PATHOLOGY AND LABORATORY MEDICINE - PART 5 pps

TROPONIN T ANTIBODIES AND ASSAY cTnT is very similar to cTnI as a biochemical marker of myocardial cell death.. As the molar concentration of cTnT in human blood is the same as the conce

could be detected in patients’ blood It was demonstrated that about the half of cTnIcirculating in patients’ blood is phosphorylated by PKA (4,43), but it is unknown yetwhat part of circulating cTnI is phosphorylated by PKC.

Phosphorylation changes the structure and conformation of the cTnI molecule, andthe affinity of interaction between the components of the troponin complex Thus, phos-phorylation can change the interaction of some antibodies with their epitopes SeveralMAbs, recognizing only phosphorylated cTnI, or vice versa, only dephosphorylated pro-tein, were described in literature during the last few years (4,43,44) If such antibodieswere to be used in cTnI immunoassay, a considerable part of the antigen in a patient’sblood would remain undetected Hence, it is preferable that the antibodies selected forthe immunoassay development should be specific to the epitopes different from the sites

of phosphorylation, so that interaction of such antibodies with the antigen will be fected by any type of phosphorylation

unaf-Oxidation of cTnI

cTnI has two cysteines at 79 and 96 positions (11) that can be oxidized or reduced invitro Oxidation/reduction changes the structure and the conformation of the proteinand thus changes the interaction of some antibodies with the number of epitopes Wu

et al (5) demonstrated that three out of nine tested commercially available assays weresensitive (higher response) to the oxidation of the antigen, whereas for others there was

no difference for the form of the protein tested Although it is still unclear in what form

—oxidized or reduced—cTnI releases from damaged cardiac tissue after AMI and lates in human blood, it is preferable that antibodies used in the assay recognize bothforms with the same efficiency

circu-Complexes of cTnI with Polyanions

As mentioned previously, cTnI is a highly basic protein with pI = 9.87 and more orless equal distribution of basic amino acid residues along the molecule At physiologi-cal pH, cTnI carries a high positive charge Electrostatic interaction is a main type ofinteraction of cTnI with other molecules Electrostatic interaction is very important forthe formation of binary complex between cTnI and highly acidic TnC (pI = 4.05 forslow skeletal isoform of TnC, expressed in cardiac tissue) Electrostatic interactioncan also be responsible for the formation of different types of complexes between cTnIand other than TnC acidic molecules circulating in blood One such known complex isthat between cTnI and heparin—a drug widely used in clinical practice to prevent bloodclotting Heparin is also widely used as an anticoagulant for the collection of plasma.Recent studies demonstrated that the effect of heparin on the interaction of cTnI withantibodies is very similar to that of cTnI—TnC complex formation In addition, similar

to the sensitivity of some immunoassays to cTnI–TnC complex formation, some cial and in-house assays are very sensitive to the presence of heparin in the sample, whereasothers show no differences for samples collected with or without heparin (4,32,45,46).Studying the negative influence of heparin on the signal level in three commercialassays, Wagner et al (47) demonstrated that the effect of heparin can be significantlydiminished by adding to the samples heparin antagonists, such as protamine sulfate orhexadimethrine bromide But it is absolutely clear that while developing new assays, it

commer-is preferable to check the antibodies to their sensitivity to heparin and select those thatgive the same response to the antigen independent of the presence or absence of hepa-rin in the sample.

Autoantibodies to cTnI

Autoantibodies to different components of skeletal and cardiac contractile systemsare described in the literature (48–50) The presence of autoantibodies in the samplecan complicate protein quantitative and qualitative measurements by immunologicalmethods because of the possible competition of the autoantibodies and the antibodiesutilized in the assay To date, there has been only one case described of autoantibodies

to cTnI in a patient’s blood Bohner et al (51) reported on a 69-yr-old coronary arterybypass graft patient with diffuse three-vessel disease that was falsely negative whenmeasured by Dade’s cTnI assay, but positive with troponin T and CK-MB assays It wasdemonstrated that the patient’s blood contained anti-cTnI autoantibodies, which com-peted for binding sites with the antibodies utilized in the assay The authors did not clar-ify the epitope specificity of autoantibodies, so we can only speculate on what part of thecTnI molecule served as an antigen for autoantibody production by the patients Werethere only one or two motifs recognized by the antibodies from Dade’s assay, or werethere other regions that could have been the target for host antibody production?ANTIBODY SELECTION

Affinity of Antibodies

Affinity of the antibodies is one of the crucial factors that should be considered whenantibodies are selected The assay sensitivity strongly depends on the affinity of theantibodies used For cTnI assays, the sensitivity is very important cTnI concentration inthe blood of AMI patients is low—usually between 0.1 and 10 ng/mL and rarely reaching

a level of 50–100 ng/mL Recent studies have shown that the detection of small changes(0.01–1 ng/mL) in the cTnI concentration in the blood of patients with unstable anginacould be very important for the detection of minor myocardial damage, and have asignificant prognostic value (52–57) Minor myocardial cell injury as detected by cTnI

is found in about 30–40% of patients with unstable angina These patients have a poorshort-term outcome (56)

At the same time, the high sensitivity of cTnI assays is very important for the earlydiagnosis of MI during the first 2–3 h after onset of the chest pain, when cTnI concen-tration in the patient’s blood just exceeds a normal level Utilization of high-affinityantibodies also decreases the assay turnaround time The original research on cTnI required24–36 h (1), whereas only 10–20 min are needed to obtain results by contemporaryassays that utilize high-affinity antibodies (58,59) Thus, the cTnI assay should be able todetect low and very low concentrations of the analyte in the sample within a short period

of time This is possible only in the case when both (capture and detection) antibodiesrecognize the antigen with high affinity

Mono- or Polyclonal Antibodies?

As was discussed above, a wide diversity of cTnI forms is released from damaged diac tissue after MI There are two approaches to extract the main part of cTnI modifica-

car-tions from blood samples The first is to use as capture antibodies generated from mals immunized with either a whole cTnI molecule or, preferably, with synthetic pep-tides corresponding to the different parts of the molecule Multipoint binding of theantigen by polyclonal antibodies should increase the avidity of antibody–antigen inter-action, and as a result increase the sensitivity of the assay But the utilization of polyclonalantibodies in the cTnI assay has two main shortcomings Antibodies should be highlycardiospecific But after animal immunization with the whole molecule or by peptides,the total pool of antibodies recognizing cTnI contains fraction that may cross-reactwith the skeletal isoform of the protein Extraction of this fraction is expensive andtime consuming Another problem is the inability of duplicating the production of goodpolyclonal antibodies, a feature that is essential for all clinical applications The solutionhere is to use several (two or three) MAbs specific to different parts of the molecule asantibodies for capture and detection Preferably, all antibodies should not be affected

ani-by any of known cTnI modifications and biochemical factors Such an approach—dual

or triple monoclonal solid phase—helps to improve the sensitivity and reproducibility(unpublished observations and ref 60) The other option is to utilize two MAbs speci-fic to the sites that are not affected by any known modification, with the epitopes located

in the stable part of the molecule as close to each other as it is possible Such approachworks well in the new generation of Access® AccuTnI™ method (32,60,62)

Epitope Mapping

The epitope location of the majority of antibodies described in literature is well mented Some mono- or polyclonal antibodies were generated after animal immunizationwith synthetic peptides (e.g., polyclonal antibodies to peptides 1–4 coming from FortronBio Science) and in this case the epitope location is restricted by peptide sequence Others(monoclonal) antibodies were obtained after mice were immunized with purified cTnI(27,63,64) or whole cardiac troponin complex (65) In this case the epitope location wasdetermined by peptide mapping (66) or, more precisely, by the SPOT technique (65,67–69) The SPOT method utilizes the library of short (10–15 amino acid residues) over-lapping peptides corresponding to the whole cTnI sequence, synthesized with steps of one

docu-to five amino acid residues The SPOT method makes possible precise epidocu-tope mappingwith the uncertainty in one or two amino acid residues

Interestingly, among MAbs generated after animals were immunized with isolatedcTnI or by whole cardiac troponin complex, 90% recognized short peptides (65) Thus, themajority of produced anti-cTnI antibodies are specific to linear motives, and not to theconformational epitopes This observation is in agreement with the present conception

of the cTnI spatial pattern, that is, the cTnI molecule does not have a complex ternarystructure This feature facilitates the production of antibodies by animal immunizationwith predetermined specificity using short peptides When the conformational epitopesare absent, the short synthetic peptides (10 to 12 amino acid residues), which have noternary structure, can be used as an appropriate immunogen for antibody production.Polyclonal and especially monoclonal antibodies, with precisely determined epitopes,are important tools in the biochemical studies of cTnI in blood, and could be very helpful

in the deliberate search of the appropriate epitopes to be used as a targets for antibodyproduction

Antibodies Recognizing cTnI from Different Animal Species

Animal models are widely used in the trials of new drugs, in the development of newmethods of surgery, and in organ transplantation In all these cases, the effect of any newdrug or technology on cardiac function and on cardiomyocyte viability should be esti-mated (70–72) Choosing between equal possibilities, it is preferable to have in the assaythe antibodies that are cross-reacting with cTnI from different animal species Such assayscould be used not only in clinical practice but also in experimental scientific work and

in the preclinical studies

TROPONIN T ANTIBODIES AND ASSAY

cTnT is very similar to cTnI as a biochemical marker of myocardial cell death Because

in the living cell they exist only as components of a heterotetrameric complex with eachother and TnC, with trace amounts of free proteins, the molar concentrations of cTnI andcTnT in cardiac tissue are equal As a consequence, after infarction, cTnT appears in apatient’s blood simultaneously with cTnI and in the comparable concentrations It reachespeak levels at the same time, and has the same time frame within which it can be detected

in a patient’s blood

As the molar concentration of cTnT in human blood is the same as the concentration

of cTnI, the cTnT assay should utilize high-affinity mono- or polyclonal antibodies, to

be able to detect very low antigen concentrations in the sample Specificity of ies to the cardiac forms of the protein is also very important The first generation cTnTassay (cTnT enzyme-linked immunosorbent assay [ELISA]) utilized detection antibodywith some cross-reactivity with the skeletal isoforms of the protein (69) As a conse-quence, this version of the assay produced falsely positive results from blood obtainedfrom patients with acute or chronicle muscle disease and chronic renal failure The false-positive results were explained by cross-reaction of antibodies with the skeletal isoform

antibod-of the protein The antibodies antibod-of the current generation antibod-of cTnT assay have correctedthis problem

The interaction between cTnT and other components of the troponin complex is nificantly lower, as compared to the interactions of the cTnI–TnC binary complex Incontrast with cTnI, in AMI patients’ blood, cTnT is present mainly as a free moleculeand its proteolytic fragments (5)

sig-cTnT undergoes rapid proteolytic degradation in ischemic and necrotic cardiac sue McDonough et al (37) reported that in the ischemic myocytes proteolytic degra-dation of cTnT results in the accumulation of fragments corresponding to the 191–298residues of cTnT sequence In necrotic tissue (in situ experiments) cTnT was rapidlycleaved by proteases, forming two main peptides with apparent molecular mass 31–33and 14–16 kDa, respectively, the products of sequential proteolysis from the N-termi-nal part of the molecule (unpublished data and ref 72) The epitopes of the antibodiesutilized in the new version of cTnT assay are only six amino acid residues apart (73), andthis feature makes this assay insensitive to the proteolytic degradation of the antigen.Stability studies of cTnT in AMI serum samples described by Baum et al (75) showedthat as measured by the new version of the cTnT assay, cTnT had no loss of immunolog-ical activity after 5 d of storage at room temperature

tis-cTnT can be phosphorylated by PKC (76,77), but we were unable to find studies onstrating the effect of phosphorylation on the interaction between antigen and anti-bodies Resembling some cTnI assays, the current version of the cTnT assay is sensitive

dem-to the presence of heparin in the tested sample, demonstrating a lower response dem-to theheparin-containing blood (serum) (45,46)

As the best among known markers of myocardial cell death, cTnI and cTnT havemuch in common as biochemical and immunochemical targets Knowledge obtained byscientists studying the forms of cTnI in blood can be helpful for those who are develop-ing new antibodies for the next generation cTnT assay

ABBREVIATIONS

AMI, Acute myocardial infarction; CK, creatine kinase; CK-MB, MB isoenzyme ofCK; cTnT, cTnI, and cTnC, cardiac troponin T, I, and C; MAbs, monoclonal antibodies;PKA, protein kinase A; PKC, protein kinase C

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From: Cardiac Markers, Second Edition Edited by: Alan H B Wu @ Humana Press Inc., Totowa, NJ

11

Interferences in Immunoassays for Cardiac Troponin

Kiang-Teck J Yeo and Daniel M Hoefner

INTRODUCTION

Cardiac troponin has largely replaced the MB isoenzyme of creatine kinase MB) as a key biochemical marker in the assessment of myocardial damage because of itshigh sensitivity (1) and cardiac specificity (2) The recent joint proposal by the AmericanCollege of Cardiology (ACC)/European Society for Cardiology (ESC) for the redefini-tion of myocardial infarction (MI) places cardiac troponin in a central role in the diag-nostic workup of MI A cardiac troponin value above the 99th percentile cut point of areference population is considered abnormal and MI is diagnosed when serial troponinsare increased in the clinical setting of acute ischemia (3) Because cardiac troponin has acornerstone role in the diagnosis of MI, has prognostic implications in patients withacute coronary syndromes (ACS) (4–6), and a role in guiding antithrombotic therapy(7–9), it is crucial that troponin assays have robust analytical performance to allow forreliable measurements, especially at low abnormal ranges

(CK-Cardiac troponin assays are two-site “sandwich” immunoassays, with a primary ture antibody and a secondary detector antibody (Fig 1A) As such, various analyticalissues may affect immunoassays, in general, giving rise to occasional false-positive orfalse-negative results in a particular patient In addition, assay imprecision in the upperreference cutoff region can dramatically affect the incidence of false-positive readings

cap-by that method This chapter reviews the effects of the presence of human anti-animalantibodies and autoantibodies, low-end imprecision, and sample matrix differences (serum

vs plasma) on cardiac troponin assays Recognizing these effects will help minimizeincorrect interpretations of this important marker in the assessment of ACS

HUMAN ANTI-ANIMAL ANTIBODIES

Exposure to animal antigens can give rise to human anti-animal antibodies (HA) thatcan cause analytical interferences with various immunoassays (10–13) The HA elicitedcan be of the immunoglobulin (Ig) classes IgG, IgM, IgA, and occasionally IgE Hetero-philic antibodies are human antibodies that arise from challenges by poorly definedanimal immunogens; historically the term was associated with IgM antibodies observedwith mononucleosis (12) If exposure to a specific animal immunogen is known, the cor-rect term should refer to the specific animal that is implicated, rather than classifyingthe antibodies as heterophilic (14) Thus, an HA elicited by exposure to mouse antigens

Fig 1 Types of HA and possible mechanisms of interferences (Adapted from Klee GG [29].)

should be termed “human anti-mouse antibody” (HAMA) The prevalence of HA haspreviously been reported to vary from as low as 0.19% (15) to as high as 80% (12).Various causes that could elicit HA include iatrogenic exposure to animal-derivedpharmaceuticals, such as murine monoclonal antibody-targeted imaging reagents, anti-cancer drugs, and vaccines HA could also arise due to noniatrogenic exposure to animalproteins that occurs during veterinary and farm work, during food preparation, or by thepresence of domestic animals in the home (12) Owing to the increasing use of murinemonoclonal antibodies for imaging and therapeutic drug targeting, the most commontype of HA reported is typically a HAMA It has been reported that even single low-dose injections of radiolabeled murine monoclonal antibodies can elicit HAMA in 41%

of patients within a period of 2 wk (16)

The first description of a falsely elevated cardiac troponin I (cTnI) was the case of a69-yr-old man with an admission cTnI of 106 ng/mL (measured on the Abbott AxSYMmethod), but with no clinical evidence of acute MI (AMI) (17) In the presence of ablocking agent containing animal immunoglobulins, the value decreased to <2.0% ofthe original This indicates the presence of multispecific heterophilic antibodies that linkthe murine monoclonal capture and secondary labeled goat polyclonal antibodies, givingrise to a false-positive result In addition, the original AxSYM cTnI assay appeared to

be more susceptible to generating a heterophilic antibody-derived false-positive resultwhen compared with other commercial immunoassays, which showed essentially nor-mal values for this specimen (17)

This differential susceptibility to HA with different troponin methods may be related

to the effectiveness of the type of blocking agents incorporated into the formulation, theformat of the immunoassay, and the type (e.g., IgG vs IgM) and concentration of thecirculating HA In addition, we and others also reported false-positive cTnI results due topresence of rheumatoid factors (RF) with the AxSYM assay (18–20) and other HA withthe AxSYM and Beckman Access methods (21) RF are usually IgM isotypic antibodiesthat can bind not only human IgG but also a wide variety of animal immunoglobulins(22–24) Enhancement of the troponin assay by Abbott due to the inclusion of effectiveblocking agents has significantly reduced much of this HA interference (15,20), although,

in an occasional patient, a false-positive result may still occur (20)

In contrast, the incidence of HA-related false-negative cTnI is less frequently tered Bohner et al reported a false-negative cTnI in a patient undergoing coronary arterybypass graft surgery who had serial evolving CK-MB and cardiac troponin T (cTnT)indicative of perioperative MI (25) Although they surmised that this interference onthe original Stratus cTnI assay was unlikely due to HA, and attributed it to circulatingcTnI autoantibodies, the presence of a type of blocking HA cannot be conclusivelyruled out

encoun-HA, once developed, are known to persist for months and can be detectable even up to

30 mo after the initial exposure (26,27) Kazmierczak et al recently reported a case studydescribing the sudden appearance of heterophilic antibodies in a patient that causedtransient false-positive CK-MB, cTnI, and cTnT measurements with various commer-cial cardiac marker immunoassays (28) Interestingly, over the course of several weeks,transient spikes of false-positive cTnI were observed interspersed with periods of normalvalues, indicating the highly variable appearance and disappearance of these heterophilicantibodies

Mechanisms of HA Interferences and Elimination

HA that bind to the Fc region of immunoglobulin are termed antiisotypic antibodies,while those that bind to the variable Fab region are termed antiidiotypic antibodies Ingeneral, antiisotypic HA are more commonly encountered than antiidiotypic HA (29)

As shown in Fig 1B,C, either type of HA can bridge the capture and secondary labeledantibodies, causing a false-positive cTn result For example, the AxSYM cTnI immuno-assay has a capture mouse monoclonal antibody/goat polyclonal-labeled second anti-body format; multispecific heterophilic antibodies can bridge the two antibodies givingrise to a false-positive result in the absence of cTnI Alternatively, it is possible that anti-idiotypic or antiisotypic HA can bind the capture antibody in a way that causes sterichindrance to the binding of the ligand, resulting in a false-negative result (Fig 1D,E).False-negative results may also arise from the presence of anti-antiidiotypic HA, whichcan bind to cTn directly and block access of the capture antibody to the ligand (Fig 1F).Various strategies have been used to reduce or eliminate interferences due to HA.Most modern commercial immunoassays have HA blockers (Table 1) incorporated inthe formulation; these may include polyclonal IgG, polymeric mouse IgG, a mixture ofanimal serum proteins, nonimmune serum, or IgG fragments (Fab/Fc) from the speciesemployed to develop the reagent antibodies (12) The efficacy of these blocking agents

is dependent on the concentration, class/subclass, specificity, and valence of the HApresent (30) Thus, it is impossible to eradicate completely HA interferences due to theoccasional presence of a unique subclass of HA that is not blocked by these agents Util-ization of capture and detector reagent antibodies developed in different species (e.g.,murine monoclonal/goat polyclonal) has been proposed to minimize the effects of HAMA(31) However, the effectiveness of this method is questionable, especially in the pres-ence of multispecific heterophilic antibodies that can cross-react with antibodies derivedfrom different animal species (17)

The format of the immunoassay, that is, one-step simultaneous vs a two-step tial incubation, may make the former more susceptible as HA could be washed awayafter the first incubation in a two-step assay (32) Another important consideration is thateven cardiac assays with the same format (e.g., AxSYM CK-MB and original AxSYMcTnI) can show differential susceptibility to heterophilic antibodies (normal CK-MB,

sequen-Table 1

Blocking Reagents and Composition

Company Blocking reagents Composition

Scantibodies Heterophilic blocking Specific murine immunoglobulins with Laboratory, Inc reagent (HBR) high affinity for heterophilic antibodies

Non Specific Antibody Nonspecific immunoglobulins that Blocking Reagent (NABR) passively blocks heterophilic antibodies Bioreclamation Inc Immunoglobulin Inhibiting Proprietary immunoglobulins formulation

Reagent (IIR) with high affinity for HA Omega Biochemicals Heteroblock reagent Active and passive blocking mixture Roche Diagnostics MAB 33 Monoclonal IgG1

Poly MAB 33 Polymeric monoclonal IgG1/Fab

falsely elevated cTnI) if a blocking agent is incorporated into the first reagent step but notthe second (17) Another strategy is to employ Fab or F(ab')2 fragments instead of intactimmunoglobulin in the design of two-site immunoassays in an attempt to eliminate theinterference of antiidiotypic HA with specificity to the Fc region The other approach

is to use chimeric murine/human antibodies as the capture or second labeled antibody(e.g., Roche Elecsys TSH and CEA assays) where the variable region is a murine/humanconstruct; this should minimize the binding of HAMA and other HA (12)

LOW-END PRECISION ISSUES

Owing to the recommendation to use the 99th percentile value (3 SD value abovethe mean) as the upper reference cutoff limit for cardiac troponin assays under the newESC/ACC guidelines (3), it is important to assess how robust these values are under real-istic day-to-day operation of the clinical laboratory Large imprecision of the assay aroundthe 99th percentile cut point will lead to increased frequency of falsely abnormal values

It is conventional to determine reference ranges using an apparently healthy populationwhere samples are measured in either a single analytical run or several runs spanning ashort period of time, and frequently with one reagent lot Thus, the 99th percentile cutpoint determined this way, although statistically valid, might be unrealistically low whenbetween-run assay imprecision is taken into account across several lots of reagents withdifferent analytical calibrations

Recently, we performed a study (33) of six common commercial cardiac troponin assays

to determine the lowest value corresponding to a coefficient of variation (CV) of 20%,which is defined as functional sensitivity The main objective of this project was to inves-tigate if the published upper reference values can be realistically attained under routineclinical laboratory working conditions Precision profiles were determined with fourpatient pools that were assayed over a period of 8–10 wk that spanned the low normal toAMI cutoff ranges We found that out of these six cTn assays, only one had an upper ref-erence limit (URL) that was twice as high as the functional sensitivity The implicationwas that most of the other cTn assays had URLs that were unrealistically low, and if usedwithout modification, would result in an increased incidence of false-positive cTn values.This could cause inappropriate diagnosis or management decisions in the clinical evalu-ation of ACS

The National Academy of Clinical Biochemistry (34) and ESC/ACC (3) have bothrecommended that cardiac troponin assays should show imprecision (CV) of £10% atthe medical decision limits In an attempt to translate the above guidelines to clinicaltrials, Apple et al (35) suggested that the lowest achievable concentration with a CV

of 10% be used as the cut point for cardiac injury They also showed, however, that(based on the information derived from package inserts) at present, no manufacturer ofcardiac troponin assays can meet the standard of £10% CV at the 99th percentile cutpoint, and challenged the industry to define this important cut point systematically Inaddition, development of future generations of cTn assays with improved low-end pre-cision should result in the convergence of the 10% CV and 99th percentile cut points.Such improvements in the precision at the low end are especially crucial for the ability

to translate the recent research finding that minor elevations of cTn are prognostic forselection of patients with unstable angina and non-ST elevation MI for invasive thera-pies (36) to the routine clinical practice

To this end, the Committee of Standardization of Markers of Cardiac Damage, a committee of the International Federation of Clinical Chemistry, has commissioned aninternational collaborative study to determine the 10% CV of various commerciallyavailable cTnI and cTnT assays Our laboratory is participating in this joint effort and hasprepared eight serum pools spanning the upper reference to AMI cutoff values Aliquotswere mailed to participating vendors who will assay these specimens over 20 workingdays with two separate reagent lots and three separate calibrations Precision profiles ofeach troponin assay will be constructed and the corresponding 10% CV value will bedefined This study is currently underway and, when completed, will provide informa-tion that will help determine whether the current 99th percentile reference and 10% CVvalues cited in various cTn assay product inserts can meet the 10% CV requirements asmandated by the ESC/ACC.

sub-IMPACT OF SERUM VS PLASMA SPECIMEN ON cTn ASSAYS

The presence of fibrin strands in plasma or in serum that is processed for analysisprior to complete clot formation is well known to cause errors in many immunoassaysystems Although this is relatively common knowledge in the field of clinical chemis-try, little has been detailed in the scientific literature in this regard Nosanchuk reported

on falsely increased levels of cTnI that were attributed to incomplete serum separation(37) During the analysis of paired serum and plasma samples, several discordant (ele-vated serum) results were observed, which were reproducible on reanalysis However,when the serum samples were recentrifuged, subsequent analysis was in agreement withthe results obtained with plasma In addition, when serum samples that deliberately con-tained microfibrin strands were tested, falsely elevated results were observed (37) Thus,

if serum is used, it is important to ensure that the clotting process is complete prior to trifugation so as to eliminate the presence of microfibrin strands For heparinized sam-ples that have been in storage for extended periods, it is essential to check that fibrinstrands, following heparin degradation, are not present prior to analysis

cen-Plasma is becoming the specimen of choice for most automated immunoassays; one

of the primary reasons for this is that it allows for a decreased turn around time, which isespecially important for potentially emergent assays such as those used to assess cardiacdamage Furthermore, fresh plasma may eliminate the problems associated with fibrin,mentioned above However, most product literature for cTn assays shows a potentialnegative bias in plasma compared to serum Recent reports also indicate that cTnI andcTnT may be falsely decreased in plasma from heparinized blood (38–40) Gerhardt et

al (39) showed that mean levels of cTnT were about 15% lower in heparinized plasmasamples when compared to serum samples In addition, when heparin was added to thesera of patients with AMI, decreased cTnT results were obtained

Troponin exists as a complex of three proteins (cTnC, cTnI, cTnT) that interact withtropomyosin, actin, and the myosin complex to form the functional unit of contraction

in cardiac and skeletal muscle Following myocardial damage, the troponins that arereleased circulate predominantly in one of three forms: the ternary complex containingall three molecular forms; the binary complex consisting of the cTnC–cTnI heterodimer; orthey may exist in free form, of which the cTnT predominates (41,42) It has been observedthat the most discrepant values for plasma vs serum cTn occurred in samples that weretaken during the early phase of myocardial damage, when cytosolic (free) cTnT may be

proportionally most elevated in respect to the cTn complexes (39) Stiegler and othersnoted similar but less dramatic plasma/serum changes for cTnT, which may be a result

of different blood collection systems and heparin concentrations as well as differentcTn complex ratios attributable to the timing of sample collection from the time of thecoronary event (40) Although the National Academy of Clinical Biochemistry recom-mends the use of plasma samples for cTn measurements (34), the consensus of the Ger-hardt and Stiegler studies (39,40) is that, until the plasma–serum bias issues can be resolved,the sample choice for cTn measurements should be serum

The mechanism by which plasma gives falsely lower results is not fully understood.Katrukha et al (43) have suggested that troponin immunoreactivity may be modifiedeither via protein conformational changes or steric hindrance of epitopes upon hepa-rin–cTnI complex formation In addition, the various types of troponin complexes maycreate or conceal various epitopes (42,44) These complexes are influenced by the avail-ability of calcium (45) and, thus, anticoagulants that bind calcium (e.g., ethylene diamine-tetraacetic acid [EDTA]) may have a marked impact on the assay Whereas heparin doesnot appear to have a complex-dissociating effect, addition of EDTA can dissociate thetroponin complexes into individual subunits (41)

DEGRADATION OF cTn AND EPITOPE STABILITY

Bodor et al (46) showed that the degree of cTnI phosphorylation in the failing hearts

of transplant patients is reduced from the level measured in normal hearts It is sible that these heart failure associated changes could affect both epitope stability aswell as antibody recognition Regarding the latter, it has been shown that certain anti-bodies will react only with the phosphorylated form of cTnI (47) In addition, oxida-tion of cTn may also occur, which can induce conformational changes to the protein (45)

plau-Wu et al (41) assessed the influence of the state of protein oxidation/reduction as sured by nine different cTnI immunoassays and found that in some assay systems theapparent concentration varied by more than fourfold, depending on the type of cTn sub-unit and the oxidation/reduction status of the measured complex Because cTnI slowlyoxidizes to form disulfide linkages after blood collection, they warn that unless stabiliz-ing agents are added, some assays may yield a differential response to the reduced vs theoxidized forms (41) Other studies have shown that oxidative modification allows myo-cardial proteins to become more vulnerable to proteolysis during ischemic episodes (48).Besides the differences observed in the immunoreactivity of subunit complexes, immu-noreactive fragments may also be generated as proteins undergo proteolytic cleavage.Degradation of cTnI may have a significant effect on its stability and immunologicalactivity, and it has been observed that cTnI fragments vary significantly in their reactiv-ity This has important analytical and clinical implications in that very little intact cTnIexists in serum samples that are typically collected 1–5 d following MI (49) While thedegradation of cTnI is associated with a loss of immunoreactivity (49), cTnC may enhancethe immunological activity of some, but not all, fragments (50) If the cTnI fragmentcontains the inhibitory region of the molecule, it is relatively resistant to proteolyticdegradation when complexed with cTnC (50) Differential degradation of cTnI is also

mea-an importmea-ant factor in assay-to-assay variability that may account for up to a 20-foldvariation in results obtained with different methods (41,51) Because of the many caveatsthat may influence immunoreactivity, it has been suggested that antibodies developed

for use in troponin assays have the epitope characteristics that are not affected by plex formation, degree of phosphorylation, pI effects, and oxidation/reduction status,and have domains that are resistant to proteolysis (42,50,52) The proteolysis-resistantregion of cTnI lies in the central part of the molecule (43,50); however, one dilemma

com-of developing antibodies to that region is that it is the amino- (N)-terminal sequence com-ofthe molecule that is suggested to generate the high cardiac specificity of the anti-cTnIantibodies (49)

SUMMARY

Current cTnI assays have variable upper reference limits as well as AMI cutoff values,with differences up to 25-fold (Table 2), owing to a lack of standardization betweendifferent commercial assays This is attributed to several factors including the use ofdifferent calibrators, use of antibodies with different epitope specificities, and the pres-ence of various molecular forms of troponin in circulation Occasionally, falsely ele-vated results may arise due to the presence of HA; this situation should be suspected inview of a nonevolving elevated cTn that either does not fit the clinical picture or theserial kinetics of an evolving AMI False-negative cTn results may be more difficult todetect, unless a gross discrepancy exists between the clinical assessment and cTn results

If interference due to HA is suspected, this could be verified by checking the

suspici-Table 2

Cardiac Troponin Assays

AMI (ROC) 99th Percentile cutoff Manufacturer Immunoassay format (ng/mL) a (ng/mL) a

Abbott AxSYM cTnI Monoclonal/goat polyclonal 0.50 2.00

Bayer ACS 180 cTnI Monoclonal/goat polyclonal 0.10 1.00–1.50 Bayer ADVIA Centaur cTnI Monoclonal/goat polyclonal 0.10 1.00–1.50 Bayer Immuno I cTnI Monoclonal/goat polyclonal 0.10 0.90

Beckman Access cTnI Monoclonal/monoclonal 0.04 0.50

BioMerieux Vidas cTnI Monoclonal/monoclonal 0.10 0.80

Biosite Triage cTnI Monoclonal/goat polyclonal 0.19 1.0

Byk-Santec Liason cTnI Monoclonal/goat polyclonal 0.03 NA

Dade Dimension RxL cTnI Monoclonal/goat polyclonal 0.07 1.50

Dade Opus cTnI Monoclonal/goat polyclonal 0.10 1.50

Dade Stratus CS cTnI Monoclonal/monoclonal 0.07 1.50

DPC Immulite cTnI Monoclonal/bovine polyclonal <1.00 d 1.00

First Medical Alpha Dx cTnI Monoclonal/goat polyclonal 0.09 b 0.40

Innotrac cTnI Monoclonal/monoclonal 0.10 b (Serum) 0.20–0.40

0.08 b (Plasma) Tosoh cTnI Monoclonal/monoclonal 0.45 1.35

Ortho Vitros ECi cTnI Monoclonal/goat polyclonal 0.10 c (Serum) 1.00 (Serum)

0.08 c (Plasma) 0.80 (Plasma) Roche Elecsys cTnT Monoclonal/monoclonal 0.01 0.10

a Package insert information or correspondence with manufacturer.

b 95th percentile cut point.

c 97.5th percentile cut point.

d 98.0th percentile cut point.

NA, Not available.

ous result with an alternative cTn assay made by a different manufacturer, or by reassay

of the specimen in the presence of HA blocking agents It is important for each tory to validate the low end of the troponin assay, that is, the value corresponding to a10% CV, so that the incidence of false abnormal results due to analytical imprecision ofthe method are minimized This is currently a very important issue to address, as recentreports have shown that minor elevations of cTn can predict benefits for patients withunstable angina and non-ST elevation MI when they are given invasive treatments (36).Recognition of the various analytical factors that can cause variable immunorecogni-tion of cTn forms/fragments in circulation should help manufacturers develop a bettergeneration of troponin assays that are less affected by these factors, resulting in moreconsistent troponin measurements across all methods

labora-ABBREVIATIONS

ACC, American College of Cardiology; ACS, acute coronary syndrome(s); AMI, acutemyocardial infarction; CK, creatine kinase; CK-MB, MB isoenzyme of CK; cTnT, cTnI,and cTnC, cardiac troponins T, I and C; CV, coefficient of variance; EDTA, ethylenedi-aminetetraacetic acid; ESC, European Society for Cardiology; HA, human anti-animalantibodies; HAMA, human anti-mouse antibody; Ig, immunoglobulin; MI, myocardialinfarction; RF, rheumatoid factors; URL, upper reference limit

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Point-of-care testing (POCT) for cardiac disease is not a recent innovation Testing

at the patient’s bedside was described in the 7th century by the Byzantine TheophilusProtospatharios Detailed technical manuals were published in the 16th century for POCT

as a routine part of the clinical workup for a range of medical conditions including chestpain (1) It is unlikely that the technique then most in vogue, examination and tasting thepatient’s urine, would satisfy current regulatory processes (or find favor with the modernclinical chemist)

ROLE OF BIOCHEMICAL TESTING

IN SUSPECTED ACUTE CORONARY SYNDROMES

The rationale for biochemical testing of patients with suspected acute coronary dromes (?ACS) is to provide an accurate diagnosis and to guide patient management.The electrocardiogram (ECG) is a poor diagnostic tool, with sensitivity as low as 41% (2).Conversely, ST-segment elevation on the ECG is at least 95% predictive of an occludedartery (3,4) The role of the admission ECG is to identify such patients; management isaimed at opening the occluded artery by primary angioplasty or thrombolysis (5) Only aminority of patients present with ST elevation acute myocardial infarction (STEMI)

syn-An audit of hospital admissions with ?ACS revealed biochemical confirmation or sion of myocyte necrosis is required in 90% of cases whereas 5% of patients with signifi-cant cardiac damage are discharged from the emergency department (6) Measurement

exclu-of the cardiac troponins (cTn), cardiac troponin T (cTnT), and cardiac troponin I (cTnI)provides a cardiospecific tool for diagnosis Initial reports demonstrated that cTn measure-ment was prognostic in patients admitted with (7) and without ST-segment elevation(8) This was followed by evidence that cTn values could be used to predict the response

to a range of therapies from low-molecular-weight heparin (9) to revascularization (10,11) The paradigm shift seen in the recent proposed guidelines for diagnosis of acute

MI (AMI) and management of non-ST ACS recognizes the importance of the role of diac marker measurements (12,13) More recently, other biomarkers such as C-reactive

car-protein (CRP) (14) and B-type natriuretic peptide (BNP) (15) have been shown to vide additional prognostic data in patients admitted with ACS.

pro-Recommendations have been made for the type and speed of service provision forcardiac marker measurement The suggested target for turnaround time (TAT) for cardiacmarkers is 60 min, with the proviso that POCT should be considered as an option (16).This requires that three conditions are fulfilled: that the technology for POCT is ade-quate, that there is evidence to support the assertion that short TAT will have clinicalbenefit, and that such a strategy is cost effective

POCT SYSTEMS FOR CARDIAC MARKERS

The cardiac markers available for routine clinical measurement by POCT comprisemyoglobin, creatine kinase (CK) and its MB isoenzyme (CK-MB), cTnT, cTnI, BNP,and CRP Prototype systems have been described for other markers including fatty acidbinding protein (17) Although these measurement systems are designed primarily foruse in the POCT environment, this distinction is artificial POCT systems are equallyusable in the emergency laboratory, in a satellite laboratory where low throughput pre-cludes the use of a large assay platform, in the main laboratory when the main assay plat-form does not support the tests required, or when a stat capability is required They can

be divided into four categories

Dry Strip Enzyme Activity Measurements

These systems have evolved from the technology used for blood glucose test strips.The basic technology is a multilayer system containing stabilized dried reagents thatare solubilized by the addition of serum or plasma and undergo a series of reactions toproduce an optically read end point They were the first examples of POCT instrumentsfor cardiac markers The first system in clinical use was the Ames seralyzer This sys-tem was capable of measuring CK as part of a range of analytes The system used serum assample and hence required serum separation by centrifugation and a sample prepara-tion step prior to analysis The analytical range is 0–1000 U/L with % coefficient ofvariation (CV) 2.9–5.5 (555–3518 U/L), and the system could be used on the coronarycare unit (CCU) (18) A similar approach was used with extension of multilayer dry filmtechnology by Kodak to the measurement CK and CK-MB with the Ektachem DT-60.This was a desktop version of a larger series of analyzers using the same chemistry forCK-MB activity measurement Again, this instrument required prior separation of serumbut no other prior preparation Experience with these systems showed good precision(% CV: 2.2–8), but agreement with other laboratory methods for CK were variable(19,20) The first true whole blood system was the Reflotron (Roche Diagnostics) Thisused lithium heparin whole blood as the sample Using a fixed volume pipet, 32 µL (range28.5–31.5 µL) is applied to the reagent strip The assay CV was 3% with a linear measur-ing range of 24 to 1400 U/L Samples values may be reported up to about 1800 U/L butare flagged Samples exceeding 1800 U/L are reported as >1400 U/L A multicenterevaluation of the instrument found the median % CV was 3.1 Agreement with conven-tional laboratory methods is good (r = 0.99), but values are not identical (21) In routineclinical use with nonlaboratory operators, greater variability is found than in the hands

of trained laboratory personnel (22)

These systems have two problems They require a reasonable degree of operatorskill, especially in pipetting blood, and the use of an independent quality control (QC)sample (a concept alien to clinicians) This has been partially or completely addressed

by the subsequent categories of devices

Dedicated Qualitative Devices

(“Stick Tests”) Primarily Oriented Toward POCT

These systems represent the first application of immunochromatographic ogy to cardiac marker testing The core technology is common to all the systems andutilizes three processes: an initial separation of cells from plasma, an immunoreactivephase, and a detection phase This is illustrated for the Cardiac T system in Fig 1 Theantibodies dissolve in the plasma and react with any cTnT present to form a double-antibody–cTnT complex The plasma containing the complex diffuses along the internalstrip by capillary action and reaches the immobilized streptavidin and synthetic troponinpeptides at the detection zone Any double-antibody–cTnT complex binds by streptavi-din biotin binding to the detection line and the gold complex is visualized as an opticalsignal The systems include a built-in QC step Excess gold-labeled antibody binds tothe synthetic peptide to act as an internal control The result is read as positive (two sig-nal lines), negative (one signal line), or assay fail (no signal lines or the test line only).Precise pipetting is not required and an applicator device is used A number of systemshave been developed but clinical validation studies are available for only two

technol-Cardiac T (Roche Diagnostics)

The initial version of this test (first-generation, TROPT®) utilized the same antibodies

as in first-generation enzyme-linked immunosorbent assay (ELISA) Troponin T but

Fig 1 Schematic of an immunochromatographic method using gold labeled optically readimmunoassay (GLORIA) format