BIOMARKER

1. Dr RAMDHAN KUMAR KAMAT
SR CARDIOLOGY IGIMS PATNA

2. BIOMARKER
2
“A biomarker is a substance used asan
indicator of a biologic state”.
Morrow and de lomos three criteria for
biomarkers
– Accurate repeated measurements at
reasonable cost
– Must provide additional information
– Should aid treatment.

3. CARDIAC BIOMARKERS
Cardiac biomarkers are protein molecules
released into the blood stream from damaged
heart muscle.
Since ECG…… inconclusive ….biomarkers
!!!!!?????
• myocardial injury
These biomarkers have a characteristic rise
and fall pattern.

4. 4
CHARACTERISTICS OF AN IDEAL CARDIAC
MARKER
 High cardiac specificity
 Pharmacokinetics of cardiac biomarker
 Easy diagnosis
 Marker should play a designed role in the
treatment and management of clinical subject

5. HISTORY OF CARDIAC BIOMARKERS
1954 - SGOT (AST)
1955 - LDH
1960 - CPK
1972 - CPK isoforms by Electrophoresis
1975 - CK - MB by immunoinhibition
1975 - Myoglobin
1985 - CK - MB Mass immunoassay
1989 - Troponin T
1992 - Troponin I

7. Types of Biochemical Markers
1- Cardiac Enzymes (isoenzymes):
Total CK
CK-MB activity
CK-MB mass
Aspartate aminotransferase (AST)
Lactate dehydrogenase (LDH)
2- Cardiac proteins:
Myoglobin
Troponins

8. PATHOPHYSIOLOGY BASIS OF BIOMARKERS
Biomarkers of inflammation .
C-reactive protein/hsCRP, Homocysteine.
Myeloperoxidase (MPO)
Soluble fragment CD40 ligand (sCD40L)
Biomarkers of plaque instability/disruption.
Pregnancy-associated plasma protein A(PAPP-A)
Choline
Placental growth factor
MMP-9
Myeloperoxidase (MPO)
Biomarkers of myocardial ischemia .
Ischemia-modified albumin (IMA)
Free fatty acids unbound to albumin (FFAu)
Heart-type fatty acid binding protein (H-FABP) (PRE
NECROSIS)
Myocardial necrosis
cTn ,
CK-MB, myoglobin
Pump failure,myocardiac stress
Nt-PROBNP,GDF-15,ST-2,ET-1

9. CLASSIFICATION OF
CARDIAC BIOMARKERS
ACCORDING TO VARIOUS STAGES
DURING CARDIAC DISEASE PROCESS

10. Marker for
inflammation hsCRP sCD40L Homocystein
Marker for plaque
Destabilization
PAPP-A LP-PLA2
Marker for
hemodynamic
stress
Natriuretic peptides
• ANP
• BNP
• Pro-BNP
• CNP
• DNP

11. 2
3
4
7
6
5
myoglobin
CK-MB
cTnT
cTnI
COMPARISON OF CTN, CK-MB , MB
1
0
0 4 8 12 16 20 24 28 32 36 40 44 48
Time after onset of AMI (hours)
Χupperlimitofreferenceinterval

12. OTHER CARDIAC MARKERS

13. Stefan Blankenberg, MD; Renate Schnabel, MD; Edith Lubos, MD, et al., Myeloperoxidase Early Indicator of Acute Coronary Syndrome and
Predictor of Future Cardiovascular Events 2005

14. 15
CARDIAC TROPONINS
 Troponin is a complex of three regulatory proteins that is integral to
non-smooth muscle contraction in skeletal as well as cardiac muscle
 Troponin is attached to the tropomyosin sitting in the groove
between actin filaments in muscle tissue
 Troponin has three subunits, TnC, TnT, and TnI
 Troponin-C has calcium binding ability and has no diagnostic
value
 Troponin-T binds the troponin tropomyosin complex,
 Troponin-I is an inhibitory protein
 The troponins found in cardiac tissue (cTn) have a different
amino acid sequence than that present in troponin of
skeletal muscles.

16. 18
Increased Troponins
 Troponin T and I are not detected in healthy individuals
 Significant increase in Troponins reflects myocardial
necrosis
 ACC/ESC has defined increase in Troponins as a
measurement above 99th percentile value of reference group
 To reduce false-positive outcomes, CV of <10% at decision limit
is recommended

17.  The pattern of release of
troponin may be
monophasic or biphasic.
 This release kinetics is
related to the distribution of
these proteins within the
myocardial cell.
 About 94-97% of these
troponins is bound to
myofibril and only 3% of
cTnI and 6% of cTnT is free
in the cytoplasm
 When the myocardial
damage occurs the cytosolic
troponins reach the blood
stream quickly resulting in a
rapid peak of serum
troponin observed during the
first few hours.

18. Studies have shown that cardiac troponins should
replace CKMB The reasons being :
 1. Troponins are highly cardiospecific especially the
TnI (100%).
 2. The prolonged elevation (4-14 days) make it a
good marker for patients admitted to the hospital
after several days of MI.
 3. cTns have greater sensitivity for minor degrees of
myocardial injury due to the cardiospecificity and their
very low concentration in serum of normal individuals.
 4. These are excellent prognostic indicator in
patients with unstable angina and is a very useful
parameter for stratifying risk in acute coronary
syndrome(ACS).
Rottbauer W, Greten T, Muller-Bard off M et al. Troponin T:A

19.  5. A single measurement of serum cTnT at the time
corresponding to the slow continuous release after AMI
(~72 hours after onset) can be used as a convenient
and cost effective non-invasive estimate of infarct
size whereas CKMB requires repetitive sampling.
 6. The early serial measurements of cTnI are a
more accurate predictor of early coronary artery
reperfusion after thrombolytic therapy as
compared to CKMB and myoglobin.

20. According to U.S. National Academy of Clinical
Biochemistry (NACB) and Joint European Society of
Cardiology and American College of Cardiology (ESC/
ACC) guidelines cTns are the most specific and
sensitive biochemical markers.

21. cTnT Versus cTnI
 Both cTnT and cTnI are almost equally good markers and
it is difficulty to say which is better because both have
some positive and negative points.
 cTnI is 100% cardiospecific and it is not elevated in
chronic renal disease, trauma and skeletal muscle
disease.
 The overall diagnostic specificity and efficiency of cTnI is
better than cTnT.

22. 24
TROPONIN T
1.Cardiac Troponin T (cTnT) is present in fetal skeletal
muscle.
2. In healthy adult skeletal muscle cTnT is absent.
3. The gene of cTnT may be re-expressed in skeletal muscle
disease. (Clin Chem. 1999;45:2129-2135)
4. Biological half life and early serum increases of cTnT are
similar to that of cTnI.

23. cTnI:
100 % cardiac specific
With greater sensitivity for diagnosing minor damage
of MI.
Appears in blood within 4 hours after onset of
infarction
peak: around 24 hours
Disappears from blood after about one week (stays
longer).
 So, useful for diagnosis of delayed admission cases
Prognostic marker (relation between level in blood &
extent of cardiac damage)

25. 27
Cardiac Troponin Release after MI

26. COMPARISON
NEJM 2002;Vol.346,No.26:2079-82
28

27.  The other advantage of cTnI may be its greater
specificity in patients of ESRD.
 However, the important advantage of cTnT is that due
to international patent restrictions there is only one
assay for its measurement, thus cTnT demonstrates a
high degree of precision at the low end of measurement
range and a relatively uniform cut-off concentration.
 In contrast, at least 18 different commercial assays for
cTnI are available leading to considerable variation in the
cut-off concentrations in the definition of a myocardial
infarction by cTnI values.
 Thus, a clinician should be aware of the cTnI cut-off
values specifically associated with the particular assay
used by the laboratory.

28.  The life-time of cTnT in blood (5- 14 days) is
some what more than that of cTnI (4-10 days).
 Although cardiac troponins are extremely specific
for myocardial necrosis, they do not discriminate
between ischaemic and non- ischaemic
etiologies of myocardial injury.
 Combining troponin with other cardiac biomarkers
may offer complimentary information on the
underlying pathobiology and prognosis in an
individual patient .
 The recommended time course for collection of
blood samples for cTn is at hospital admission, 6
and 12 hours later but when it is used along with
an early marker like myoglobin (two-marker
strategy) then at hospital admission, 4, 8 and 12.

29. WASH-OUT PHENOMENON.
 Patients with ST-segment elevation myocardial
infarction who achieve an effective reperfusion have
a greater and earlier peak plasma concentration of
troponin, followed by a faster return to normal – the
so-called “wash-out phenomenon” – compared with
those patients having no significant reperfusion.
 In this event, two blood samples should be collected –
at the time of the patient's admission to hospital,
and 90 min later – and the enzyme plasma
concentrations compared.
 The ratio between the concentrations at these two
points can be used to discriminate between
successful and unsuccessful reperfusion. In general,
the greater the ratio (at least 5), the more likely it
is that reperfusion has occurred.

30. AFTER CABG
Increase more than 5 x 99 percentile URL during first 72
hrs

31. Tn IN ESRD
 The cardiac troponin especially cTnT pose diagnostic
challenges in patients of chronic renal failure.
 Frequent cTnT elevations (30 to 70% of end stage renal
disease (ESRD) patients compared with <5% in similar
patients of cTnI) are seen in patients of renal failure in the
absence of clinical suspicion of ACS .
 The putative mechanisms for chronic elevation of troponin
in chronic renal disease patients include
 endothelial dysfunction,
 acute cardiac stretch,
 microinfarction and
 left ventricular hypertrophy.
 uremic toxicity

32.  Increasing evidence suggests that chronically elevated troponin
levels indicate a worse long-term prognosis for cardiovascular
outcomes in this patient population
 False positives have been reported with use of troponin- T in
ESRD patients but not as much with troponin-I
 CK: plasma concentrations are elevated in 30-70% of dialysis
patients at baseline, likely secondary to
1.skeletal myopathy,
2. intramuscular injections and
3. reduced clearance.
 CK-MB: 30-50% of dialysis patients exhibit an elevation in the MB
fraction >5% without evidence of myocardial ischemia
 Therefore, the most specific marker for suspected AMI in ESRD
patients is Troponin-I.

33. 35
CARDIAC TROPONINS
• Troponin is a complex of three regulatory proteins that is integral to
non-smooth muscle contraction in skeletal as well as cardiac muscle
• Troponin is attached to the tropomyosin sitting in the groove
between actin filaments in muscle tissue
• Troponin has three subunits, TnC, TnT, and TnI
– Troponin-C has calcium binding ability and has no diagnostic
value
– Troponin-T binds the troponin tropomyosin complex,
– Troponin-I is an inhibitory protein

35. 40
TROPONIN ASSAYS
 TropT (Roche Diagnostics, Germany)
 Trop I (Siemens Healthcare Diagnostics)
 Troponin T
 99th percentile limits - 0.01 ng/mL
 assay ranges - 0.01-25 ng/Ml
 (Troponin I)
 99th percentile limits -0.04 ng/mL
 assay range -0.04-40 ng/mL
 Reference limits based on the 99th percentile for a
healthy population are 0.01 ng/mL (Troponin T) and
0.04 ng/mL (Troponin I)

36. Creatine kinase
(CK) is a
cytosolic enzyme
CK is a dimer composed of two subunits B (brain type)
and M (muscle type), resulting in three isoenzyme:
CK-BB (CK1) : is of brain origin, found in blood only when BBB is damaged
CK-MB (CK2) : it is relatively specific for myocardial origin
CK-MM (CK3) : it is found primarily in skeletal muscle
CK-MB

37. Total CK (sum of CK-MM, CK-MB & CK-BB)
non specific to cardiac tissue (available in skeletal ms.)
CK-MB (CK-2) activity
more specific than total CK
BUT: less specific than troponin I (available in sk. Ms)
appears in blood: within 4-6 hours of onset of attack
peak: 12 - 24 hours
returns to normal: within 2 - 3 days (no long stay in blood)
Advantages: - useful for early diagnosis of MI
- useful for diagnosis reinfarction
Disadvantages: not used for delayed admission (more than
2 days)
not 100% specific (elevated in sk.ms damage)

38. •CK-MB mass
- appears one hour earlier than CK-MB activity (more
sensitive)
- So, useful for diagnosis of early cases & reinfarction
- BUT: not for diagnosis of delayed admission cases
& less specific than troponin I
•Relative index = CK-MB mass / Total CK X 100
more than 5 % is indicative for MI

39. 44
DRAWBACKS
• False positive (for MI) CK-MB elevation can be seen in:
– Significant skeletal muscle injury
– The MB fraction is determined to be expressed during the
process of muscle regeneration
– Cardiac injury for reason other than MI
• Defibrillation
• Blunt chest trauma
• Cocaine abuse
The search for cardiac specificity continues…

40. Small-size heme protein found in all tissues mainly assists in oxygen
transport
It is released from all damaged tissues
Its level rises more rapidly than cTn and CK-MB.
Released from damaged tissue within 1 hour
Normal value: 17.4-105.7 ng/ml
Timing:
• Earliest Rise:
• Peak
• Return to normal:
1-4 hrs
6-9 hrs
12 hrs
Myoglobin

41. CONDITIONS FOR MYOGLOBIN
INCREASE :
Acute myocardial infarction
Skeletal muscle damage, muscular
dystrophy, inflammatory myopathies
Renal failure, severe uremia
Shock and trauma

42.  Clinical usefulness of myoglobin :
*if myoglobin concentration remains within the reference range
8 hours after the onset of chest pain, AMI can be ruled out
essentially.
*because of its rapid clearance by the kidney, a persistently
normal Mb concentration will rule out reinfarction in patient
with recurrent chest pain after AMI
*Rapid monitor of success of thrombolytic therapy
DRAWBACKS
 Due to poor specificity, myoglobin levels do not always predict
myocardial injury

43. 2
3
4
7
6
5
myoglobin
CK-MB
cTnT
cTnI
COMPARISON OF CTN, CK-MB , MB
1
0
0 4 8 12 16 20 24 28 32 36 40 44 48
Time after onset of AMI (hours)
Χupperlimitofreferenceinterval

44. Ischemia Modified Albumin (IMA)
A novel marker of ischemia, is produced when circulating serum
albumin contacts ischemic heart tissues
IMA can be measured by the albumin cobalt binding (ACB)
assay that is based on IMA's inability to bind to cobalt
Mechanism- due to structural change in the amino terminal end
of albumin
IMA levels rise within 6 hours
remain elevated for 12 hours

45. Drawbacks
IMA levels raised in non- cardiac ischemia
Modification to n- terminal end may also be induced
by extracellular hypoxia, acidosis etc,
Conclusion
FDA in 2010 has approved a multimarker approach for
using the combination of ECG, the cTnI, and the IMA levels
achieving a sensitivity of 95% for ACS

46. Heart type fatty acid binding protein is a very stable low
molecular weight (14-15kDa) in the cytoplasm of myocardial
cells.
FABPs are involved in active fatty acid metabolism where it
transports fatty acid from cell membrane to mitochondria for
oxidation.
Small size of H-FABP facilitates rapid diffusion through
interstitial space, appearing as early as 1-3 hrs after onset and
peaking within 6hrs. It return to normal levels with in 12-24hrs.
Normal levels : 1.6 – 19 ng/ml
H-FABP

47. NATRIURETIC PEPTIDES
The natriuretic peptides (NP) are a group of
structurally similar but genetically distinct peptides.
NPs are identified as regulatory diuretic-natriuretic
substances responsible for salt and water
homeostasis
Lowers blood pressure.

48. The NP family
includes
ANP : -atrial
natriuretic peptide
(28 a.a.)
N-terminal
proANP (98 a.a.)
BNP : brain
natriuretic peptide
(32 a.a.)
N-terminal
proBNP (76 a.a.)
CNP : C-type
natriuretic peptide
(22 and 53 a.a.)

49. Fig. Schematic representation of the ANP and BNP precursors with
sequence numbering defining low-molecular-mass forms, N-terminal forms
and high-molecular-mass precursors

50. OTHER CARDIAC MARKERS

51. Homocysteine
Intermediary amino acid formed by the conversion of
methionine to cysteine
Moderate hyperhomocysteinemia occurs in 5-7% of the
population
Recognized as an independent risk factor for the
development of atherosclerotic vascular disease and venous
thrombosis
Can result from genetic defects, drugs, vitamin deficiencies,
or smoking

52. Homocysteine implicated directly in
vascular injury including:
• Intimal thickening
• Disruption of elastic lamina
• Smooth muscle hypertrophy
• Platelet aggregation
Vascular injury induced by leukocyte
recruitment, foam cell formation, and
inhibition of NO synthesis
Homocysteine

53. Elevated levels appear to be an independent risk factor,
Screening recommended in patients with premature CV
disease (or unexplained DVT) and absence of other risk
factors
Treatment includes supplementation with folate, B6 and
B12
Homocysteine

54. C-REACTIVE PROTEIN
CRP is an acute-phase protein produced by the liver
Pentameric structure consisting of five 23-kDa identical
subunits
Plasma levels can increase rapidly to 10000x levels
High-sensitivity CRP (hs-CRP) assays

56. CRP previously known to be a marker of high risk in
cardiovascular disease
More recent data may implicate CRP as an actual
mediator of atherogenesis
Mechanism of CRP-mediated atherogenesis:
Once ligand-bound, CRP can:
• Activate the classical compliment pathway
• Stimulate phagocytosis
• Bind to immunoglobulin receptors
• Endothelial dysfunction via ↑ NO synthesis
• ↑LDL deposition in plaque by CRP-stimulated macrophages

59. Clinical Uses
• Screening for cardiovascular risk in otherwise
“healthy” individuals
• Predictive value of CRP levels for disease
severity in pre-existing Coronary artery disease
Elevated levels predictive of
• Long-term risk of first MI
• Ischemic stroke

60. Limitations of CRP
Low specificity
No evidence that lowering CRP levels decreases CV risk
• Industry and FDA staff guidelines 2005 had given clinical cut off value
as less than 1 mg/l as safe levels with hs-CRP tests
CRP Risk for CVD
Less than 1.0 mg/L
1.0-2.9 mg/L
Low
Intermediate
Greater than 3.0 mg/L High

61.  miRNAs are appx. 20-25 nucleotide long non coding RNAs, that negatively
regulate or inhibit gene expression by binding to sites in the untranslated
regions of targeted messenger RNAs.
miRNA

62.  miRNA are found to be involved in almost every
biological process, from cellular differentiation and
proliferation to cell death and apoptosis
 Many different types of miRNA can be detected in
circulating blood and these miRNA are present in
remarkably stable form that even withstand repetitive
freezing/thawing cycle and are protected against Rnases.
 Thousands of miRNAs have been described in human to
date which ehibits tissue specific pattern of expression.

63.  miRNAs that regulates cardiovascular system can be
divided into 4 groups :
1.miRNA regulating endothelium function and
angiogenesis : miR126, miR17-92 cluster, miR130a,
miR221, miR21
2. cardiac myocyte specific mRNA : miR208a
3. cardiac myocyte and skeletal muscle miRNA : miR1,
miR133a, miR499
4. smooth muscle miRNAs :miR143, miR145
miRNAs hold promise as very specific and accurate
marker of cardiac dysfunction.

64. CYTOKINES
Variety of interleukins (TNF, IL-1, IL-6, IL-8, IL-12, IL-
18) are responsible for the atherosclerosis events.
Regulated through the T cell-mediated and
monocytes.
At present there are no standardized assay,
reference interval studies.

65. MYELOPEROXIDASE (MPO)
Released from aggregated neutrophils.
Increased in CAD,ACS.
Is one of the best prognostic marker
At present no standardized assay method, nor consistency validation
of the test.
Further, Type of specimen collect have shown variations.
Normal level: 640 pmol/L

66. LIPOPROTEIN ASSOCIATE
PHOSPHOLIPASE A2 (Lp-PLA2)
Previously known as PAF ( platelet activating factor)
Synthesised from monocytes and lymphocytes.
Known to form lipid fragments which are most
atherogenic which increase endothelial adhesion.
FDA approved for the assay.
Threshold for high risk: >200μg/L

67. PREGANACY ASSOCIATED
PLASMA PROTEIN A(PAPP-A)
It is an metaloprotimase.
Produced from the plaque which are prone to rupture.
Marker for the adverse CV events.
At present no standardized assay, consistency validation
is present.
By two approach, median value for free PAPP-A is 0.18
mIU/L.

68. CHOLINE
Is released after stimulation by phospholipase D.
Touted as a test of prognosis in pt with chest
discomfort.
Not available

69. UNBOUND FREE FATTY ACID
(FFA-U)
Marker for ischemia.
Ischemia increase the small unbound
fraction.
Studies revealed mixed result.
Not available

70. NOURIN
Small protein released by stressed
myocytes.
Induce changes of inflammatory cytokines
and attract neutrophils.
Preliminary studies have made attempt to
validate its use.

71. 30 a.a glycoprotein.
Prognostic marker in hemorrhagic and sepsis.
Recently, early biomarker for rule out the AMI with
normal cTn.
The test method has to undergo many trials and
head-to-head comparison with cTn.
COPEPTIN