., Willerson, J.T. and Glynn, R.J. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N. Engl. J. Med. 359, 2195–2207 (2008).

Rifai, N. and Ridker, P.M. Proposed cardiovascular risk assessment algorithm using high-sensitivity C-reactive protein and lipid screening. Clin. Chem. 47, 28–30 (2001).

Saenger, A.K., Beyrau, R., Braun, S., Cooray, R., Dolci, A., Freidbank, H., Giannitis, E., Gustafson, S., Handy, B., Katus, H., Melanson, S.E., Panteghini, M., Venge, P., Zorn, M., Jarolim, P., Bruton, D., Jarausch, J. and Jaffe, A.S. Multicenter analytical evaluation of a high-sensitivity troponin T assay, Clin. Chim. Acta 412 (2011). 748–745.

Sealey, J.E. Plasma renin activity and plasma prorenin assays. Clin. Chem. 37, 1811–1819 (1991).

Stampfer, J.J., Malinow, M.R., Willett, W.C., Newcomer, L.M., Upson, B., Ullmann, D., Tishler, P.V. and Hennekens, C.H. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US Physicians. J. Am. Med. Assoc. 268, 877–881 (1992).

Stampfer, J.J., Sacks, F.M., Salvini, S., Willett, W.C. and Henneckens, C.H. A prospective study of cholesterol, apolipoproteins and the risk of myocardial infarction. N. Engl. J. Med. 325, 373–381 (1991).

The Lp-PLA2 Studies Collaboration Lipoprotein-associated phospholipase A2 and risk of coronary disease, stroke and mortality: collaborative analysis of 32 pro-spective studies. Lancet 375, 1536–1544 (2010).

Thygesen, K., Alpert, J.S. and White, H.D. Universal definition of myocardial infarction. J. Am. Coll. Cardiol. 50, 2173–2195 (2007).

Vaidya, H.C. Myoglobin: an early biochemical marker for the diagnosis of acute myocardial infarction. J. Clin. Immunoassay 17, 35–39 (1994).

Vaidya, H.C., Maynard, Y., Dietzler, D.N. and Ladenson, J.H. Direct measure-ment of creatine kinase-MB activity in serum after extraction with a monoclo-nal antibody specific to the MB isoenzyme. Clin. Chem. 32, 657–663 (1986).

Van Leeuwen, M.A. and van Rijswijk, M.H. Acute phase proteins in the monitoring of inflammatory disorders. Baillieres Clin. Rheumatol. 8, 531–552 (1994).


Van Nieuwenhoben, F.A., Kleine, A.H., Wodzig, K.W.H., Hermens, W.T., Kragten, H.A., Maessen, J.G., Punt, C.D., Van Dieijen, P., Vusse, VanDer and Glatz, J.F.C. Discrimination between myocardial and skeletal muscle injury by assessment of the plasma ratio of myoglobin over fatty acid-binding protein. Circulation 92, 2848–2854 (1995).

Wieczorek, S.J., Wu, A.H.B., Christenson, R., Rosano, T., Hager, D., Bailly, K., Dahlen, J., Chambers, B.S. and Maisel, A. A rapid B-type natriuretic peptide (BNP) assay accurately diagnoses left ventricular dysfunction and heart failure – a multi-center evaluation. Am. Heart J. 144, 834–839 (2002).

Wei, C.M., Heublein, D.M., Perrella, M.A., Lerman, A., Rodeheffer, R.J., McGregor, C.G.A., Edwards, W.D., Schaff, H.V. and Burnett, J.C. Natriuretic peptide system in human heart failure. Circulation 88, 1004–1009 (1993).

Wu, A.H.B. (ed), Cardiac Markers, 2nd edn, (Humana, Totowa, NJ, 2003).Yin, W.H., Chen, Y.H., Wei, J., Jen, H.L., Huang, W.P., Young, M.S., Chen, D.C.

and Liu, P.L. Associations between endothelin-1 and adiponectin in chronic heart failure. Cardiology 118, 207–216 (2011).

Yun, D.D. and Alpert, J.S. Acute coronary syndromes. Cardiology 88, 223–237 (1997).

Zuo, W.M., Pratt, R.E., Heusser, C.H., Bews, J.P.A., de Gasparo, M. and Dzau, V.J. Characterization of a monoclonal antibody specific for human active renin. Hypertension 19, 249–254 (1992).

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