5

Cardiac Troponin: Clinical Role in the Diagnosis of Myocardial Infarction

FRED S.APPLE and ALLAN S.]AFFE

CASE REPORT

The patient, a 63-year-old Caucasian female, was hospitalized on . j April 2002 though 10 April 2002 for a non-ST segment elevation myocardial infarction (non-Q-wave MI per chart documen­tation). She had a negative adenosine stress test after the initial event. Her seru m cardiac-specific troponin 1 (cTnI) concentration 24 hours after her onset of chest pain was l.4 I-Ig/L (upper limit of normal is 0.3 ng/ml.), and her creatine kinase (CK) MB level was 12.5 I-Ig/L (upper limit of normal 6 .0 nglmL) .Three days post-event her cTnl level was 0 .5 I-Ig/L and her CK-MB level was 4.5 I-Ig/L (Fig. 5-1) . MB refers to one of the isoenzyme forms of CK found in Seru m . The form of the enzyme that occurs in brain (BB) does not usually get past th e blood-brain barrier and therefore is not normally present in the serum. The MM and MB forms account for al­most all of the CK in serum. Skeletal muscle con­tains mainly MM, w ith less than 2% of its CK in the MB form. MM is also the predominant myo­cardial creatine kinase and MB accounts for 10%-20% of creatine kinase in heart muscle.

At 72 hours after presentation , the patient ex perienced new-onset ch est p ain , described as a burning pain in the left shoulder. arm , and epi­gastrium.The electrocardiogram (ECG) demon­strated only nonspecific T-wave abnormalities and was not different from the one obtained at the time of her initial presentation . Normal si­nus rhythm was now present. Nitroglycerin provided some reli ef. Rased on new symptoms, along with recurring T-wav e abnormalities and

54

an increasing c'Tnl ,diagnosis of reinfarction (ex­tension of her initial event) was made. The cTnI concentration on the day of her suspected rein­farction (day 4) was increased to 1.8 I-Ig/L with a corresponding CK-MB value of 1:3.6 ug/L, Car­diac catheterization revealed a 95% distal left anterior descending stenosis , a 95% mid-right coronary artery narrowing, and a ~O % occluded circumflex proximally. Stents were placed in both the distal and proximal right coronary ar­tery.The rest of her hospital stay was unevent­ful , and she was discharged home on day 7 . At 3·month follow-up , the patient was participat­ing in a cardiac rehabilitation program and do­ing well.

DL<\.GNOSIS

The term acute myocardial infarction (AMI) is defined as an imbalance between myocar­dial oxygen supply and demand , resulting in injury to and the eventual death of myocytes , When the blood supply to the heart is inter­rupted ,"gro ss ne crosis" of the myocardium re­sults. Abrupt and total loss of co ro na ry blood flow usually results in a clinical syndrome known as ST segment elevation ANII (STE AMI or Q-wave M1; diagnostic by electrocardio­gram) because of the characteristic electrocar­diographic changes that occur. Partial loss of coronary perfusion, if severe, also can lead to necrosis , which is generally less ex te ns ive, and this type of infarction is usually termed non-ST elevation myocardial infarction ( NST EMJ or

Ca rd ia c Troponin in MI Diagnosis 55

24 48 72 96

12

10 ::r::J 3 EE til8OJ .s..s :2

m6i: ::!: l­ x:u (J4

2 120

Time (hrs)

Figure 5·1. Time-course of changes in serum car­diac troponin I and creatine kinase MB (CK-MB) follow ing myocardi al infarct ion and subsequent rcmfarction during hospitalization . Cardiac-spe cific rrop onin 1 (cT NI), op en squares ; CK-MB, filled cir­cles. Reprinted from Apple and Murakami (2005).

non-Q-wavc MI; not diagnostic by e tecrrocar­diugram). There is considerable overlap be­twcvn the pathophysiology and the pattern of necrosis of the tw o entities .Other even ts of less severity may be missed entirely o r if detected may be diagnosed as angina that can range from stable to unstable .

'Ole term acute coronary syndrome (ACS) encompasses most of th e patients defined so far in th is chapter who present wi th unstable is­chemic heart disea se. Most of these syndromes Occur in response to an acute event in the coro­nary artery when c irculation to a region of th e heart is obs tructed . If the obstruction is high grade an d persists, then ne cros is usually en sues. Since necrosis tak es som e time to develop , it is apparent that th erapy, including opening the blocked coronary artery in a timely fashion, of­ten can prevent some of the death of myocar­dial tis sue. These syndromes are usually, but not always , associated with chest disc omfort.

Previousty, the diagnosis of AMI established by th e World Health Organizati on required at least tw o of the following criteria: a history of chest p ain, evolutionary changes on the ECG, or elevation s of serial ca rdiac biomarkers (init ially ddined as a twofold increase of total serum CK Or CK-MB) . However, it was rare for a diagnosis ofAMI to be made in the absence of biochemi ­cal evidence of myocardial injury.A 2000 Euro­pean Soc iety of CardiologyI American College of Card iology (ESC/ACC) consensus conference has codifie d the role of biomarkers, with spe­cific focus on cardiac troponins, by advocating

that th e diagnosis be based o n evidence o f myo­cardial injury based on biomarkers of cardiac injury in the appropriate clinical situati on.

For these guidelines, ei ther of th e following criteria satisfies th e d iagn osis for an acute , evolving, or recent MI.The first is a ty pical rise or gradual fall of card iac troponin, or more rapi d rise and fall of CK-MB, w ith at least o ne of th e following: (1 ) ischemic sympto ms; (2) de­velopment of pathologic Q waves o n th e ECG ; (3) ECG change s indicative o f ischemia (ST seg­ment elevation o r depression); or (4 ) coro nary artery intervention (e .g., coronary angioplasry) . For th e second, there should be pathologi c find­ings of an AMI as identified at au topsy. The guidelines recognized the reality th at neither the c lini cal presentation nor the ECG had ade­quate sensit iviry and specificity, but that the tro­ponin markers, in particular, could provide both , These guidelines do not suggest th at all el­evations of these biomarkers should d icit a di­agnosis of AMI, o nly those associate d w ith the app rop riate clini cal (ischemic presentation) and ECG findings. When e levat io ns of card iac tropunin are observed that are not du e to acute ischemia, the clini c ian is obligated to search for another etiology for th e cardi ac injury.

Patients w ith ACS can be categori zed into four groups . Firs t , there is the group of patients who present early to th e emergency room, wi thin 0 to 4 hours after th e onset o f ches t pain, and w ho lack diagnostic ECG evidence of AMI. These patients require rapid laboratory testing for evidence of cardiac injury. Thus , use­ful laboratory markers of card iac injury are th ose that are released rapidly from the heart and are highly specific for cardiac myocrye damage. These assays must be rapid and sensi­tive enough to detect even the small changes within the re ference interval th at can occur in blood early after the onset of symptoms.

The second patient group presents 4 to 48 hours after the onse t of chest pain but without diagnostic evidence of A..lIJI by ECG. This group of patients also requires se rial monitoring of cardiac biomarkers and ECG changes.

Th e third group is patients who present still later, more than 48 hours after the onset of ches t pain , and also lack diagnosti c ECG changes . The ide al biomarker of myo cardial injury for this group would have to be one that persists in the circ ulatio n for several days to provide a late di­agnostic time window. The shortfa ll of such a marker might be its inability to distinguish re ­current injury from the prior, older injury.

S6 Nl TCLEICACIDSAND PROTEIN STRUC1l 1REAND FUNCTION

TIle fourth group is those who present to the emergency department at any time afte r the on­set of chest pain with clear ECG evidence of AMI. In thi s group , detection with serum bio­markers of myocardial injury is not necessary ini­tially. Many uf these patients may qualify for reperfusion therapy at a time before blo od mark­ers of cardiac injury have increased , and therapy should not be withheld if these crite ria arc met. Subsequently, specific and sensitive myocardial markers could be employed to monitor the suc­cess of reperfusion during the 60- to 90-minute period after therapy. Rapid assays providing early serial values followed by interpretation of the markers ' patterns of appearance are often help­ful in determining subsequent management.

BIOCHEMICAL PERSPECTIVES

Cardiac Troponin I and T

The contractile proteins of the myofibril in­clude three troponin regulatory proteins . The troponin complex includes three protein sub­units, troponin C (the calcium-binding compo­nent) , troponin I (the inhibitory component), and troponin T (the tropomyosin-binding com­ponent). The subunits exist in a number of iso­forms .The distribution of these isoforrns varies between cardiac muscle and slow- and fast­twitch skeletal muscle. Only two major iso­forms of troponin C are found in human heart and skeletal muscle. These are characteristic of slow- and fast -twitch skeletal muscle . The heart isoform is identical with the slow-twitch skele­tal muscle isoform. Isoforrns of cardiac-specific troponin T (cTn'T) and cTnI also have been identified and arc the products of unique genes All cardiac troponins are localized primarily in the myofibrils (94%-97%), with a smalle r cyto­pla sm fract ion (3%-6%) .

Cardiac troponin subunits I and T are en­coded by different genes than the respective skeletal muscle isoforms and have different amino acid sequences, giving them unique car­diac specificity. cTnJ has never be en show n to be expressed in normal, reg en erating , or dis­eased human or animal skeletal muscle. By con­trast , small amounts of cTnT are expressed as one of four identified isoforms in ske le tal mus­cle during human fetal development, in regener­ating rat ske le tal mu scle, and in diseased human skeletal muscle. cTnT isoform expression has been demonstrated in skeletal muscle specimens

obtained from patients w ith muscular dystrophy, polymyositis, dermatomyositis, and end-stage re­nal disease. Thus, care is necessary to choose an­tibody pairs for cardiac assay use that do not detect the isofor rns reexpressed in noncardiac tissue. The commercial assay used in clinical practice only detects the heart cTnT form.

Cardiac troponin I exists as a part of the tro­ponin T-I-e ternary complex as a structural and regulatory component of the myofibril. Follow­ing myocardial injury. multiple forms of card iac troponins are elaborated both in tissue and in blood (Fig. 5-2). These include the T-I-C ternary com plex , IC binary complex, free I, and multiple modifications of these three forms resulting from oxidation, reduction, phosphorylation, and dephosphorylation, as well as both e-and N-ter­minal degradation. \Vhat is elaborated likely re­flects the nature of the injurious stimulus, blood flow that determines how long the protein re­mains in the tissue prior to reaching the circula­tion, the timing of the insult (i.e ., forms may change as the tissue damage evolves), and per­haps genetics. Depending on which fragments are elaborated, the selection of antibodies used to detect cTnJ (i.e ., different antibody configura­tions) can lead to substantially different recogni­tion patterns . It is now clear that assays need to be developed with the antibodies that recognize epitopes in the stable region of cTnJ an d ideally demonstrate an equirnolar response to the differ­ent cTnJ forms that may circulate in the blood.

Creatine Kinase Isoenzymes and Isoforms

Three cytosolic isoenzyrnes (CK-.\1M, CK-MB, CK-BB) and one mitochondrial isoenzyme (CK­Mt) of CK have been identified .Three different genes have been identified that encode for and are spe cific for CK-M, CK-B, and CK-Mt sub­units . Alth ough CK-MM is predominant in both heart and skeletal muscle, CK-MB has been shown to be more specific for th e myocardium, which contains 10% to 20% of its total CK activ­ity as CK-MB, compared to amounts varying from 0% to 7% in skeletal muscle.

Early studies involving animal hearts or spec i­mens obtained at autopsy from human hearts suggested a uniform distribution of CK·MB rang­ing from 5% to 50% of the total CK act ivity. How­ever, it has been shown by Ingwall and colleagues (1985) that the proportion of CK-MB was 6% to 15% lower in the surrounding normal areas of tissue than in infarc ted myocardtum in

Figure S-2. Schema tic of cardiac troponin rand T release following myocardial cell necrosis into the circulation , demonstrating [he multiple forms that exist in the blood. c'Tn., cardiac-sp ecific troponin I;cTnT, cardiac-specific troponin T

Cardiac Troponin in /1.11 DitlR,wsis

I . I

' II

!

I

j' I ! I·

57

Blood

to th e adaptation by the skeletal muscle during regular training and after acute exercise , result­ing in increased CK-MB tissu e concentrations. The mechanism responsible for increased CK­MB in skeletal muscle following chronic muscle disease or injury is thought to be due to the re ­generation process of muscle, with reexpres­sion of l.K-B genes similar to those found in the heart . thus giving rise to increased CK-MB levels in skeletal muscle. Thus, skeletal muscle can be­come like heart muscle in its CK isoenzyme composition, with up to 50% CK-MB in some patients with severe polymyositis.

Cardiac Troponins

The first assay (a radioimmunoassay) that me a­sured cTnI used polyclonal anti-c'Tnl antibodies. The first monoclonal enzyme-linked immunosor­bent assay, anti-c'Tnl antibody-based immunoas­say, was described by Bodor and co-workers (1992). Numerous manufacturers have now de­veloped monoclonal antibody-based diagnostic immunoassays for the measurement of cTnI in serum.Assay times range from 5 to 30 minutes.

IMMUNOASSAY PERSPECTIVES FOR MONITORING CARDIAC BIOMARKERS

Bound 95-98%

8 Free 2-5%

Cytoplasm

humans . Wh en studied more completely in hu­mans , CK-MB concentrations ranged from 15% to 24% of to tal CK in mvocardial tissue obtained from patients with left ventricular hypertrophy (LYH) due to aortic stenosis, from patients with coronary artery disease (CAD) without LVH, and from patients with CAD and LVH due to aortic stenosis. In contrast , patients with normal left ventricular tissue had a low percentage of CK-MB «2%).These data suggest that changes in the CK isoenzyme distribution are dynamic and occur in hypertrophied and disea sed human myocardium. Diseased cell s also have less total CK per cell .

Normal skeletal muscle usually contains very little CK-MB. Levels as high as 5%- 7% have been reported in some muscles, but less than 2% is much more common. Severe skeletal muscle in­jury following trauma o r surgery can lead to ab­SOlute elevations of CK-MB above the upper reference (normal) limit of CK-MB in serum . In­creases in serum total CK and CK-MB in se veral patient groups o fte n present a diagnostic chal­lenge to the cl inician . Persistent elevations of serum CK-MR resulting from chro nic mu scle disease occur in patients w ith muscular dystro­phy, end-stage renal disease . and polymyositis (a generative disease of ske le tal muscle) as well as in healthy subjects who undergo extreme exer­cise or physical acti vities .Th e increase in serum ( :K-l\lB in runners , for example , may be related

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58 NUCLEIC ACIDS AND PROTEIN STRUCTUREAND FUNCTION

Table 5-1. Cardiac Tropo nin Assays Cleared by the Food and Drug Administration Assay LLD 99th ROC lO% CV

Abbott AxSYMADV" 0.02 0 .04 0 .4 0.1 6

Architect " 0 .00 9 0.012 0 .3 0 .032

Bayer ACS 0 .03 0.1 1.0 0 .:;5

Ce nt aur 0.02 0 .1 1.0 0 .3';

Beckman Access 0 .01 0 .04 0 .5 0 06

Biosit e Triage 0 .19 0 .19 O. j 0.5

Dade RxL' 001 007 0 .6-1'; 0 .14

CS' 0 .03 005 0 .6 -1. '; 0.06

DPe Immulite 0 .1 0 .2 1.0 0 .6

i-STAT i-STAT 003 O.OH ND 0 .1

Ortho Vitros 0 .02 0.08 0 .4 0 .12

Roche Elecsys" 0 .01 0 .01 0.1 0 .03

Reader 005 <0 .05 0.1 NA

Tosoh AlA" 0 .06 0 .06 0 .3 1-0.64 0.06

LI.n , lowe r limit of detection ;99 th, 99t h percentile refe ren ce limit ; ROC, receiver operator ch ara ct eri st ic cu rve optimized cutoff: 10% CV, lowcst concentration to provide a total imprecision of 10%.

"Sec o nd generation.

"Third ge neration

N.\ . no t available

As shown in Table 5-1, over a dozen assays have been cleared by the Food and Drug Administra­tion (FDA) for patient testing within the United States on cen tral laboratory and point-of-care (POC) or near-bed side testing platforms. In addi­tion to these quantitative assays, several assays have been FDA-cleared for the qualitative deter­mination of cTn!.

In practice, two obstacles limit the ease for sw itching from one cTn! or cTnT assay to an­other. First , there is currently no primary refer­ence cTnl material available for manufacturers to use for standardizing their assays . Second , be­cause of the different epitopes recognized by the different ant ibodies used, assay concentrations fail to agree . Whil e standardization of assays re­mains elus ive, harmonization of cTnl concentra­tion s by different assays has been narrowed from a 2G-fold difference to a 2- to 3-fold difference.

Se veral adaptations of the Roche Diagnostics cTnT immunoassay have be en described , result ­ing in an FDA-cleared third-generation assay available worldwide. Two monoclonal anticar­dia c troponin T antibodies are used in the third ­ge ne rat ion assay. Skeletal muscle TnT is no longer a potential interferent, as was found in th e first -generation enzyme-linked immunosor­be nt cTnT assay. In contrast to cTn l, no stan­dardizati on bias exists for cTnT because the sam e ant ibo d ies (M 11>M7) are used in both the

centra! laboratory and POC quantitative and POC qualitative as say systems.

In 200 1> quality specifications for cardiac troponin assays were published.These specifi­cations were int ended for use by the m anufac­turers of commercial assays and by clinical laboratories utilizing troponin assays.The over­all goal was to attempt to establish uniform cri­teria so all assays could objectively be evaluated for their analyt ical qualities and clinical perfor­mance. Both analytical and preanalytical factors were addressed . The following recommenda­tions have been proposed:

1. The antibody spedficity (which epitope locations are id entified) needs to be de ­lineated. Epiropcs located on the stable part of the cTnJ molecule should be a prio rity.

2. Assays ne ed to clarify whether different eTnJ forms (i.c ., binary vs. ternary complex) arc recognized in an equirnolar fashi on by the ant ibodies used in the assay.Specific rel­ative resp onses need to be described for the following cT nl forms : free cTnl; the l-C binary complex; the T-I-<: ternary complex; and oxidized, reduced, and phosphorylated isoforrns of the three cTnl forms.

3. The effects of different anticoa gu lant s on binding of e I'nl need tv be addressed.

Ca rd ia c Troponiu ill VI ni'7f!, l/osis 59

I. The source of materi al used to calib rate cI 'n assays. specifically for cTnl, sho uld he tra ceable.

While clinicians and lab oratorians continue to publish guidelines sup port ing turnaround time s ( TATs; def in ed as th e time fro m blood draw to report ing of the resul t to a health cart' provider) of k ~s than 60 minutes fo r cardiac biomark ers, th e largest 'IAT stu dy publ ished to

date dc rno nstrateu that TAT expect ations arc not b eing met in a large pro portion of hospitals . A survey study of 7020 cardiac troponin an d 436R C. K-\ 1B det erminations in 159 hospitals demonstrated that the median and 'JOth per­centik TATs for troporun and CK-MB were as fol­lows , respectively: ~4 .5 min , 129 min : 82 min , 131 min. Less th an 25'7.. of hosp itals w en: able to meet the TAT in less than 6U minutes. Prelimi­nary data has sh own that implementation of poe ca rd iac troponin testing can de crease TATs to less th an 30 minutes in cardiology c ritical care and short-stay units . These data highlight the continued need tor laboratory services and health ca re prov iders to w ork together to de ­velop better processes to meet a TAT th at is less than 60 minutes as requested hy physician s .

Reference Intervals for Cardiac Troponins and Creatine Kinase Isoenzymes

If po ssible , each lab oratory should determine a 99th percentile of a reference group for ca rdiac troponin assays using the specific ass ay used in clin ical practice o r validate th e assay based on findings in the literature. Further, accep table im­precision (coefficie nt of va riatio n, %CV) of each card iac troponin assay (as well as for CK-MB mass assay) has been defined as 10% or lower CV at th e Y9th percentile reference limit. Unfor­tunatety, the majority of lab orat ories do not have the re sources to perform adequately powered reference interval stud ies. Therefore, clinical lab­Orato ries have to rely on the pe er-reviewed pub­lished lite rature to establish reference intervals . When reviewing re fe re nce stud ies, caution must be tak en When comparing th e findings reported in the manufacturer's ap p roved pa ckage inserts With the findings reported in journals because of differences in total samp le size , distributions by gender and ethnici ry, age ranges , and the statistic Used to calculate the 99th percentile given.

There is no established guide line se t to mandate a c onsiste nt evaluation o f th e 99th

p ercentile refe ren ce limit for cardiac troponins. Th e largest and most diverse reported refer­ence in te rval study to d ate showed pl asma ( he ­parin; used for anticoagulation of blood) 99 th percentile reference limits for eight card iac tr o­ponin assays (seven cTn!, one cTnT) and seven CK-:vll:l ma ss assa ys (Tabl e 5-2). These studies w ere performed in 6 96 healthy adults (age ran ge 18 to R-i yea rs) stra tified by ge n der an d cthni.iry.The dat a demon strate severa l issu es . First , two cTn! ass ays showed a 1.2- to 2 .'i-fold higher 99th percentile for males versu s females. Second , rw o c'Tnl assays demonstrated a 1.1- to LR-fold highe r 99th pe rcentile for Afr ica n Amer­icans versus Ca uc asians .Third, the re was a 13 ­told differenc e between th e lowest ve rsus th e highest measured cTnl 99th p ercentile limi t . T he lack o f cardiac troponin assay sta ndardiza ­tion ( the re is no primar y reference material avail able) and th e differences in antibody epi­tope rec ognition between ass ays (di ffe ren t as­says use d ifferent antibod ies ) give r ise to subst antially di screpant results .

Alth ough many stu dies have addressed the to­tal imprecision of cTn assays , the man ufacturers ' pack age inserts prefer to publish imp rec isio n data primarily based on Within-run o r within-day precision.Again , there is nu consistent spec ifica ­tion regarding the precision value that should be reported in the pa ckage insert . Published findings demonstrating th e total imprecision for 13 commercial ass ays have indicated none of the assa ys w ere ab le ex perimentally to achieve a 10'% CV (to tal imprecision) at th eir 99 th per­centile cutoff.

Therefore, to avoid th e potential for false ­positi ve diagnost ic cri teria based on cTI1 moni ­toring at the 99th percentile , a group of experts in both the laboratory medicine and cardiology communities has endorsed the concept that W1 ­

til ca rd iac troponin assa y imprec ision improves at the low concentrations, the lowest concentra­tions to attain a 10% CV (C.V is the same as pre­cision) sh ould be used as a modified ESC/ACC diagnosti c cutoff for detection of myocardial in ­ju ry. This concept has been endorsed by seve ral cardiology and laboratory medicine groups. The ultimate goal w ill be to have a LI cTn assays atta in a 10% CV at the 99 th percentile reference limit .This approach should reduce false-positive analytic results from lack of imprecision values between the 10% CV cuto ff and th e 99th per­cen t ile . However, all biomarker increases above the 99th percentile should be int erpreted ca u­tiously, especially in th e high-risk patient, and

i l

I I

I i l I I

I

I I

60 NUCLEIC ACIDS AND PROTEIN STRUcrcREAN D FUNCTION

Table 5-2. Heparin -Plasma 99th Percentile Reference limits (ug/L) by Gende r and Race for Card iac Trop oninA ssays Cleared by th e Food and Dru g Administ ration

/I " A bbott Beck man oaae OeD RodJet n* Tosoh nf Bay er n il DPC

All 696 0.8 0.08 0 .06 0.10 <0.0 1 473 0.07 403 0.15 28 1 0.21

Male 3 15 0.8 0. 10 0 .06 0.11 <0.0 1 223 0 .07 187 0.17 115 0.2 1

Female 38 1 0.7 0.04 0 .06 0.09 <0.01 2 50 <0.06 216 0.14 166 <0.2

P .739 .034 .985 .0 17 .534 .52 1 ,44 1 .21

Caucasian 400 0 .8 0.07 0.04 0.11 <0.0 1 2 15 <0.06 193 0.17 166 0.2 1

African- 218 0.5 0 . 08~ 0.Q3 0 .10 <0.0 1 196 0.17 156 0.17 91 <0 .2 American

"Num ber of samples test ed in th e Abbort, Beckman , Dad e-Behring. OCD,and Roche assays .

'The Roche assay is th e only Card iac-Specific trop onin T assay on th e market; all o the r assays are for Cardiac-Spe cific tro porun I.

*Num be r o f samples test ed in the Toso h assay.

INumbe r of samples tes ted in th e Bayer assay.

'Numbe r of sam ples tested in the DPC assay.

' Significant ly di fferent (P =.05) fro m Caucasians based on mean concent rations .

DPC = Diagnostics Produc ts Corp oration

OC D = Or tho-Clinica l Diagnost ics

followed with se rial samples over a 6- to 12-hour p eri od after presentation.

Myocardial Infarction Detection Rates and Cardiac Troponins

Advan ces in dia gn ostic technology for the de ­vel opment o f im pro ved low-end analyt ical detection of card iac tr opon ins have impacted the prevalence ofAlvll detection.Accumulating dat a sug gest th at the more sens it ive cardiac troponin test s result in greate r rat es of MI dia g­nosis and grea te r ra tes o f card iac tropon in pos­itivity, com pa red to CK-MB. Smalle r Mis w ill be detected. Clin ica l cases th at w ere ea rlie r classi­fied as unst able angi na will be given a dia gno­s is of MI (due to an increased cTn ) , and now procedure-related troponin increases (i.e ., fol­lowing angio p lasry) will be labeled MI.

Th e im portance of sma ll tropon in increases has been co nfir med by their associatio n w ith a poor prognosis. Based o n seve ra l studies th at co mpa re d CK-MB and ca rdiac troponin assays in patients w ith ACS, a subs tantia l in crease in rate of MIs ranging from 12% to 127% w as de­te c ted . In o ne study o f 171 9 patients with ACS presenting to rule in/rule out Ml. a subse t (5'!,>} of c'Tnl-negat ive but CK-MB-positive patients re­veal ed the pot entially underlying false -p os itive MI rat e w he n using CK-MB as a standa rd for MI detec tion. Th is was likely du e to rel ease of CK­MB fro m skele ta l mu scle , in th e abse nce o f myo­ca rd ial in jury. Fur ther, a subset ( 12%) o f

c'I'nl-positive, CK-MB-ne ga tive patien ts dem on­stra ted a subse t of Mis th at would n ot have bee n detected without cardiac troponin moni­toring. All these data taken toge ther su pport the imp lem ent ation of ca rd iac troponin in place of, not in comb ina tio n wi th , CK-MB. This fac t w ill th en have an impact on the prognosis of Alvll overa ll.

Creatine Kinase MB

CK-.\1B can be measured in numerous w ays. Irn­munoassays de vel oped in rec ent yea rs h ave im­proved on the ana lyt ica l and clini ca l sen sitivlry and speciflc iry of th e earlier immunuinhibiti on and im rnun oprecipitat ion assays. T hese ass ays now : (1 ) m easure CK-MB directl y and p rovide mass measurements , (2) are eas ily autom at ed , and (3) provide rapid results (.:5:30 minutes). Mass assays re liably measure low CK-MB con­ce ntra t ions in both samp les wi th lo w to tal en­zyme ac tivity « 100 l 'IL) and wi th hi gh total en zyme acti vity ( > I0,000 LII.) . Furthermore , no interferences fro m other protein s have been documented . The majori ty of c o mm erci a lly available irnmuno assays that use monoclonal ant i-CK-MB anti bo d ies a re the same as those listed in Table 5-2 for card iac troponin ass ays. Exc ellent con cordan ce has been show n be­tween mass concentrati on and ac t ivity assays, A primary referen ce material is comm erci ally availab le to ass ist in ha rmo nizat io n . If use d for assay standa rdizatio n, then th is mat eri al allows

61 Ca rd ia c Tropon in in MI Diagnosis

concentrations to be reported w ithin 20% of eac h o the r.

As has been recognized for yea rs for total CK, all CK-MB assays demonstrate a significant 1.2­to 2.6-fo ld higher 99 th percentile for males ver­sus females . Several assays show ed h igher, up to 2.7-fold , concentrations for African-Americans vers us Ca ucasians . These data demonstrate that clinical laboratories mu st co ns ide r estab lishing different CK-MB reference cutoffs for at least me n versu s w omen.

CLINICAL UTlllZATION OF CARDIAC BIOMARKERS

Use in Patients with Acute Coronary Syndrome

Th e ideal marker of myocardial in jury should: (I ) provide ea rly detection of injury, (2) allow rapid diagnosis of cardiac injury, ( 3 ) serve as a risk stra t ificatio n tool in patients with ACS, (4) assess the success of reperfusion after throm­bolytic therapy, (5) detect reocclusion an d rcl nfarction, (6) determine the timing of an in­farction as w ell as infarct size , and (7) detect procedural-related perioperativc MI during car­diac or noncardiac surge ry.At present , the per­fee t biomarker tu sati sfy all these needs does not exist . It is the fun cti on of the laboratory to pro vide advice to physicians ab out cardiac bio­marker charact eristics .

Patients p re sent to emergency departments or other primary care providers with a multi­tude of clinical signs and symptoms for which the differential diagn osis of AMI ( heart attack) is cons id e re d . Figure 5-3 demonstrates the com­plete sp e ctru m of clini cal presentations of such a patient. Tills entire spectrum of clinical pre­sen tatio ns has be en designated ACS.The corner­stone of th e redefinition of MI is predicated on cardiac biomarkers, specifically cTnl or cTnT. The foUowing are de sign ated as biochemical in­dicators for de tecting myocardial necrosis :

1. A maximal concentration of cTnl or cTnI ex c eeding the d ecision limit, defined as the 99th percentile of values for a refer­en ce c ontrol group , on at least one occa­sio n during the first 24 hours after the in d ex clinical eve nt.

2. . A maximal valu e of CK-MB (preferably mass) exceeding the 99tJl percentile of values for a reference control group on two succes sive samp les o r a maximal

value exceeding twice th e upper re fer­ence limit during the first hours afte r th e in dex clinical event .Alth ough th e consen­sus document sta tes values for troponin and CK-MB should ris e and faU , e ithe r a rising or a failin g pattern should be con­side red diagnostic . Values that remain ele­va ted w ithout ch ange are rarely du e to MI .

3. In th e absence o f availability of a ca rd iac troponin or CK-MB assay, total CK greater th an two times the upper reference limit may be employed.

In addition to the ESC/ ACC consensu s docu ­ment for redefining MI, th e ACC/American Heart Assoc iation guidelines for management of unstable angina recommend monitoring cardiac troponin in patients with ACS as a way of differentiating unstable angina (define d as when cardiac tr oponin is within the 99th per­centile reference limit) and non-Sf segme nt­e leva tion MI (de fined as when card iac troponin is increased above the 99th percentile refer­ence limit) .

Severa l markers sho uld no longer be used to evaluate cardiac disease , in cl uding aspart ate amin otrans fe ra se, total CK, total lact ate dehy­drogenase (LDH), and LOB isoenzymes. Due to their wide tissue di stribution, these markers have poor sp ecifici ty for the detection of car­diac injury. Because total CK and CK-MB have se rved as standards for so man y years, so me lab­o rato ries ma y continue to measure them to al­low for comparisons to cardiac troponin over time, before discontinuing use of CK and CK­MB. In addition, the use of total CK in develop' ing countries may be th e preferred or only alternative for financial reasons. However, it should be clear that, for monitoring ACS pa­tients to assist in clinical classification , cardiac troponin is the preferred b iomarker.

For the majority of patients, blood sh ould be obtained for testing at ho spital admission (0

hours), at 6 to 9 hours, and again at 12 to 24 hours if the earlier specimens are normal and the clinical index of suspicion is high . For pa­tients in need of an early d iagnosis th at would parall el a rapid triage protocol, a rapidly appear­ing biomarker su ch as myoglobin h as been sug­gested to be added to serial cardiac troponin monitoring. In practice, it appears th at the ma­jority of hospitals throughout the world do not use these markers .

Several general clinical impressions can be made regarding cTnI and cTnT. First , the ea rly

62 NUCLEIC ACIDS AND PROTEIN STRUCnrREAND FUNCTIO!\:

Stages of Vascular Inflammation • Proinflammatory Cytokines

• IL-B

• Plaque Destabilization • MPO

Plaque Rupture • sCD401

• Acute Phase Reactants • hs-CRP

• Ischemia • IMA

• Necrosis • eTnT

• eTN;

• Myocardial Dysfunction BNP

• NT-proBNP

Figure 5-3. Complete spectrum of acute coronary pathophysiological process from ini­tiation of atherosclerosis to cell death. Biomarkers released at different stages include in­terleukin 6 (IL-{)),myeloperoxidase (MPO) ,soluble CD40 ligand (s<.;D40L), high-sensitivity C-reactive protein (Hs-CRP), ischemia-modified albumin (lMA), cardiac troponins I and T (cTnT and cTnI , respectively), B-type natriuretic peptide (BNP) , and N-terminal proBNP (NT-proBNP). Of these, only the troponins and BMPsare myocardial specific.

release kinetics of both cTnI and cTnT are simi­lar to those of CI,...-MB after AMI, with increases above the upper reference limit seen at 2 to 6 hours. The initial increase is due to the :,% to 6% cytoplasm fraction of troporun (C K-MB is 100% cytoplasmic). Second, cTnl and cTnT can remain increased up to 4 to 14 days after AMI. The mechanism is likely the ongoing release of troponin from the 94% to 97% myofibril-bound fraction since the halt-life in clearance studies of either the native protein or of c omp lex es is in the range of 2 hours. The long interval of car­dia c troponin increase has resulted in its utiliza­tion in pl ace of the I.DH isoenzyme assay in the detection of late-presenting AMI patients ,Third, the very low to undetectable cardiac troponin values in serum from patients without cardiac disease (no rmal, healthy reference population) permits use of lower dis criminator concentra­tions co mpared to CK-MB for the determination of myocardial injury. Finally, card iac tissue speci­ficity of cTnl and cTnT should eliminate false clinical impression of AMI in patients wi th in­creased CK-MB concentrations following skele­tal muscle injuries.

Clinica l usc of the percentage relative index [%Rl; %Rl = (C K-MB ma ss/Total CK activ­ity X 100)] or %CK-MB (CK-M13 activity/Total CK x 100) aids in th e interpretation of CK-MB concentrations for th e detection of ANlI. While

not absolute', an increased %CK-MB or %R1 points to the heart as the source of CK-MB in serum. However, the %R1 and %CK-MB should not be used for interpretation When the total CK activity remains within the reference inter­val.Any concomitant skeletal muscle injury will decrease the sensitivity of the relative index for the detection of cardiac events.

As increases in cardiac troponin detect any form of myocardial injury, nonischemic mccha­nisms of injury are also responsible for card iac troponin release from the heart, causing in­creases in circ ulating troponin. Table '5 -:3 shows a list of potential etiologies that have been re­sponsible for increases in non-ischemic damage to th e heart.Thus ,whenever cardiac troponin is monitored, it is important to follow the serial pattern of a rising or a falling pattern of th e bio­marker. An increased cTn pattern that remains relatively unchanged and is not indicarive of this serial trend is likely not an Ml.

Strategies for the Role of Cardiac Troponin for Risk Assessment

Numerous prospective and retrospective clini­cal studies have evalu ated and compared the utility of measurements of c'Tnl and cTnT for risk stratification or clinical o utco mes assessm ent of patients with ACS with possible myocardial

I

Ca rd ia c Troponin i n !HI D iagnosis 63

Table 5-3. Ele \'at i~ns ofTrop on ins without Overt Ische mic Heart D ise~se _ Trau ma (inc ludi ng co ntusion, ablation , pacing, ca rd ioversion)

Co ngest ive he art failure , acu te and chronic '

Aort ic val ve di sease and hype rtrop hic obstruct ive cardiomyopathy with sign ifican t le ft ventricular hype rtro phy'

Hypertension J Hypot e nsion . o ften with arr hyt hmias

Postope rat ive noncardiac sur ge ry pa tie nts who seem to do well "

Ren al fa ilure '

Cri tica lly ill pat ient s ,esp ecially with d iab et es, respira to ry failure'

Drug to xiciry,suc h as ad riarnyc in , 5-fluorouracil, herceptin, sn ak e venoms'

Hypothyro idism

Co rona ry vasospasm . incl ud ing ap ical ba llooning synd ro me

Inflammatory dis eases such as myocardi tis (e .g ., with parvovirus B19, Kaw asa ki di sease , sarcoidosis , smallpo x vaccination , o r myocardial extension of bact erial endocarditis)

Postpe rc utan eous coronary intervention pat ients who appear w ith out complicat ion'

Pulm onary emb olism, se vere pulmonary hypertension'

Se psis'

Burn s , e speci ally if tot al burn surface area >30%'

Infiltrative d iseases , incl ud ing amyloidosis, hemochromatosis, sarcoidosis , and scl e rode rma'

Acute neuro logi cal d isease , including cerebrovascula r accident, subarachno id bleeds '

Rhabdo myo lys is with ca rdiac in jury

Transplant vascu lopathy

Vital ex ha ust ion

"Design a t io ns imply prognost ic in fo rm al ion has been rep orted.

ischemia in the emergency department. Patients presenting with a complaint of chest pain or other symptoms suggesting ACS have been as­signed to blood-sampling protocols including only a single draw at presentation as well as to several serial blood samplings over a 12- to 24­hour period following presentation. Overall. in the approximately 18,000 patients studied, at 30 days the odds ratio for an adverse outcome was 3.4 for increased troponin. As both cTnT and cTnI offer powerful risk assessment, cTn moni­toring needs to be included in current practice guidelines not 00.1)' regarding diagnosis and management of ACS patients, but also as useful risk stratification tools. It is recommended to draw two samples on patients with ACS who do not rule in for AMI:one at presentation and one at 6 to 9 hours following presentation. This will allow for an increase in ei ther cardiac troponin to occur above baseline in a patient presenting With a very recent acute coronary lesion. How­ever, it should be noted that a normal cardiac troponin does not remove all risk.

Seve ral studies have now documented that assays with lower limits of detection are able to

identify more patients with ACS with poor prog­nosis who may be candidates for early invasive procedures. In on e representative study (Venge e t al., 2000), two assays were compared to assess clio.ical performance in unstable patients with CAD. While both assays showed patients with normal cTn.l levels had a significantly better prognosis than patients with increased Levels,a cohort of 11% of patients (n = 98) with a poor prognosis was identified only by the second­generation assay with a Lower limit of detection. Invasive treatment only reduced clinical events in the group of patients with increased cTn.l. Thus, each troponin assay, I or T, needs to evalu­ate th e stratification of patients at low-end con­centrations to avoid the potential of analytical inaccuracies leading to inappropriate manage­ment decisions and therapy. These high-risk pa­tients have been sh own to benefit from aggressive therapies, including low molecular weight heparin, lIbIlIIa glycoprotein platelet in­hibitors, and an early interventional strategy.

Clinicians arc often confronted with a cLinicaJ r , history of a patient without overt CAD and a low probability of myocardial ischemia. However, as a

Nn":LEICAUDSAND PROTEIN STRl.:CTUREAND RNCnUN64

I I·

precautionary reflex , se rial cardiac biomarkers, specifically cTn, are o rdered . The 20% of sus­pected ACS patients who clinically do not rule in for !\11 but display an increased cTn represent those nonischemic pathologies suc h as my­ocarditis, blunt che st trauma, or chemo th erapeu­tic agents tor which th e mechanism s of in jury are well defined (Table 5-3) as well as the unex­pected finding of myo cardial injury, for which patients have been show n to have increased cTn, but the mechanism of release is not clear. These obse rvations have led to important and novel investigations involvin g pati ents wi th non­ischemic heart disease and the role for ca rdiac troponin as a diagnostic and prognostic tool. The conditions show n in Table 5-3 th at are indi­cated by an asterisk demonstrate that, in addi­tion to ca rdiac troponin being indicative of ca rdiac injury, the data have also indicated that increased cardiac troponin is useful as a prog­nostic tool for assessing risk of death an d MI.

Monitoring Reperfusion Following Thrombolytic Therapy

Release of cTnl or cTn 'I' from myocardium into the blood following AMI and after th e w ashout th at acc ompa nies suc ce ssfu l reperfusion gen er­ates an ex cellent sign al com pa re d to no de­tect able baseline levels prior to myocard ial damage .Th e initial rap id release of cardia c tro­ponin subunits I and T following successful rcperfusion is most likely derived from the solu­ble cytosolic myocardial fraction (6% cTnT, 3% cTnI).The clinical utility of cardiac biomarkers for monitoring reperfusion following throm­bolytic th erapy has not gained favor as a ro u­tine fo rm of testing for determining the suc cess or failure of reperfusion therapy becau se it ca n­not dist ingu ish 11MI 2 from TIMI j flow, which is a c ritic al issue in regard to p rognosis. (T IM! is th e tim ed interve nt ion in myocardial infarct ion. TIM! 2 and 3 refer to th e extent of flow th rou gh coronary ves sels, w ith TIM! 2 referring to par­tial flow and TIMI 3 to comp lete flow.)

It is accep ted th at the kin etics of myocardial protein appearance in th e circ ulatio n depends on infarct perfusion . Early repe rfusion causes an ea rlier increase above the upper refer ence lim it and an earlier and greater enzyme peak af­ter reperfusion. However, once the peak has oc­cu rred , there is no difference in the time of cleara nce of e nzymes. In addition, enhan ced washo ut identifies w hether an artery is patent or closed but can not distingui sh between nor­

mal and abno rma l coron ary perfusion, which is anoth e r ke y prognostic parameter. Further, it is difficult to asse ss the amo unt of irreversible myoc ardial in ju ry by infarc t sizing because of the larg e va riab ility in the amo unt of enzyme wash out th at ap pears afte r rep erfusion .

Strategies Using Multimarkers

The re is a growing body of evide nce su gges ting tha t dlfferent cardiac biomark ers provide inde­pendent and com plemen tary information about pathophysiology, diagnost ics, prognostics, and response to therapy in patients with ACS. Th us, it is probable that muI timarker stra tegies or bio­chemical profiling may be used to cha rac te rize ind ividual patients presenting wi th ACS. For ex­amp le , in o ne multicenter study, cTnl , CK-MB, and myogl obin muItimarker analysis identified positive patients earlier and provided a better risk stratification for 3o-day mortality than cen­tral laboratory an alysis of CK-MB alone (20% vs. 3%, respectively) .

Estimation of Infarct Size

Older studies ha ve sho w n that one can use the int egrat ed values for total or CK-MB to estimate th e biochemical ex tent of infa rction . Further stu dies verified th at the amo unt of ca rd iac dam­age is the p rimary determinant o f prognosis. Suc h determinations have been co rrelated with morphological infarct size. Some have used the peak CK-MB value as a sur rog ate for the inte­grated data. Rcperfuslon cha nges the release ra­tio (the percentage of ma rker that appears in th e blood rel at ive to the amo unt depleted from myocard ium). making infarct sizing p roblematic­in the modern era.Additional data from both ex­perimental and patient-related data h ave sug­ges te d that the 72-ho ur tropo nin measurement co rrela tes w ith scinrigraphically-d erc r rn incd in­fract s ize .Th e data are stronger fo r troponin T th an for I, although the principles are probab ly sim ilar for both analytes .At p rese nt, it is not rec­ommended tha t se rial monitor ing o f ca rdiac tro ponin or CK-MB be used for infarct sizing ,

Reinfarction

Figure 5-1 de mo nstrates th e seria l p attern s for card iac tropon in I and CK-.\1B both during th e in itia l infa rc t and re infa rc tio n, as described fo r th e initial case presentation . Alth ou gh th e number of reinfarcti on cases pres ented in th e

7

Car d ia c Tr oponin in Mf Diagnosis 65

literature is few e r than 20, the findings demon­stra te that CK-MB analysi s is no longer clinically relevant, or cost-effective, in the differential diagnosis of myocardial reinfarction in patients with ACS when cardiac troponin monitoring is available . We encourage others to test this hy­pothesis to be ab le to dispel the theoretical ra­tionale for use of CK-MB te sting in addition to cT n testing, ex ped itin g the cost-effective ad ap­tation favoring only cTn monitoring in testing for Ml or rcinfarcti on.

QUESTIONS

1. Diagram the rising and falling serial bio­marker profiles for cardiac troponins com­pared to CK-MB.

2. Define the ESC/ACC consensus guidelines for the redefinition ofAMI.

3. Review the role of cardiac biomarkers for infarct sizing and monitoring the success of reperfusion following therapy.

4. List the multiple forms of cardiac tro­ponin I that may exist in the circulation.

5. Define the two major concerns that have been responsible for substantial concen­tration differences when measuring car ­diac troponin I by different com m e rcial irnrnunoassays.

6 , Explain how the determination of refer­ence intervals can have an impact on the number of MI cases that are defined in a g iv en population of patients with ch est pain. Explain the role of cardiac troponin deter­minations in risk-stratifying patients who present with ACS.

BmUOGRAPHY

Alpert JS,Thygesen K,Antman E, ct al. : Myo cardial infarction redefined-a consensus document of th e joint European Society of Cardio logy/ Ameri­can College of Cardiology Committee for the re­definition of myocardial infarction.] Am Col! Card iol 36:959-959, 2000,

Apple FS:Tissue specificity of cardiac troponin I, card iac rroponin T, and creatine kin ase MB. CUn Cbim A cta 284:151 -159, 1999.

Apple FS, Falahati A, Paulsen PR, et al.: Improved detection of minor ischemic myocardial injury with measurement of serum c ardiac rroponin I. Glirl Chem 43!2047-2051 , 1997.

Apple FS, Murakami MM: Cardiac troponin and c re­atin e kinase MB monitoring during in-h ospital

myocardial reinfarction . Cl in Chern 51:460- 463, 2005.

Apple FS, Quist HE, Doyle PJ, et al.: Plasma 99th p ercentile re fere nce l.i.m.its for cardiac troponin and creatine kin ase MB mass for use with Euro­pean Socie ty of Cardiology/American Coll ege of Cardio logy co nsensus recommendations. CUn Ch ern 49:1 331-1 336 , 2003.

Apple FS, Wu AHB, Jaffe AS: European Society o f Cardio logy an d American College of Card io logy gu idelines for red efinition of myocardial infarc ­tion: how to usc existing assa ys clinically and for clinical tri als . A m Heart ] 144:981-986, 2002.

Bodor GS, Porter S, Landt Y,er al. : Development of monoclonal an tibod ies for an assay of ca rd iac troponin-I and preliminary results in suspected cases of myocardial infarction . Cl i n Cbem 38:220 3-221 4,1 992.

Fishbein MC, Wang T, Matija sevic M, et al. : Myocar­dial tissue troponins T and I : an immunohisto­chemical study in experimental models of myocardial ischemia. Cardiovasc Pathol 12: 65-71,2003.

Ingwall JS, Kramer MF, Fifer MA, et aI.:The creatine kinase system in normal and diseased human my­ocardium.N Engl] Med 313:1050-1054,1985.

Jaffe AS,LandtY, Parvin CA, e t al .:Comparative sensi­tivity of cardiac troponin I and lactate dehydroge­nas e isoenzymes for diagnosing acute myocardial infarction. CUn Cbem 42:1770- 1776, 1996.

Jaffe AS, Ravkilde J , Roberts R, et al.: It 's time for a change to a troponin st andard . Circulation 102:1216- 1220, 2000 .

Katrukha AG, Bereznikova AV, Filtaov Vl., et al. : Degradation of ca rd iac troponin l : implication for reliable imm unode te ctio n . Clin Cbem 44: 2433-2440 ,1 998

Katus HA, Looser S, Hallermayer K, et al.: Develop­ment and in vitro characterization of a new im­munoassay of card iac troponin T. Clin Cbern 38:386-:W3, 1992

Lin JC ,Appk FS, Murakami MM,et al.: Rates of pos­itive cardiac tr op onin 1 and c reatine kinase MB among patients hospitalized for suspected acute coronary sy nd ro mes. Gin Chern 50:3 :)3 -338 , 2004.

Ottani F, Galvani M, Nic olini A, e t al.: Elevated car­diac troponin levels predict th e risk of adverse outcome in patients with acute coronary syn ­dromes.Am Heart] 40:917-927, 2000.

Panteghiru M, Gerhardt \\:~ Apple FS, et al.: Quality specifications for cardiac troponin assays. Clin Cbem Lab Med 39: 174-178,2001.

Venge P, Lagerquist B, Diderholm E, et 31. on behalf of the FRISC II Study Group : Clinical perfor­mance of three cardiac troponin assays in pa ­tients with unst able coronary artery disease (a FRISC n Subs tudy).A m ] CardioI89:103'5-1041 , 2000.

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~.LINlCAL STUDIES

IN MEDICAL BIOCHEMISTRY

Third Ldition

Edited by

Robert H. Glew

and

Miriam D. Rosenthal

OXFORD U N IV E RSITY PRLSS

2007

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