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 >1mm) or nuclear
imaging techniques (reduced coronary artery blood ﬂow).
Acute Coronary Syndromes
Patients 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 (>1mm) 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
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
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.
A 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.
Hypertension is deﬁned when the systolic blood pressure
exceeds 140mm Hg and the diastolic blood pressure
exceeds 90mm 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.
CARDIAC TROPONIN (T AND I)
The 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 37kDa.
Cardiac troponin I (cTnI) functions to inhibit calcium-
dependent ATPase and has a mass of 24kDa. Troponin C
is so named because it has four binding sites for calcium,
and weighs 18kDa. 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.
The 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, 6h and 12h to deﬁnitively rule out AMI and at 2–3h
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: 0–0.03ng/mL (10% CV); cTnI: varies with
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).
820 The Immunoassay Handbook
After 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 6–12h 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.
Both 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.
All 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
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
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
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
821CHAPTER 9.12 Cardiac Markers
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
Assays 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 Sample
Serum or heparinized plasma.
Frequency of Use
Troponin has become the standard biomarker for use in
acute coronary syndromes.
CREATINE KINASE AND THE MB
CK 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.
Mitochondrial 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.
Total CK: males 0–200U/L, females 0–160U/L.
Relative index: <2.5%.
Total 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 4–6h after the onset of chest pain, peak at 18–24h,
and return to normal with 72h (Fig. 5). If there are no
further episodes of ischemic injury, the activity declines in
a mono-exponential fashion.
The 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.
Either 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).
822 The Immunoassay Handbook
Desirable Assay Performance
The assay for CK-MB should be sensitive to below 1ng/mL
and have a dynamic range of at least 100ng/mL. The accept-
able precision is about 10% at the cutoff concentration.
Type of Sample
Serum or heparinized plasma.
Frequency of Use
If 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 is an iron-containing protein with a molecular
mass of 18kDa. 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.
After 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
6h after infarction peaking within 5–18h. 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
24h 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.
Myoglobin 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
The 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
Desirable Assay Performance
The myoglobin assay should have a sensitivity of <5ng/mL
and a dynamic range of at least 500ng/mL. The acceptable
imprecision is about 10% at the cutoff concentration.
Types of Sample
Serum, plasma, or urine.
Frequency of Use
Myoglobin 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
Fatty 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
The clinical performance of free FABP is similar to that of
myoglobin. Both are low molecular weight proteins that are
released within 3–6h 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.
The 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
* Provisional cutoffs.
823CHAPTER 9.12 Cardiac Markers
All assays for FABP are based on a two-site immunometric
Frequency of Use
Commercial 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
The 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
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.
Adult male Apo AI: 94–176mg/dL.
Adult female Apo AI: 101–198mg/dL.
Adult male Apo B: 52–109mg/dL.
Adult female Apo B: 49–103mg/dL.
Adult Apo B/A1 ratio: <0.29 below average risk.
0.29–1.30 average risk.
>1.30 above average risk.
Patients 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.
The 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
Many 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 Sample
Frequency of Use
Lipoprotein (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.
Several 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 30mg/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-
of diet and exercise. Recent clinical studies have shown, how-
ever, that plasma concentrations of Lp(a) can be lowered by
824 The Immunoassay Handbook
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.
The 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 800kDa. 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.
Assays 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 Sample
Fasting serum or plasma.
Frequency of Use
Assays 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 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.
Total homocysteine: ≤10µmol/L desirable.
>10 to <15µmol/L intermediate (low to high).
15 to <30µmol/L high.
30µmol/L very high.
Homocysteinemia 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 2mg for B6, 5µg for B12, and 200µg for
folic acid. To increase the folate concentration in the gen-
eral population, grain products are supplemented with
folate in the USA.
Total 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
Standardized assays should be used, and the analytical per-
formance goals are imprecision of <5%, <10% bias, and
<18% total error.
Type of Sample
Fasting serum or plasma.
C-reactive protein was ﬁrst described in 1930. It is an acute-
phase protein that is released into the circulation during
825CHAPTER 9.12 Cardiac Markers
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 118kDa. In patients suffering acute
inﬂammation, CRP is released into blood within 24h to
exceptionally high levels (>1000 times the normal range).
Normal sensitivity assay: <10mg/L.
High-sensitivity assay: <1.0mg/L low cardiovascular risk.
See Fig. 6.
1.0–3.0mg/L average risk.
3.1–10.0mg/L high risk.
>10.0mg/L very high risk.
The 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 <130mg/dL
but hsCRP of ≥2mg/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.
If 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)).
826 The Immunoassay Handbook
However, for CVD risk, high-sensitivity CRP assays are
necessary. The limit of quantitation necessary for this use
is ≤0.3mg/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
C-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
Standardized assays should be used, and <10% impreci-
sion throughout the analytical measurement range is
Types of Samples
Serum and heparinized plasma.
PHOSPHOLIPASE A2 (LP-PLA2)
Lp-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.
Patients with moderate CVD risk (two or more risk factors):
≤200ng/mL Lp-PLA2 LDL-C goal<130mg/dL.
≥200ng/mL Lp-PLA2 LDL-C goal<100mg/dL (reclas-
sify patient as high risk).
Patients with high CVD (with CHD or CHD risk
≤200ng/mL Lp-PLA2 LDL-C goal<100mg/dL.
≥200ng/mL Lp-PLA2 LDL-C goal<70mg/dL (reclassify
patient as very high risk).
Lp-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
<130mg/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-
of Lp-PLA2 and hsCRP may be complementary to identify
individuals at increased risk for ischemic stroke.
The 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.
The 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 Samples
Serum 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 6h (not including incubation on the microplate).
827CHAPTER 9.12 Cardiac Markers
B-TYPE NATRIURETIC PEPTIDE (BNP)
The 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.
The 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 are
BNP: <100pg/mL no HF.
100–300pg/mL suggests HF is present.
>300pg/mL mild HF.
>600pg/mL moderate HF.
>900pg/mL severe HF.
NT-proBNP: <50 years old: <300pg/mL HF unlikely.
>450pg/mL high probability
>50 years old: <300pg/mL HF unlikely.
>900pg/mL high probability
of HF (in the absence of
The 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.
BNP is not stable beyond 24h at 4°C. NT-proBNP is
stable for 72h 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>75years of age. The natriuretic peptides have
substantial circadian variation that is not related to HF.
Assays 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
preproBNP (134 aa)
proBNP (108 aa) signal peptide (26 aa)
NT-proBNP (1-76) BNP (77-108)
FIGURE 7 Derivation and secretion into blood of BNP and NT-
proBNP from myocytes.
TABLE 1 New York Heart Association Classiﬁcation of HFa
Class 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
A No objective evidence of CVD,
B Objective evidence of minimal CVD,
C Objective evidence of moderately severe CVD, and
D Objective evidence of severe CVD.
aFunctional capacity and objective assessment. In Dolgin (1994).
828 The Immunoassay Handbook
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 Sample
BNP: whole blood or plasma collected in EDTA only.
NT-proBNP: serum, heparin, or EDTA plasma.
Frequency of Use
BNP 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 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.
≤17.8ng/mL: low risk of HF.
17.8–25.9ng/mL: intermediate risk of HF, mortality, and/
or hospitalization (should be interpreted with caution as
these values lie within the reference interval).
>25.9ng/mL: high risk of HF and mortality.
Increased 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.
Falsely 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 (>2.5g/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 3.5h
to obtain the results.
An 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 450nm—
the absorbance is proportional to the galectin-3 in the
Type of Sample
Serum and EDTA plasma.
SOLUBLE ST2 (SST2)
ST2 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
>35ng/mL (males and females) is predictive of all cause
mortality from HF.
Increased 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.
ST2 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.
An 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
829CHAPTER 9.12 Cardiac Markers
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 Sample
Angiotensin 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.
Plasma renin activity (pH 6.0):
renin (supine): 0.2–2.8ng/mL/h.
renin (upright): 1.5–5.7ng/mL/h.
The above values are only indicative for a sodium intake
of 100–150mEq per 24h.
The 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).
Because 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
The 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 Sample
Plasma, using EDTA as anticoagulant, as heparin inter-
feres with angiotensin I production.
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FIGURE 8 Distribution of sST2 plasma concentrations in healthy
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