1. Speaker: Undhad Vishal V.
Reg. No. : 04-1443-2010
Major Advisor: Minor Advisor:
Dr. D. J. Ghodasara Dr. A. M. Thaker
Associate Professor Professor and 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.  Measurement of circulating cardiac troponin
concentrations is considered the “gold standard”
for the non-invasive diagnosis of myocardial
injury.
 It has replaced traditionally used cardiac
biomarkers such as creatine-kinase and its
isoenzymes due to its high sensitivity and
specificity for the detection of myocardial injury.
(Anita, 2008)

5.  Troponin is a complex of three regulatory proteins
that is integral to muscle contraction in skeletal and
cardiac muscle, but not smooth muscle.
 Cardiac troponin I (cTnI) and cardiac troponin T
(cTnT) are uniquely expressed in the myocardium
and have been widely recognized as highly sensitive
and specific serum markers for the noninvasive
diagnosis of increased mayocyte permeability or
necrosis.16,18,28,29
Definition

6.  In 1950’s Clinical reports that transaminases released
from dying myocytes could be detected via laboratory
testing aiding in the diagnosis of myocardial
infarction.
 First cTnI in 1987 and later cTnT in 1989 were used as
biomarkers of cardiac cell death.
(Katus et al., 1989)
history

7. Biochemistry

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

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

10.  The first data on the three-dimensional structure of
troponin C appeared in 1985, and a detailed
structure with 2 Å resolution was published in 1988.
(Satyshur et al., 1988)
 Troponin C has a dumbbell-like structure and made
up of
1. Two Globular Domains
a. N-Terminal globular domain
b. C-Terminal globular domain
2. Long central α-helix
Troponin C

11. (Filatov et al., 1999)
Three-Dimensional Structure of Troponin C

12.  cTnI has an extra 30 amino acid sequence at the N
terminal portion of molecule making it absolutely
specific to cardiac muscle.
 Troponin I is the inhibition of actomyosin ATPase
activity. This inhibition is enhanced in the
presence of tropomyosin and is completely
abolished in the presence of fully Ca2+ -saturated
troponin C.
(Perry, 1999)
Troponin I

13.  In the absence of Ca2+ the inhibitory sites of
troponin I interact with actin, whereas in the
presence of Ca2+ these sites interact with troponin
C.
 The orientations of the polypeptide chains of
troponin I and troponin C are antiparallel.
(Farah et al., 1994)

14.  In most muscles troponin T is present in the form of
multiple isoforms.
(Jin et al., 1992)
 Human cardiac muscle contains four troponin T
isoforms, three of which are expressed in the fetus,
and one isoform is characteristic for adult heart.
(Anderson et al., 1995)
Troponin T

15.  In the isolated state or inside the thin filament
troponin T has the form of a comma or rod with a
length of 185-205 Å.
(Flicker, 1982)
 Troponin T is located in the groove of the actin
helix and is extended along the filament.
 There are three tropomyosinbinding sites in the
troponin T structure.
 Troponin T kinase is involved in phosphorylation
of troponin T in vivo.

16. (Filatov et al., 1999)

17. physiology

18.  In the 1960s it was hypothesized that the contraction of
striated muscle is regulated by a special protein complex
located on actin filaments this complex was called native
Tropomyosin
(Ebashi et al., 1968)
 Native Tropomyosin consists of two parts
1. Tropomyosin
2. Troponin
(Bailey, 1946)
 TROPOMYOSIN is a single long protein strand that winds
around the actin filament.
 TROPONIN is a globular complex of three proteins, and is
found in clumps around the Tropomyosin protein.

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

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.htm

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 Pi,
cocking of the myosin head occurs
Myosin head (high-energy
configuration)
Thick
filament
Myosin head
(low-energy
configuration)
ADP and Pi (inorganic
phosphate) released
Thin filament
Sequential Events of Contraction

24.  In animals without induced myocardial damage
cTnT is undetectable (<0.01 µg/L) in serum.
(O’Brien et al., 1997)
 In the normal dog and cat mean plasma cTnI
concentrations is 0.02 ng/ml and 0.04 ng/ml
respectively.
(Sleeper et al., 2001)
Normal range of Cardiac Troponin

25.  In clinical healthy cows serum cTnI concentration
of ≤ 0.04 ng/ml have been reported.
(Jesty et al., 2005)
 In clinically healthy horses cTnI concentrations of
≤ 0.015 ng/ml were reported.
(Begg et al., 2006)

26.  Human studies showing that levels of cTnT in the
neonates were significantly higher than those in
children of older age.
(Clark et al., 2001)
 Younger broilers exhibited a significantly higher
cTnT concentration than the older chicks, thereby
supporting the fetal origin hypothesis of this marker
protein in the younger age group.

27.  In the rat, an age and gender dependent variation
in serum cTnI was found. Male rats aged six and
eight months had a 10-fold greater serum cTnI
than age matched females and three-month-old
rats.
(O’Brien et al., 2006)
 In healthy cows age, body weight, lactation, and
pregnancy status had no effect on baseline cTnI
concentration.
(Anita, 2008)

28.  TnI and TnT is 40% and 10-30% structural
distinction from the skeletal Troponin I and
troponin T isoforms respectively.
(Mair, 1997)
 It is important to recognize that an increase in the
concentration of serum cardiac troponins indicate
myocardial damage but does not indicate
mechanism.
(Alpert et al., 2000)
Specificity and sensitivity

29.  Both cTnT and cTnI have been used as biomarkers to
detect drug-induced cardiac injury in humans and
animals.
(O'Brien et al., 2008)
 Most intracellular cTnI and cTnT is bound to the
myofibrils in the cardiac myocyte however a small
percentage exists in a cytosolic pool (6-8% of cTnT
and 3-4% of cTnI) with the majority of the remaining
troponin found in the sarcomere.
(Bhayana et al., 1995)

30. Myocardial injury
Transient release
Reversible ischemia Irreversible ischemic necrosis
Persistent release
Troponin releases
Cytosolic pool Sarcomere of the myocyte
(Boswood, 2004)

31.  Following myocardial damage, TnT and TnI are
released in different forms.
 TnT is primarily found as the intact T:I:C complex,
free TnT, and smaller immunoreactive fragments.
TnI is released.
 TnI is released primarily as the intact T:I:C: and
I:C complex. The binary form of cTnI:C appears to
be the predominant molecule.
(Lowbeer, 2007)

32.  Cardiac troponin I is susceptible to proteolysis by
serum proteases as well as phosphorylation and
oxidation after it is released into the bloodstream.
 The rate of degradation of cardiac troponin
depends on different factors such as size of the
cTnI fragments released and their complex
formation with other troponin subunits.
(Organ et al., 2000)

33.  In people, free cTnI has a lower stability than the
binary or ternary forms. The N-terminal as well as
C-terminal regions of troponin I are rapidly
cleaved during proteolysis.
 Immunoassay that employs antibodies against the
stable core region of the protein will increase
analytical performance .
(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.  A good relationship between cTnI concentration
and the extent and severity of myocardial injury as
shown by histopathologic evaluation of cardiac
tissue in people and laboratory animals has been
observed.
(Hasic et al., 2003)
 Minimum serum concentrations of 1.3 ng/ml for
cTnI and 0.35 ng/ml for cTnT were necessary for
myocardial cell injury to be detected
histologically.
(Bertsch et al., 1997)

39.  Loss of cTnI immunolabeling was also identified,
even in the absence of histologic proof of
myocardial necrosis or degeneration.
(Tunca et al., 2008)
 Immunolabeling for cTnI was more sensitive than
routine HE staining for the recognition of
peracute myocardial necrosis in both
experimental animals and humans.
(Tunca et al., 2008 )

40. (Tunca et al., 2008 )

41. Immunofluorescent Labeling 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 ++++ ++++ ++++
(Hochholzer et al., 2010)

43. In Human
 Ischaemic causes
• Coronary embolis and 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 (Iwan et al., 2006)
Cardiac contusion (Schober et al., 1999)
Cardiomyopathy (Baumwart et al., 2007)
Babesiosis (Lobetti et al., 2002)
GDV (Schober et al., 2002)
Chronic mitral valve disease (Bakirel and Gunes, 2009)
Cats
Hypertrophic cardiomyopathy (Herndon et al., 2002)

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

47.  Concentration of cTnI is generally not increased
in renal failure patients in the absence of
myocardial damage.
(McLaurin et al., 1995)
 It has been clearly established that troponin levels
are increased in patients with renal failure, even in
the absence of clinically suspected myocardial
ischemia.
(Francis and Tang, 2004)
Causes of Elevated Troponin in renal failure

48.  cTnT (53%) remains elevated to a higher degree than
cTnI (17%) in patients with CKD in the absence of
clinical acute myocardial necrosis.
(Kontos et al., 2005)
 However in most ESRD patients without acute
myocardial ischaemia, cTnT is only slightly elevated
with a majority of the results below 1.0 µg/L, and
concentrations above 3.0 µg/L are extremely rare.
(Lowbeer, 2007)

49.  It is possible that the cTnT assay is so sensitive
that it detects subclinical myocardial cell injury
whereas the cTnI assays are less Sensitive.
(Lowbeer, 2007)
 cTnI was superior than cTnT for the diagnosis of
myocardial ischaemia in patients with renal failure
 Several mechanisms have been proposed for this
increase troponin in renal diseases , although no
definite explanation exists.

50.  Heterophile antibodies
 Human anti-animal antibodies
 Autoantibodies
 Rheumatoid factor
 Fibrin clots
 Hemolysis Interference
 High concentration of AKP
 Immunocomplex formation
 Analyzer malfunction
Causes of False Positive Troponin Results

51.  Heterophile and Human anti-animal
antibodies
 Heterophile antibodies are antibodies that are
produced against poorly defined antigens and are
generally weak antibodies with multispecific
activities.
 Human anti-animal antibodies are produced
against well-defined antigens and may have
specificities for a wide range of animal proteins
such as mouse, rabbit, rat, and others.
(Kricka, 1999)

52.  Rheumatoid factor (RF)
• RF may also be a cause of interference with cTnI
immunoassays.
• Positive RF has been found in varying percentages
in other connective disease disorders such as
systemic lupus erythematosis, systemic sclerosis
and polymyositis.
(Moore and Dorner, 1993)
 Fibrin clots
• Excess fibrin has been reported to be a cause of
falsely elevated cTnI results.
• Non-specific binding of the antibody to fibrin.

53.  Hemolysis Interference
• A recent study showed that hemolysis can lead to
false positive cTnI results.
(Hawkins, 2003)
 Alkaline Phosphatase Interference
• cTnI may be effected by high concentrations of
ALP.
• If ALP value of 129 U/L than cTnI concentration
was falsely elevated to 4.3 ng/mL.
(Dasgupta et al., 2001)

54.  As the homolgoy between troponins is about 95%
among mammals, commercial diagnostic kits
designed for use in humans also provide excellent
results in other animals including poultry.
(O’brien et al., 1997)
 The Cardiac isoforms of troponin ( cTnI) and
troponin T ( cTnT) can be specifically recognised
by monoclonal antibodies which do not cross
react with the skeletal muscle isoforms.
(Katus et 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.  There is only one clinical (Roche Diagnostics,
Basel, Switzerland) and one research (Bioveris
Europe, Whitney, Oxford, UK) vendor for the
single cTnT assay available with relatively uniform
cutoff concentration.
(Tate et al., 2002)
 At least 18 different commercial assays for
measurement of cTnI are available with
considerable variation in the cutoff
concentrations.

58.  Studies have shown that troponin I results may
vary by a factor of 100 fold from one assay and
manufacturer to another.
 Antibody used by a particular manufacturer may
be directed against different epitopes of troponin
I, which result in assay-to-assay variation in
detected levels of cTnI.
 This analytical variability which leads to wide
variations in lower detection limits, upper
reference limits and diagnostic cut points.
(Krishnaswamy et al., 2001)

59.  Recently, an ultrasensitive cTnI assay (Erenna
immunoassay system) has been described with an
approximate 50-fold improvement in sensitivity
over many currently utilized troponin
immunoassays.
(Todd et al., 2007)

60. (Hochholzer et 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 - Vit B12 and 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.by GVK
BioSciences, Hyderabad
WB/S/P 15 min 0.2
Cardicheck Troponin I
Monozyme Diagnostics
Hyderabad
S/P 25-30 min 0.5
Cardi trop Plus Ameritek, USA WB/S/P 10-15 min 1
Troponin I
ANI Biotech OY, Finland
By Rapha Diagnostics-Thane ,
Maharashtra
S
WB
10-15min
10-15min
0.3
0.3
Biocard Troponin I
ANI Biotech OY, Finland Mkted
by Ranbaxy
WB/S 10-15 min 0.3
Cardiac STATus -
Troponin I
Spectral Diagnositcs Inc -
Canada
WB/S/P 15 min 0.15
Peerless Troponin I
Mktd.by Peerless Biotech. Mfg
by Veda-Lab, France
S/P 15-20 min 1
Life Sign MI Troponin
I
PBMC-USA, Mkted by Premier
Med Corp (PMC) - Daman
WB/S/P 15 min 0.5
Troponin T Market Player
Trop T
Mfg.& Mkted by 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. It does not indicate the mechanism responsible for
the necrosis
It is not an early biomarker of cardiac necrosis
The troponins persist in blood for a long period of
time after an initial injury, may be of no value in
detecting reinfarction.
(kemp et al., 2004)
There is currently no standardization of cTnI assays
with different commercial assays giving
numerically different results.

73. Sensitivity, specificity and persistent cardiac
biomarker.
cTn concentration related with severity of
myocardial damage.
cTnI and cTnT has high prognostic, diagnosis and
therapeutic value than traditional cardiac marker.
Test is less time consuming and less costly
Conclusion

74. Further investigation using a larger sample size is
needed to fully understand the effect of storage on
cTnI recovery.
(Anita, 2008)
Studies are needed to elucidate the specific
pathogenic mechanisms causing elevated cTnT in
chronic renal failure.
(Lowbeer, 2007)
Future prospects

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