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Laboratory diagnosis of myocardial

infarction and acut coronary syndrome
Classical markers: CK LDH isoen mes m oglobin Classical markers: CK, LDH isoenzymes, myoglobin.
New markers: Troponin I, Troponin T, significance
of point of care testing diagnostic algorithms of point of care testing, diagnostic algorithms.
2009.III. 24.(Tuesday)
15.00-17.00
Pathobiochemical changes during MI
Formation of myocardial
infarction

Pump failure
Ions (eg. potassium)
Myocardial ischemia

Energy deficit gy

Disturbed metabolic control

Substances
released from
Metabolic
disturbance

Reversible damage

Irreversible damage
released from
cells
(lactate)
Irreversible damage

Cell death

Membrane defect

Cell necrosis
Macromolecules
(eg. enzymes)
WHO criteria for the diagnosis of MI WHO criteria for the diagnosis of MI gg
1/ Ischaemic symptoms
(Severe prolonged chest pain) ( p g p )
2/ ECG changes consistent with ischaemia
3/ Biochemical markers 3/ Biochemical markers
The exclusion of MI can not be established on a
single biochemical test, but for the proof of the g , p
disease a single test can be suitable.
Characteristics of the ideal
biochemical marker
1/ Hi h iti it d ifi it 1/ High sensitivity and specificity
2/ Early marker 2/ Early marker
3/ Stays elevated for several
days
(large diagnostic window) (large diagnostic window)
4/ The assay can be performed y p
quickly
Biochemical markers in Biochemical markers in
the diagnosis of MI the diagnosis of MI
Early markers(They are not specific but their level
isalreadyelevated4-6hoursafter theonset) is already elevated 4-6 hours after the onset)
Exclusion tests: myoglobin, (CK-MB isoforms?)
Definitivemarkers(specific but their values Definitive markers(specific, but their values
elevates later) diagnostic tests:
cTnI cTnT or CK-MB (mass) cTnI, cTnT, or CK MB (mass)
The 2 troponins are equivalent
Old” markers: „Old markers:
Total CK, CK-”MB” activity,
AST LD LDisoenzymes AST, LD, LD isoenzymes
Cardiac marker concentrations
after AMI
MYOGLOBIN MYOGLOBIN
•Globular, monomer, hem containing protein
•Function: oxigen supply of striated muscle (O-binding protein)
•Inthe cytoplasmof musclecellswithMW: 17800 •In the cytoplasm of muscle cells with MW: 17800
•Duetoitsabundancyandlowmolecular weight it is Due to its abundancy and low molecular weight it is
released into blood rapidly (as early as 1 hour) after cell
damage of heart or sceletal muscle damage of heart or sceletal muscle.
Peaks after 6-9 hours
After 16-24 hours returnstonormal values After 16 24 hours returns to normal values
•If myoglobin remains within the reference range 10
hoursafter chest painonset AMI canberuledout with hours after chest pain onset, AMI can be ruled out with
high probability (high negative predictive value).
T i i fil Tropomyosin-actin filaments
Slide 8 Slide 8
Release of cardiac
i i MI troponins in acute MI
6% i T 6% troponin T
3% troponin I
are in the cytoplasm
theremainingis the remaining is
structurally bound
and released continuously
by proteolytic degradation yp y g
into the circulation.
Troponinsremainelevated Troponins remain elevated
for up to 10-14 days.
How troponin get released into circulation? How troponin get released into circulation?
cTnI (209 amino acid) cTnI (209 amino acid)
N-, and C terminal
proteolysis
Fragmented cTnI
(30-110 core-fragme
In the blood:
•Intact/fragmented •Intact/fragmented
•free cTnI/ cTnI-C-T
complex complex
METHODS FOR THE DETERMINATION OF TROPONIN
Q i i i ( l ) i Quantitative immunoassays (serum, plasma) using
Troponin I and Troponin T specific monoclonal
tib di : antibodies:
ELISA
MEIA MEIA
Luminescent immunoassay
Quick tests (anticoagulated whole blood):
Immune-chromatography Immune chromatography
(qualitative/quantitative)
Sensitivity values of different Sensitivity values of different
Troponin assays Troponin assays
H
Pontatlanság
Teszt
/
a
analitikai
(ng/ml)
functional
(ng/ml)
Ref.tartomány/ cut off
(ng/ml) (% percentil)
cut off AMI WHO
(ng/ml)
10% pontatlanság
(ng/ml)
Szenzitivitás Cut off
( p )
Byk-Sangtec
LIAISON Troponin I
0.005 0.03 0.034 (99%) 0.06
Abbott
AxSYMTroponin I
0.3 nincs adat 0.5 (95%) 2.0 nincs adat
Roche
Elecsys Troponin T
0.01 0.03 0.01 (99%) 0.1 nincs adat
DADE Behring
Stratus Troponin I 0.03 nincs adat 0.06 (95%) 0.6-1.5 nincs adat
RxL DimensionTroponin I
0.04 nincs adat 0.05 (97%) 0.6-1.5 nincs adat
Beckman Coulter
Access ACCU TnI
0.006 0.03 0.04 (99%) 0.09
DPC
Immulite/ TurboTroponin I 0.1 nincs adat 1,0 (98%) nem specifikált nincs adat
Immulite 2000
0.1 nincs adat 1,0 (98%) nem specifikált nincs adat
ACC criteria 2002
Introduction of two decision limits for cTroponins:
Below 1. cut off: exclusion myocardial injury
Above2cut off: suggestsAMI Above 2 cut off: suggests AMI
In-between: suggests myocardial injury
The time course of „old” plasma
k ft di l i f ti markers after myocardial infarction
Enzyme Start to
rise
Peak
elevation
Duration
of rise
(hours) (hours) (day)
CK-MB 3-8 16-24 1-4
CK (total) 4-10 20-30 3-6
LD 12-24 30-60 7-14
LD
Case 1 Case 1
A 61-year-oldwomanpresentedwithbackandchest A 61 year old woman presented with back and chest
pain. She had 3 episodes of stabbing chest pain over
the past year. The pain did not radiate down the arms p y p
and was thought to be atypical for MI. She took statin
for her hypercholesterolaemia.
Her laboratory results were as follows:
CK 6094 U/L (30 140) CK 6094 U/L (30-140)
LDH 654 U/L (85-180)
Which can be the potential causes of the CK
elevation ? elevation ?
Total CK can be elevated Total CK can be elevated
• AMI
i i j ti • i.m. injection
• Traumatic damage of skeletal muscle g
• Hypothermia
E i • Exercise
• Intoxication
• Dose-related side effect in statin treatment
CK :Creatine kinase
Humancreatinekinase(CK) issynthesizedbyanumber of Human creatine kinase(CK) is synthesized by a number of
different genes(14 and 19 chromosoma)
Therespectivegeneproductsarecalled The respective gene products are called
CK-M muscle
CK-B brain CK B brain
CK-Mi mitochondrion
Thetotal CK activityinserumcanbecomposedof the The total CK activity in serum can be composed of the
activity of dimeric iosoenzymes CK-MM,CK-MB,CK-
BB, and postsynthetically modified form of macro-CK
In healthy individuals total CK activity consists mainly of
CK-MMwhile the other CK isoenzymes and variants
are present in trace
Tissue distribution of CK isoenzymes
CK-MM: skeletal muscle cardiacmuscle
Tissue distribution of CK isoenzymes
CK MM: skeletal muscle, cardiac muscle
CK-MB: cardiac muscle, tounge, (skeletal muscle)
CK BB b i i i l CK-BB: brain, uterus, gastrointestinal tract,
lung, prostata,
NormallytheCK-MB fractionis<6%intheserum the Normally the CK MB fraction is < 6% in the serum, the
rest is CK-MM (~95%).
Myocardium: 60% CK-MM, 40% CK-MB
Skeletal muscle: 97% CK-MM, 3% CK-MB
CK-electrophoresis CK electrophoresis
Case 2.
A 64-y-old Caucasian man had been receiving
treatment for hypercholesterolemiawiththe3- treatment for hypercholesterolemia with the 3
hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) reductase inhibitor simvastatin ( )
for 3 wk.
His serum creatine kinase (CK) was measured ( )
as part of the routine follow-up of statin
treatment.
He had no chest pain and denied any recent
pain similar to his normal anginal pain. p g p
PAST MEDICAL HISTORY
The patient had suffered from angina for 12 y. He
had hypertension and epilepsy yp p p y
He walked with the aid of crutches due to weakness
in his left leg and had a neuropathic bladder. Both of
h di i h db ib d d these conditions had been attributed to a cauda
equina syndrome which had been operated on with a
lumbar laminectomy3ypreviously lumbar laminectomy 3 y previously.
Hereceivednospecifictreatment for hisangina. He received no specific treatment for his angina.
Laboratory results
Patient Reference
CK : 396 IU/L 15-195 IU/L CK : 396 IU/L 15 195 IU/L
CK-MB: 38 IU/L < 25 IU/L
9.6% < 6 %
WHAT ADVICE WOULD YOU GIVE TO THE
PATIENT’S PHYSICIAN REGARDING THE PATIENT S PHYSICIAN REGARDING THE
INTERPRETATION OF THIS RESULT?
CK MB l ll i d i hi h i di l CK-MB elev. are usually associated with ischemic myocardial
damage
Although the patient had multiple risk factors for an ischemic g p p
cardiac event, on this occasion there was no typical history of
such.
Statintreatment isassociatedwithincreasesinCK but thisis Statin treatment is associated with increases in CK, but this is
attributed to the skeletal (MM) isoenzyme only.
Repeat sample
carry out an electrocardiogram (ECG)
stopthesimvastatintreatment stop the simvastatin treatment
Exclude spurious analytical cause of the raised CK-MB
investigate the possibility of a nonmyocardial cause of the g p y y
raised cardiac isoenzyme fraction
Why should we think about spurious
analytical causes related to CK-MB analytical causes related to CK-MB
results?
Methods for determination of CK isoenzymes Methods for determination of CK isoenzymes
1. Electrophoresis. Detects all CK izoenzymes,
2 Immunoinhibition Quick reliable doesnot 2. Immunoinhibition. Quick, reliable, does not
differentiate among CK-BB, macro CK,
i h d i l C dC l b mitochondrial CK and CK-MB, results can be
expressed as percentage of total CK activity
3. CK-MB mass concentrationby immunoassay,
specific precise relativelyexpensive certain specific, precise, relatively expensive, certain
assays are time-consuming
Makro CKs
Type 1.
Makro CKs
Complex of a CK isoenzyme (most frequently CK-BB) with
immunoglobulins.
Frequently occurs in women above 50,
In severe gastrointestinal and vascular diseases, adenomas,
i carcinomas
Type 2.
Oligomeric mitochondrial CK
Mostly in severe malignant or liver diseases
!Both macro CKs interfere with the immuno-inhibition method of
CK-MB determination.
HOW WOULD YOU EXCLUDE A SPURIOUS
CAUSE OF THE RAISED CK-MB?
Exclusion of spurious causes of
CK-MB elevation
perform CK-MB sandwich („mass”)
i (CK MB 23 /L l 7) immunoassay (CK-MB 23 µg/L, normal <7)
electrophoresis (CK-MB 8.5%) (macroCK) p ( ) ( )
troponin I (result not available at initial
cons ltation) asfo ndnormal (<01 g/L) consultation) was found normal (<0.1 µg/L),
suggesting the patient had not suffered a
myocardial event
Which conditions may be associated
i i C i A ? with truly raised CK-MB beside AMI?
True increase in CK-MB without AMI
Relative contribution: <6%,
muscular dystrophy,
h bd l i rhabdomyolysis,
malignant hyperthermy
Relative contribution: >6%
cardiac surgery, g y
athlets (e.g. Marathon runners), when the increase in CK-MB
attributes to a high propotion of regenerating skeletal muscle fibers,
which likefetal skeletal muscle arerelativelyrichinCK-MB which, like fetal skeletal muscle, are relatively rich in CK MB
Final diagnostic possibilities
Possibly raised CK-MB related to leg
k weakness
Possibly raised CK-MB secondary to statin y y
treament ? (Statin-related increases in CK
mainlyaffect MM isoenzyme) mainly affect MM isoenzyme)
Serum cardiac markers at
presentation and during follow-up
MARKER DAY 0 DAY 4” DAY 29 DAY 120
CK (IU/L) 396 530 407 405
CK-MB [IU/L (%)] 38 (9.6) 37 (7.0) 28 (7.0) 28 (7.0)
CK-MB ( µg/L) 23 25 14 15 CK-MB ( µg/L) 23 25 14 15
AST (IU/L) 20 24 17 -
LDH (IU/L) 639 570 580 -
Troponin I ( µg/L) <0.1 <0.1 - -
CK MM d CK MB i f CK-MM and CK-MB isoforms
Results of posttranslational modification
A C t i l l i i l d ff f th A C-terminal lysine is cleaved off from the
M-subunit by a plasmatic carboxypeptidase y p yp p
CK MM1 CK MM2 CK MM3 CK-MM1, CK-MM2, CK-MM3(CK-MM3/CK-MM1>1)
CK-MB1, CK-MB2(CK-MB2/CKMB1>1.5 AMI) C , C ( )
Determination of CK isoforms by electrophoresis.
„Old”not really useful enzyme
k i l i AMI markers in classic AMI
GOT/AST
glutamate-oxaloacetate transaminase glutamate oxaloacetate transaminase
aspartate transaminase
Ref.interval.:< 40U/L Ref.interval. < 40U/L
Tissue distribution: Tissue distribution
cardiac muscle,
liver, ve ,
skeletal muscle,
kidneys, d eys,
erytrocytes
LD (l t t d h d ) LD (lactate dehydrogenase)
Composes of 4 peptide chain (H és M subunit)
Isoenzyme Tissue
LD-1;HHHH;H
4
LD-2;HHHM;H
3
M
Cardiacmuscle, erythrocytes and kidney ,
brain
l l l k l h d LD-3;HHMM;H
2
M
2
Spleen,lung,leukocytes, lymph nodes,
endocrine glands (thyroid, pancreas, adrenals)
LD-4;HMMM;HM
3
Liver skeletal muscle LD 4;HMMM;HM
3
LD-5;MMMM;M
4
Liver, skeletal muscle
The relative amount of LD isoenzymes in normal serum: The relative amount of LD isoenzymes in normal serum:
LDH2 > LDH1 > LDH3 >LDH4 ~ LDH5
LDH1/LDH2 ratio < 0.8 LDH1/LDH2 ratio 0.8
After MI: the total LD is elevated, LD1/LD2 >1
Diff dg haemolysis
LD (l t t d h d ) i LD (lactate dehydrogenase) isoenzymes
Where should be the
biochemical markers of AMI tested?
TAT (sampling to result report): •TAT (sampling to result report):
•max. 60 min
•analytical part max 30 min •analytical part max. 30 min
•If this is not feasible Point of Care •If, this is not feasible Point of Care
Test
•POCT sample: anticoagulated whole venous
blood blood
CARDIAC READER
Test principle- Reaction
Sample area
Glassfiber fleece
p
Reaction
Glassfiber fleece
Removal of cells
Plasma
Detection zone
Plasma
Plasma Plasma
Marker: cTnT
Mioglobin
CARDIAC READER
T i i l D i Test principle- Detection
Control:
Gold labelled anti-marker MAB2
(monoclonal antibody 2) bounds to
th th ti tid the synthetic peptide
Negative reaction:
Only the biotin labelled anti- Only the biotin labelled anti
marker MAB1 bound by
streptavidin
Positive reaction:
Biotin labelled anti-marker
MAB1-marker- gold labelled
anti-marker MAB2 complex
bound by streptavidin
Marker: cardial Troponin T Marker: cardial Troponin T
Myoglobin
CARDIAC READER
Test parameters
Cardiac T Quantitative Cardiac M Cardiac T Quantitative
• Heparinized whole blood
• Measuring range:
/
Ca d ac M
• Heparinized whole
blood
0.1-2.0 μg/L
• Measuring time: 12 min
• Measuring range:
30-700 μg/L
• Measuring time: 8 min • Measuring time: 8 min
Easy to use
1 2 3
How to perform the qualitative assay
Obt i th bl d D 150 l (± 15 l)
Apply the sampleto the test
Obtain the blood
sample
Draw up 150 μl (± 15 μl)
blood into the
dispensing aid
Apply the sampleto the test
strip and read result after
15 minutes.
c) No control line
a) Single line (control
line) = negative test
result
b) 2 lines (control and
test) = positive test
result
c) No control line
= invalid test result!
Repeat test!
Case 3.
A 77-year-old man presented to the Emergency Department
with chest pain His vital signs were within normal limits with chest pain. His vital signs were within normal limits.
ECG results on presentation
-right bundle branch block g bu d e b c b oc
-no acute or evolutionary changes consistent with AMI or
ischemia.
PE not detected.
Case 3 Case 3.
Case 4 Case 4.
A 76-year-old man with type
1 diabetes mellitus,
di i i h d l diminished mental status,
and low blood glucose (1.6
mmol/L) presented to the mmol/L) presented to the
Emergency Department
with the chief complaint of p
weakness.
Next day ECG - atrial
fib ill ti id f ld fibrillation, evidence of old
septal myocardial
infarction infarction.
No ECG signs of new AMI.
Case 4 Case 4.
Case 5 Case 5.
An 82-year-old woman
presented to the Emergency
D i h l i f Department with complaints of
non-radiating chest pain on
the left side and shortness of the left side and shortness of
breath.
She had chronic obstructive
pulmonary disease and an old
MI, multiple angioplasty in the
t f past few years.
ECG - no evidence of acute
MI MI.
Case 5 Case 5.
Case 6
An 88 year old woman presented
Case 6.
An 88-year-old woman presented
to the Emergency Department
after falling in her home. She was g
admitted with a fracture in the
left femur.
Medical history:
-atrial fibrillation
no other known cardiac disease -no other known cardiac disease
-ECG at pres.: ST-T abnorm.,
possible anterolateral ischemia p
-ECG follow up: no evidence of
AMI
Ni d f h i l d i i -Nine days after hospital admission:
open surgery of the femur
Case 6 Case 6.
Case 7 Case 7.
A 74-year-old woman was admitted to the inpatient rehab unit because
of progressive weakness and difficulties in walking.
EMG: inflammatory myopathy, ECG: hypokinetic left ventricular wall
Case 8 Case 8.
A 76-year-old man with type I
diabetes mellitus fell on the
ground resulting in an admission ground, resulting in an admission
to the hospital for an
intertrochanteric fracture of the
l f hi H i h h f 49 left hip. He was in the hosp. for 49
days.
ECG (initial): old infarct with
anterolateral ischaemic changes
ECG follow up: no changes
consistent with a new AMI.
During hosp. stay:
-open surgery
bradycardia bradycardia
DVT
Case 8 Case 8.