Cardiac troponin (Dr.Vishal)

1. Speaker: Undhad Vishal V.
Reg. No. : 04-1443-2010
Major Advisor: Minor Advisor:
Dr. D. J. Ghodasara Dr. A. M. Thaker
Associate Professor Professorand Head
Dept. of Vet. Pathology Dept. of Vet. Pharmacology
And Toxicology
College of Veterinary Science and Animal Husbandry
Anand Agricultural University
Anand -388 001

2. ♥Introduction
♥Definition
♥History
♥Biochemistry
♥Physiology
♥Sensitivity and Specificity
♥Causes of Elevated Troponin Levels
♥Causes of False Positive Troponin Results
♥Tests To Measure Cardiac Troponin
♥Advantages
♥Limitations
♥Conclusion

3. Traditional biomarkers
Myoglobin
CK
CK-MB
LDH
SGOT
Cardiac auscultation
Radiography
ECG
Echocardiography
Myocardial damage
Infectious
Toxic
Nutritional
Traumatic
Introduction

4. Measurementofcirculatingcardiactroponin
concentrationsisconsideredthe“goldstandard”
forthenon-invasivediagnosisofmyocardial
injury.
Ithasreplacedtraditionallyusedcardiac
biomarkerssuchascreatine-kinaseandits
isoenzymesduetoitshighsensitivityand
specificityforthedetectionofmyocardialinjury.
(Anita, 2008)

5. Troponinisacomplexofthreeregulatoryproteins
thatisintegraltomusclecontractioninskeletaland
cardiacmuscle,butnotsmoothmuscle.
CardiactroponinI(cTnI)andcardiactroponinT
(cTnT)areuniquelyexpressedinthemyocardium
andhavebeenwidelyrecognizedashighlysensitive
andspecificserummarkersforthenoninvasive
diagnosisofincreasedmayocytepermeabilityor
necrosis.16,18,28,29
Definition

6. In1950’sClinicalreportsthattransaminasesreleased
fromdyingmyocytescouldbedetectedvialaboratory
testingaidinginthediagnosisofmyocardial
infarction.
FirstcTnIin1987andlatercTnTin1989wereusedas
biomarkersofcardiaccelldeath.
(Katuset al., 1989)
history

7. Biochemistry

8. •Troponin C
–Binds calcium
•Troponin I
–Binds actin
•Troponin T
–Binds tropomyosin
Troponin I
Troponin C
Troponin T
Tropomyosin
Actin
Types of Troponin

9. Troponin CTroponinITroponin T
Molecular
mass
(kd)
18 22.5 37
AA residues 161 181-211 185-205
Isoforms
and
specificity
Two isoforms
(fast and slow
skeletal)
3Isoforms:2
unique for
skeletal
muscle, 1 for
cardiac
muscle
Multiple
isoforms
(Filatovet al., 1999)

10. Thefirstdataonthethree-dimensionalstructureof
troponinCappearedin1985,andadetailed
structurewith2Åresolutionwaspublishedin1988.
(Satyshuret al., 1988)
TroponinChasadumbbell-likestructureandmade
upof
1.TwoGlobularDomains
a.N-Terminalglobulardomain
b.C-Terminalglobulardomain
2.Longcentralα-helix
Troponin C

11. (Filatovet al., 1999)
Three-Dimensional Structure of TroponinC

12. cTnIhasanextra30aminoacidsequenceattheN
terminalportionofmoleculemakingitabsolutely
specifictocardiacmuscle.
TroponinIistheinhibitionofactomyosinATPase
activity.Thisinhibitionisenhancedinthe
presenceoftropomyosinandiscompletely
abolishedinthepresenceoffullyCa
2+
-saturated
troponinC.
(Perry, 1999)
Troponin I

13. IntheabsenceofCa
2+
theinhibitorysitesof
troponinIinteractwithactin,whereasinthe
presenceofCa
2+
thesesitesinteractwithtroponin
C.
Theorientationsofthepolypeptidechainsof
troponinIandtroponinCareantiparallel.
(Farah et al., 1994)

14. InmostmusclestroponinTispresentintheformof
multipleisoforms.
(Jin et al., 1992)
HumancardiacmusclecontainsfourtroponinT
isoforms,threeofwhichareexpressedinthefetus,
andoneisoformischaracteristicforadultheart.
(Anderson et al., 1995)
Troponin T

15. Intheisolatedstateorinsidethethinfilament
troponinThastheformofacommaorrodwitha
lengthof185-205Å.
(Flicker, 1982)
TroponinTislocatedinthegrooveoftheactin
helixandisextendedalongthefilament.
Therearethreetropomyosinbindingsitesinthe
troponinTstructure.
TroponinTkinaseisinvolvedinphosphorylation
oftroponinTinvivo.

16. (Filatovet al., 1999)

17. physiology

18. Inthe1960sitwashypothesizedthatthecontractionof
striatedmuscleisregulatedbyaspecialproteincomplex
locatedonactinfilamentsthiscomplexwascallednative
Tropomyosin
(Ebashiet al., 1968)
NativeTropomyosinconsistsoftwoparts
1.Tropomyosin
2.Troponin
(Bailey, 1946)
TROPOMYOSIN isasinglelongproteinstrandthatwinds
aroundtheactinfilament.
TROPONINisaglobularcomplexofthreeproteins,andis
foundinclumpsaroundtheTropomyosinprotein.

19. Thin filaments made up from the proteins actin,
tropomyosinand troponin
An actin molecule is a globular protein with binding site
to which the myosin cross-bridges can attach
Groove hold tropomyosinmolecules
Smaller, calcium-binding troponin molecules stuck to
tropomyosin
Thinfilaments

20. Thick filaments made up from the protein 200 to 500
myosin molecules
2 entwined golf clubs
Arranged in a bundle with heads directed outward in a
spiral array around the bundled tails
Thick filaments

21. Actin myosin contraction
http://www.sci.sdsu.edu/movies/actin_myosin.html

22. Myosin cross bridge attaches to
the actin myofilament
1
2
3
4 Working stroke—the myosin head pivots and
bends as it pulls on the actin filament, sliding it
toward the M line
As new ATP attaches to the myosin
head, the cross bridge detaches
As ATP is split into ADP and P
i,
cocking of the myosin head occurs
Myosin head (high-energy
configuration)
Thick
filament
Myosin head
(low-energy
configuration)
ADP and P
i(inorganic
phosphate) released
Thin filament
Sequential Events of Contraction

24. Inanimalswithoutinducedmyocardialdamage
cTnTisundetectable(<0.01µg/L)inserum.
(O’Brien et al., 1997)
InthenormaldogandcatmeanplasmacTnI
concentrationsis0.02ng/mland0.04ng/ml
respectively.
(Sleeper et al., 2001)
Normal range of Cardiac Troponin

25. InclinicalhealthycowsserumcTnIconcentration
of≤0.04ng/mlhavebeenreported.
(Jestyet al., 2005)
In clinically healthy horses cTnI concentrations of
≤ 0.015 ng/ml were reported.
(Begget al., 2006)

26. HumanstudiesshowingthatlevelsofcTnTinthe
neonatesweresignificantlyhigherthanthosein
childrenofolderage.
(Clark et al., 2001)
Youngerbroilersexhibitedasignificantlyhigher
cTnTconcentrationthantheolderchicks,thereby
supportingthefetaloriginhypothesisofthismarker
proteinintheyoungeragegroup.

27. Intherat,anageandgenderdependentvariation
inserumcTnIwasfound.Maleratsagedsixand
eightmonthshada10-foldgreaterserumcTnI
thanagematchedfemalesandthree-month-old
rats.
(O’Brien et al., 2006)
Inhealthycowsage,bodyweight,lactation,and
pregnancystatushadnoeffectonbaselinecTnI
concentration.
(Anita, 2008)

28. TnIandTnTis40%and10-30%structural
distinctionfromtheskeletalTroponinIand
troponinTisoformsrespectively.
(Mair, 1997)
Itisimportanttorecognizethatanincreaseinthe
concentrationofserumcardiactroponinsindicate
myocardialdamagebutdoesnotindicate
mechanism.
(Alpert et al., 2000)
Specificity and sensitivity

29. BothcTnTandcTnIhavebeenusedasbiomarkersto
detectdrug-inducedcardiacinjuryinhumansand
animals.
(O'Brien et al., 2008)
MostintracellularcTnIandcTnTisboundtothe
myofibrilsinthecardiacmyocytehoweverasmall
percentageexistsinacytosolicpool(6-8%ofcTnT
and3-4%ofcTnI)withthemajorityoftheremaining
troponinfoundinthesarcomere.
(Bhayanaet al., 1995)

30. Myocardial injury
Transient release
Reversible ischemia Irreversible ischemic necrosis
Persistent release
Troponin releases
Cytosolicpool Sarcomereof the myocyte
(Boswood, 2004)

31. Followingmyocardialdamage,TnTandTnIare
releasedindifferentforms.
TnTisprimarilyfoundastheintactT:I:Ccomplex,
freeTnT,andsmallerimmunoreactivefragments.
TnIisreleased.
TnIisreleasedprimarilyastheintactT:I:C:and
I:Ccomplex.ThebinaryformofcTnI:Cappearsto
bethepredominantmolecule.
(Lowbeer, 2007)

32. CardiactroponinIissusceptibletoproteolysisby
serumproteasesaswellasphosphorylationand
oxidationafteritisreleasedintothebloodstream.
Therateofdegradationofcardiactroponin
dependsondifferentfactorssuchassizeofthe
cTnIfragmentsreleasedandtheircomplex
formationwithothertroponinsubunits.
(Organ et al., 2000)

33. Inpeople,freecTnIhasalowerstabilitythanthe
binaryorternaryforms.TheN-terminalaswellas
C-terminalregionsoftroponinIarerapidly
cleavedduringproteolysis.
Immunoassaythatemploysantibodiesagainstthe
stablecoreregionoftheproteinwillincrease
analyticalperformance.
(Lowbeer, 2007)

34. Cardiac
Marker
Half life
(hours)
Increase
(hours)
Peak
(hours)
Normalization
(Days)
CK 17 3-12 12-24 3-4
CK-MB 13 3-12 12-25 2-3
CK-MB
mass
13 2-6 12-24 3
Myoglobin 0.25 2-6 6-12 1
cTnI 2-4 3-8 12-24 7-10
cTnT 2-4 3-8 12-96 7-14

35. Cardiac markers: Approximate Levels vs. Time of onset post MI

37. Cardiac Marker Sensitivity Specificity
Mayoglobin +++ +
CK ++ ++
CK_MB +++ +++
Troponin I ++++ ++++
Troponin T ++++ ++++

38. AgoodrelationshipbetweencTnIconcentration
andtheextentandseverityofmyocardialinjuryas
shownbyhistopathologicevaluationofcardiac
tissueinpeopleandlaboratoryanimalshasbeen
observed.
(Hasicet al., 2003)
Minimumserumconcentrationsof1.3ng/mlfor
cTnIand0.35ng/mlforcTnTwerenecessaryfor
myocardialcellinjurytobedetected
histologically.
(Bertschet al., 1997)

39. LossofcTnIimmunolabelingwasalsoidentified,
evenintheabsenceofhistologicproofof
myocardialnecrosisordegeneration.
(Tuncaet al., 2008)
ImmunolabelingforcTnIwasmoresensitivethan
routineHEstainingfortherecognitionof
peracutemyocardialnecrosisinboth
experimentalanimalsandhumans.
(Tuncaet al., 2008 )

40. (Tuncaet al., 2008 )

41. ImmunofluorescentLabeling of Cardiac Troponin I (cTnI)
in Doxorubicin-induced Cardiomyopathy
Control Doxorubicin

42. Marker of
cardiac
necrosis
Prognostic
impact
Diagnosis
impact
Therapeutic
impact
CK-MB +++ +++ ++
Myoglobin ++ ++ ++
Troponin ++++ ++++ ++++
(Hochholzeret al.,2010)

43. In Human
Ischaemiccauses
•Coronary embolisand spasm
•Coronary dissection
•Aortic dissection
•Transplant vasculopathy
Myopericarditis
•Rheumatic fever
•Systemic vasculitis
Cardiac surgery
Causes of Elevated Troponin Levels

44. Infiltrative diseases of the myocardium
•Amyloidosis
•Sarcoidosis
Miscellaneous Causes
•Tachyarrhythmia
•Hypertension
•Congestive heart failure
•Renal failure
•Drug toxicity (e.g. Adriamycin, 5-fluorouracil, etc)
•Pulmonary embolism with right ventricular
infarction

45. In animals
Dogs
Acute myocardial damage (Iwanet al., 2006)
Cardiac contusion (Schoberet al., 1999)
Cardiomyopathy (Baumwartet al., 2007)
Babesiosis (Lobettiet al., 2002)
GDV (Schoberet al., 2002)
Chronic mitral valve disease (Bakireland Gunes, 2009)
Cats
Hypertrophic cardiomyopathy(Herndon et al., 2002)

46. Cattle
Idiopathic pericarditis (Jestyet al., 2005)
Traumatic reticulopericarditis(Guneset al., 2008)
Calf
Foot and mouth disease (Guneset al., 2008)
Experimentally induced endotoxemia
(Peek et al., 2008)
Poultry
Ascites (Maxwell et al., 1995)

47. ConcentrationofcTnIisgenerallynotincreased
inrenalfailurepatientsintheabsenceof
myocardialdamage.
(McLaurinet al., 1995)
Ithasbeenclearlyestablishedthattroponinlevels
areincreasedinpatientswithrenalfailure,evenin
theabsenceofclinicallysuspectedmyocardial
ischemia.
(Francis and Tang, 2004)
Causes of Elevated Troponin in renal failure

48. cTnT(53%)remainselevatedtoahigherdegreethan
cTnI(17%)inpatientswithCKDintheabsenceof
clinicalacutemyocardialnecrosis.
(Kontoset al., 2005)
HoweverinmostESRDpatientswithoutacute
myocardialischaemia,cTnTisonlyslightlyelevated
withamajorityoftheresultsbelow1.0µg/L,and
concentrationsabove3.0µg/Lareextremelyrare.
(Lowbeer, 2007)

49. ItispossiblethatthecTnTassayissosensitive
thatitdetectssubclinicalmyocardialcellinjury
whereasthecTnIassaysarelessSensitive.
(Lowbeer, 2007)
cTnIwassuperiorthancTnTforthediagnosisof
myocardialischaemiainpatientswithrenalfailure
Severalmechanismshavebeenproposedforthis
increasetroponininrenaldiseases,althoughno
definiteexplanationexists.

50. Heterophileantibodies
Humananti-animalantibodies
Autoantibodies
Rheumatoidfactor
Fibrinclots
HemolysisInterference
HighconcentrationofAKP
Immunocomplexformation
Analyzermalfunction
Causes of False Positive Troponin Results

51. Heterophile and Human anti-animal
antibodies
Heterophileantibodiesareantibodiesthatare
producedagainstpoorlydefinedantigensandare
generallyweakantibodieswithmultispecific
activities.
Humananti-animalantibodiesareproduced
againstwell-definedantigensandmayhave
specificitiesforawiderangeofanimalproteins
suchasmouse,rabbit,rat,andothers.
(Kricka, 1999)

52. Rheumatoid factor (RF)
•RFmayalsobeacauseofinterferencewithcTnI
immunoassays.
•PositiveRFhasbeenfoundinvaryingpercentages
inotherconnectivediseasedisorderssuchas
systemiclupuserythematosis,systemicsclerosis
andpolymyositis.
(Moore and Dorner, 1993)
Fibrin clots
•Excessfibrinhasbeenreportedtobeacauseof
falselyelevatedcTnIresults.
•Non-specificbindingoftheantibodytofibrin.

53. HemolysisInterference
•Arecentstudyshowedthathemolysiscanleadto
falsepositivecTnIresults.
(Hawkins, 2003)
AlkalinePhosphataseInterference
•cTnImaybeeffectedbyhighconcentrationsof
ALP.
•IfALPvalueof129U/LthancTnIconcentration
wasfalselyelevatedto4.3ng/mL.
(Dasguptaet al., 2001)

54. Asthehomolgoybetweentroponinsisabout95%
amongmammals,commercialdiagnostickits
designedforuseinhumansalsoprovideexcellent
resultsinotheranimalsincludingpoultry.
(O’brienet al., 1997)
TheCardiacisoformsoftroponin(cTnI)and
troponinT(cTnT)canbespecificallyrecognised
bymonoclonalantibodieswhichdonotcross
reactwiththeskeletalmuscleisoforms.
(Katuset al., 1995)
Tests for measurement of cTn

55. Sample collection for Test
Whole Blood
Serum
Plasma
Anticoagulant
EDTA
Heparin
Sample

56. Significant decrease in cTnI recovery when cTnI
samples were stored at room temperature (23°C),
4°C and -80°C for 2 days.
However storage at -80°C for 7 and 14 days had no
significant effect on cTnI recovery.
Storage at -20°C for 7 days had no significant
effect on cTnI recovery.
(Anita, 2008)
Storage of samples

57. Thereisonlyoneclinical(RocheDiagnostics,
Basel,Switzerland)andoneresearch(Bioveris
Europe,Whitney,Oxford,UK)vendorforthe
singlecTnTassayavailablewithrelativelyuniform
cutoffconcentration.
(Tate et al., 2002)
Atleast18differentcommercialassaysfor
measurementofcTnIareavailablewith
considerablevariationinthecutoff
concentrations.

58. StudieshaveshownthattroponinIresultsmay
varybyafactorof100foldfromoneassayand
manufacturertoanother.
Antibodyusedbyaparticularmanufacturermay
bedirectedagainstdifferentepitopesoftroponin
I,whichresultinassay-to-assayvariationin
detectedlevelsofcTnI.
Thisanalyticalvariabilitywhichleadstowide
variationsinlowerdetectionlimits,upper
referencelimitsanddiagnosticcutpoints.
(Krishnaswamyet al., 2001)

59. Recently,anultrasensitivecTnIassay(Erenna
immunoassaysystem)hasbeendescribedwithan
approximate50-foldimprovementinsensitivity
overmany currentlyutilizedtroponin
immunoassays.
(Todd et al.,2007)

60. (Hochholzeret al., 2010)

61. Cardiac Panel Test Card

65. Numerous Parameters
Cardiac–Troponin I, CK-MB-Adrenal -Cortisol
Skeletal Muscle troponin -Insulin
Thyroid-T4, T3, TSH -VitB
12and folate
Reproductive-Progesterone, Estradiol, Testosterone
Centaur CP-Automated Immunoanalyzer

66. RAMP Cardiac Marker System

67. Market for Cardiac Troponin in India
Product Company Specimen
Duration
minutes
Sensitivity
ng/ml
Troponin I Market Players
Alma-TI
Mfg & Mktd.byGVK
BioSciences, Hyderabad
WB/S/P 15 min 0.2
CardicheckTroponin I
MonozymeDiagnostics
Hyderabad
S/P 25-30 min 0.5
CarditropPlus Ameritek, USA WB/S/P10-15 min 1
Troponin I
ANI Biotech OY, Finland
By RaphaDiagnostics-Thane ,
Maharashtra
S
WB
10-15min
10-15min
0.3
0.3
BiocardTroponin I
ANI Biotech OY, Finland Mkted
by Ranbaxy
WB/S 10-15 min 0.3
Cardiac STATus-
Troponin I
Spectral DiagnositcsInc -
Canada
WB/S/P 15 min 0.15
Peerless Troponin I
Mktd.byPeerless Biotech. Mfg
by Veda-Lab, France
S/P 15-20 min 1
Life Sign MI Troponin
I
PBMC-USA, Mktedby Premier
Med Corp (PMC) -Daman
WB/S/P 15 min 0.5
Troponin T Market Player
Trop T
Mfg.& Mktedby Roche
Diagnostics -Mumbai
WB/S/P 15 min 0.1

68. Advantage

69. High specificity
High sensitivity
High concentration in cardiac muscle
Persists for several hours
Concentration related with the size of infarction
area.

70. High prognostic, diagnosis and therapeutic
impact than other cardiac marker.
Test is less time consuming
Test is relatively less costly

71. Limitations

72. Itdoesnotindicatethemechanismresponsiblefor
thenecrosis
Itisnotanearlybiomarkerofcardiacnecrosis
Thetroponinspersistinbloodforalongperiodof
timeafteraninitialinjury,maybeofnovaluein
detectingreinfarction.
(kempet al., 2004)
ThereiscurrentlynostandardizationofcTnIassays
withdifferentcommercialassaysgiving
numericallydifferentresults.

73. Sensitivity,specificityandpersistentcardiac
biomarker.
cTnconcentrationrelatedwithseverityof
myocardialdamage.
cTnIandcTnThashighprognostic,diagnosisand
therapeuticvaluethantraditionalcardiacmarker.
Test is less time consuming and less costly
Conclusion

74. Furtherinvestigationusingalargersamplesizeis
neededtofullyunderstandtheeffectofstorageon
cTnIrecovery.
(Anita, 2008)
Studiesareneededtoelucidatethespecific
pathogenicmechanismscausingelevatedcTnTin
chronicrenalfailure.
(Lowbeer, 2007)
Future prospects

75. A “gold standard” assay for the analysis of cTnI
will develop with uniform cut off value.