|Cardiac markers and point-of-care testing: the perfect fit
|Each year, more than six million Americans visit
clinics and emergency departments reporting chest discomfort. These patients must be
evaluated accurately, quickly, and efficiently to optimise outcomes, conserve healthcare
resources and minimise the likelihood of litigation for improper diagnosis. It has been
reported that each year up to 70 per cent of patients admitted to coronary care units for
suspected acute myocardial infarction (AMI) are later discharged with a different
diagnosis costing billions of dollars.
Biochemical markers are of increasing importance in diag-nostic strategies for ruling in
and ruling out AMI, particularly when electrocardio-graphic(ECG) findings do not allow a
diagnosis. During the 1990s, physicians showed great interest in the utilisation of
biomarkers to facilitate the diagnosis and treatment of patients with acute coronary
syndromes. Today, cardiac markers are a multimillion-dollar industry. In the Western
world, serial testing for cardiac markers are performed on nearly every patient presenting
with chest pain.
markers are of increasing importance in diagnostic strategies for ruling in and ruling out
acute myocardial infarction (AMI), particularly when electrocar-diographic (ECG) findings
do not allow a diagnosis. Point-of-care (POC) or near-patient testing allows
for diagnostic assays to be performed at the site of patient care delivery. The
biochemical markers that are commonly used by physicians to aid in the diagnosis of AMI
are myoglobin, CK-MB, troponin I, and troponin T. Currently available POC assays possess
comparable diagnostic performance to laboratory based cardiac marker assays providing the
opportunity for POC testing to evolve into the standard-of-care for evaluating more than
six million American patients with chest pain. Full acceptance of this relatively new
technology will not be realised until users reach a comfort level where utilisation of
these devices is foolproof and they have faith in the results.
Point-of-care (POC) or near-patient testing allows for diagnostic assays to
be performed at the site of patient care delivery such as the ED, chest pain evaluation
center, or intensive care unit (I C U ). Compared with centralised laboratory testing, POC
testing provides for rapid clinical decision making by reducing the time spent ordering
tests, collecting and transporting samples, as well as retrieving data. Increasingly, POC
tests for cardiac markers are being researched, developed, and put into clinical use. Most
of these new POC devices have common attributes in that they use small quantities of whole
blood; return results in 10 to 30 minutes; and demonstrate accuracy and precision
comparable to laboratory based analysers.The
Acute Myocardial Infarction and Cardiac Markers Typically, AMI is the result of a sudden
occlusion that decreases blood flow to a portion of the myocardium, causing cell death. In
the majority of cases, the mechanism accountable for the occlusion is a focal rupture of
the surface of an atherosclerotic plaque causing a thrombus to form at the point of
The World Health Organisations (WHO) criteria for diagnosing AMI was developed in
1979 and specifies that two of the following three criteria must be met:
characteristic chest pain
diagnostic ECG changes
elevation of serial enzyme changes.
Many of the biochemical markers (described below) that came into use after the publishing
of this criteria are not themselves enzymes, such as myoglobin and the troponins. This has
lead to a call for revision of the criteria.
Serial measurement of biochemical markers is now universally accepted as an important
determinant in AMI diagnosis. In addition, new biochemical markers have demonstrated
significant benefit in the risk stratification of patients presenting with unstable
angina, as well as in outcome prediction and therapy selection.
The ideal cardiac marker is one that has high clinical sensitivity and specificity,
appears early after AMI, remains abnormal for several days after AMI, and can be assayed
with a rapid turnaround time. Today, there is no single marker capable of meeting all
these criteria necessitating a multi-analyte approach. The biochemical markers that are
commonly used by physicians to aid in the diagnosis of AMI are myoglobin, CK-MB, troponin
I, and troponin T. As is depicted in the graph below, cardiac markers follow a specific,
predictable pattern of release kinetics following a coronary event. The differences in the
time that it takes each marker to reach peak concentration has made it standard practice
for clinicians to make use of at least two different markers in tandem; an early marker,
and a later one.
Myoglobin: Myoglobin is a 17.8 kDa oxygen binding heme protein present in both cardiac and
skeletal muscle. Cell injury during AMI releases myoglobin into the blood circulation. As
a small molecule, myoglobin diffuses quickly into the circulatory system and can thus be
detected soon after an AMI, reaching twice unaffected serum concentrations within two to
three hours after AMI and peaking within four to six hours. Quick release into the blood
stream makes myoglobin a valuable, early screening test for AMI. However, as it is not
cardiac specific, elevated levels should be interpreted with caution if the patient being
evaluated has renal dysfunction or skeletal muscle injury.
Creatine Kinase and CK-MB:
Creatine kinase is an important enzyme regulator of high energy phosphate production and
utilisation within contractile tissues. CK has different isoforms (isoenzymes) depending
on the tissue from which it originates: CK-BB, the brain component; CK-MM, the skeletal
muscle compo-nent; and CK-MB, produced primarily in the myocardium. Total CK, being made
up of these three molecular forms originating from a variety of tissues, is a poor
indicator of AMI. However, CK-MB (86 kDa), is more specific to coronary events and has
been the gold standard among biochemical markers for diag-nosis of AMI. Serially measuring
CK-MB is an established criteria for myocardial cell injury when ECG is inconclusive with
levels reaching twice the unaffected concentrations within five to six hours after the
onset of chest pain and peaking in 12 to 24 hours. As with myoglobin, there are a number
of non-AMI related pathologies (e.g. urinary tract infection, seizures, congestive heart
failure) that may result in increased levels of CK-MB, hence caution must be used in the
interpretation of results.
The Troponins: In striated muscle, the thin filament contains a calcium regulating protein
complex containing three polypeptides, troponin C, I, and T (all < 30 kDa). Only TnT
and TnI are sensitive and specific for cardiac damage.
Troponin T, which has a muscular tissue component, may elevate in muscle injury
or renal failure whereas troponin I offers greater specificity for AMI assessment because
it is exclusive to heart muscle tissue. TnI kinetics following AMI are similar to CK-MB in
that it takes four to eight hours to increase above the upper reference limit, peaks
between 14 and 36 hours and may remain elevated three to seven days after the event. TnT
mimics the early release kinetics of TnI, but may remain elevated for as long as three
weeks. Late clearance of troponin make it a good marker for diagnosing AMI at later stages
but reduce its utility in assessing recurrent AMIs.
In 1999, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)
released evidence and suggestions on the use of biochemical markers for the triage
diag-nosis of acute coronary syndromes. For hospitals with a protocol for rapid AMI ruling
out, in the absence of definitive ECG evidence, there is a sampling frequency recommended
for the detection of AMI by biochemical markers.
Using upper reference limits as a diagnostic threshold, the negative predictive value for
myoglobin is approximately 100% four hours after admis-sion allowing for early exclu-sion
of AMI. Positive results for troponin lead to triage of a patient to the appropriate level
of care, without the need for necessarily completing the sequence of blood samples.
of Care Technology
Antibodies have been in use as diagnostic reagents for over 50 years, but the modern day
immunoassay, now one of the fastest growing technologies in clinical diagnostics, owes
itself to the work of Yallow and Berson.
Point of care immunoassay devices can be classified as either qualitative or
quantita-tive. Qualitative POC devices, such as home-pregnancy tests, only yield a
positive/negative result depending on whether the analyte of interest meets or exceeds a
predefined threshold. If the test is positive, the result is visibly recognised, usually
as a change in colour. Quantitative POC devices analyse the specimen and provide a number
correlating to the amount of analyte in the sample. The choice of which type of assay to
use depends upon the needs of the user.
Qualitative: Spectral Diagnostics (www.spectral-diagnostics.com, Toronto, Canada)
Cardiac STATus combination test employs solid-phase chromatographic immunoassay technology
to simultaneously detect the presence of Troponin I, CK-MB, and myoglobin above an
established cutoff level in human blood, serum and plasma samples. After a specimen has
been dispensed into the sample well, plasma or serum is transferred into a region
containing monoclonal antibody-dye conjugates and biotinylated polyclonal anti-bodies.
These antibodies bind to their respective analytes in the sample to form complexes, which
migrate through the reaction strip. The antigen/antibody dye complexes are then captured
by immobilised streptavidin in the capture area. Additional protein dye conjugates not
bound in the test area are later captured in the control area. Visible purplish horizontal
bands will appear in the test and control areas if the level of the analytes is above the
The Cardiac STATus combination assay provides a qualitative analytical test result. The
qualitative nature of this assay does not provide information about change either
the rise or fall in the concentration of the analytes with single testing.
Fixed Quantitative: The Dade Behring (www.dade-behring. com, Deerfield, IL)
Stratus CS, a fluorometric enzyme immunoassay analyser based on solid-phase radial
partition immunoassay technology, performs quantitative measurements of CK-MB, myoglobin,
and troponin I in whole blood samples collected using lithium heparinate as anticoagulant.
The samples are processed automatically by the analyser, which provides the option of
centrifugation if heparinized plasma is not dispensed directly into the sample cup for
analysis. In this procedure, dendrimer linked monoclonal antibody is added to the centre
portion of a square piece of glass fibre paper in the TestPak. This antibody recognises a
distinct antigenic site on the cardiac marker of interest. Sample is then added onto the
paper where it reacts with the immobilized anti-body. After a short incubation, a
conjugate consisting of enzyme labeled monoclonal antibody directed against a second
distinct antigenic site on the cardiac marker is pipetted onto the reaction zone of the
paper. During this second incubation period, enzyme-labeled antibody reacts with the bound
cardiac marker forming an antibody-antigen labeled antibody sandwich. The unbound labeled
antibody is later eluted from the field of view of the analyser by applying a substrate
wash solution to the centre of the reaction zone. The enzymatic rate of the bound fraction
increases directly with the concentra-tion of analyte in the sample. The reaction rate can
then be measured by an optical system that monitors the reaction rate via front surface
fluorescence. All data analysis functions are performed by the micro-processor within the
The Stratus CS is designed for emergency departments and other locations where
quick and effective diagnosis is required. It is also suited for low volume cardiac
testing performed in a clinical laboratory.
Portable Quantitative: The RAMP System by Response Biomedical (www.respon-sebio.com,
Vancouver, Canada) consists of the following components: (i) a portable fluorescence
reader that is used to quantify antibody antigen complexes, and (ii) a disposable
cartridge that houses an analyte-specific immunochromato-graphic strip.
The RAMP assay cartridge contains a mylar-backed nitrocellulose strip on which the assay
runs. On the membrane strip are two populations of latex particles each in dependently
fluorescently labeled and tagged with specific monoclonal anti-bodies to generate two
independent signals during the assay reaction. One of these is the test reaction and the
other is an independent internal standard. An internal standard has been incorporated into
the RAMP tests that run concurrently with every test assay to compensate for test-to-test
variations in membrane and sample properties.
The RAMP Reader incorporates two detectors, one for each wavelength of dye incorporated in
the latex particles. When the reaction in the cartridge is complete, the reader scans the
test strip, causing the dyes to fluoresce at two independent wavelengths that can be
detected by the reader. The RAMP reader determines the absolute concentration of analyte.
To perform this task, the reader calculates the ratio between the concentrations of latex
particles in the test and internal standard reactions, refers to an analyte-specific
calibration curve, and converts this ratio to analyte concentration.
To perform a test, the operator places an anticoagulated whole blood sample into the well
of a test cartridge and inserts the cartridge into the reader. After application, the
sample is drawn by capillary action along the membrane through the contact and internal
standard zones. The analyte present in the sample will interact and bind with the
antibodies at the contact zone. The readers two detectors separately measure the
amount of fluorescence emitted by the complexes bound on the detection and internal
standard zones. To establish a quantitative reading, the reader calculates the ratio
between the two readings. By calculating the final test result as a ratio between the two
measurements, the RAMP system automatically accounts for variations in sample and membrane
properties. A blank or abnormal reading from the internal standard automatically
invalidates the test, acting as an automatic quality control.
Within 10 minutes from cartridge insertion into the reader the assay is complete, and the
test result appears on the LCD readout. The result can also be stored, printed, or
uploaded to a laboratory or hospital information system.
The RAMP System is being developed for use in all POC settings, and due to its compact
size, and battery operation, it is a truly portable testing system. The RAMP system is
proceeding to clinical trials for FDA submission in 2001. The system is designed to
accommodate a broad spectrum of analytes, of which myoglobin will be the first to go to
trials. Tests for CK-MB and Troponin I will follow soon after.
The ultimate goal of POC testing is to improve patient outcomes. Full acceptance of this
new technology will not be realised until a comfort level is reached by users where
utilisation is fool-proof and they have faith in the results.
New POC assays possess comparable diagnostic performance to laboratory based cardiac
marker assays providing the opportunity for POC testing to evolve into the standard of
care for evaluating over six million American patients presenting with chest discomfort.
The decision as to which device to use, which markers to employ and when to perform the
assays must be made by each individual institution.
Recommendations by societies such as the IFCC will assist in the decision making process
but there are no clearly obvious answers. New biochemical markers will continue to be
developed as new information is acquired on the pathophysiology of AMI and as new
therapeutic strategies develop.
>> Supplied by Joanne Stephenson & Paul Harris, PhD, Response
Biomedical Corporation, British Columbia, Canada. For more details phone (604) 681-4101.