cardiac markers and renal function

7/30/2019 Cardiac Markers and Renal Function

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1

Interpretation of Laboratory Tests:

A Case-Oriented Review of Clinical

Laboratory Diagnosis

Roger L. Bertholf, Ph.D.

Associate Professor of Pathology

University of Florida Health Science Center/Jacksonville


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2

Case 1: Oliguria and hematuria


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R. Bertholf American Society of Clinical Pathologists 3

Case 1: Oliguria and hematuria

A 7-year-old boy was brought to the pediatrician because of vomiting

and malaise. On physical examination he was slightly flushed, and had

some noticeable swelling of his hands and feet. The patient was

uncomfortable, and complained of pain in his tummy. He had a slightfever. Heart was normal and lungs were clear. His past medical history

did not include any chronic diseases. The mother noted that he had a

severe sore throat about two weeks ago, but that it had cleared up on

its own. The child was not taking any medications. There were no

masses in the abdomen, and lymphadenopathy was not present. Thechild had some difficulty producing a urine specimen, but finally was

able to produce a small amount of urine, which was dipstick-positive

for blood and protein.


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Questions. . .

What is the differential diagnosis in this case?

What laboratory tests might be helpful in

establishing the diagnosis?


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What do the kidneys do?

Regulate body fluid osmolality and volume

Regulate electrolyte balance

Regulate acid-base balance

Excrete metabolic products and foreign substances

Produce and excrete hormones


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The kidneys as regulatory organs

The kidney presents in the highest degree the phenomenon

of sensibility, the power of reacting to various stimuli in a

direction which is appropriate for the survival of the organism;

a power of adaptation which almost gives one the idea that its

component parts must be endowed with intelligence.

E. Starling (1909)


7/77R. Bertholf American Society of Clinical Pathologists 7

Review of Renal Anatomy and Physiology

The kidneys are a pair of fist-sized organs that are

located on either side of the spinal column just

behind the lower abdomen (L1-3).

A kidney consists of an outer layer (renal cortex)

and an inner region (renal medulla).

The functional unit of the kidney is thenephron;

each kidney has approximately 106 nephrons.



Renal anatomy

Cortex

Medulla

Pelvis

To the bladder

Capsule



The Nephron

Renal artery

Glomerulus

Bowmans capsule

Proximal tubule

Distal tubule

Collecting duct

Henles Loop

Afferent arteriole



Glomerular filtration

Glomerlular

capillary

membrane

Vascular space Bowmans space

Mean capillary bloodpressure = 50 mm Hg

BC pressure = 10 mm Hg

Onc. pressure = 30 mm Hg

Net hydrostatic = 10 mm Hg

2,000 Liters

per day(25% of cardiac output)

200 Liters

per day

GFR 130 mL/min



What gets filtered in the glomerulus?

Freely filtered

H2O

Na+, K+, Cl-,

HCO3-, Ca++,

Mg+, PO4, etc.

Glucose

Urea

Creatinine

Insulin

Some filtered

2-microglobulin

RBP

1-microglobulin

Albumin

None filtered

Immunoglobulins

Ferritin

Cells



Then what happens?

If 200 liters of filtrate enter the nephrons each day,

but only 1-2 liters of urine result, then obviouslymost of the filtrate (99+ %) is reabsorbed.

Reabsorption can be active or passive, and occurs

in virtually all segments of the nephron.


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Reabsorption from glomerular filtrate

% Reabsorbed

Water 99.2

Sodium 99.6Potassium 92.9

Chloride 99.5

Bicarbonate 99.9

Glucose 100

Albumin 95-99Urea 50-60

Creatinine 0 (or negative)


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How does water get reabsorbed?

Reabsorption of water ispassive, in response to

osmotic gradients and renal tubular permeability.

The osmotic gradient is generated primarily byactive sodium transport

The permeability of renal tubules is under the

control of the renin-angiotensin-aldosterone

system. Thedriving force for water reabsorption, the

osmotic gradient, is generated by theLoop of

Henle.


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The Loop of Henle

Proximal tubule Distal tubule

Descendinglo

opA

scendingloop

In

creasingosmolality

Renal Cortex

Renal Medulla

Na+

Na+

Na+

H2ONa+

1200 mOsm/Kg

300 mOsm/Kg


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Regulation of distal tubule Na+

permeability

JGA Renin Na+

BP

Angiotensinogen

Angiotensin I

Angiotensin II

Angiotensin III

vasoconstriction

AldosteroneAdrenal cortex

Na+ Na+


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Regulation of H2O reabsorption

Pituitary

ADH (vasopressin)

Plasma

hyperosmolality

H2OH2O

Renal Medulla (osmolality 1200 mOsm/Kg)


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Summary of renal physiology

Filtration - Reabsorption + Secretion = Elimination

GFR (Filtered but not reabsorbed or secreted)

TRPF (Filtered and secreted)


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Measurement of GFR

694.0

)24(

p

huu

C

VC

Clearance

Cu = Concentration in urineVu(24h) = 24-hour urine volume

Cp = Concentration in plasma

0.694 = 1000 mL/1440 min


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Compounds used to measure GFR

Should not be metabolized, or alter GFR

Should be freely filtered in the glomeruli, but neither

reabsorbed nor secreted

Inulin (a polysaccharide) is ideal

Creatinine is most popular

There is some exchange of creatinine in the tubules

As a result, creatinine clearance overestimates GFR byabout 10% (But. . .)

Urea can be used, but about 40% is (passively)

reabsorbed


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Relationship between creatinine and GFR

P

lasmacreatin

ine

GFR (mL/min)

1

2

3

4

5

6

00 20 40 60 80 100 120 140


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Measurement of TRPF

Para-aminohippurate (PAH) is freely filtered in the

glomeruli and actively secreted in the tubules.

PAH clearance gives an estimate of the total

amount of plasma from which a constituent can be

removed.


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Creatinine

O

N

CH3

HN

NH

HO

O CH3

NH

NH2

- H2

ON

Creatine Creatinine

1-2% of creatine is hydrolyzed to creatinine each day


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Jaffe method for creatinine

O

N

C HH N

NH

O N N O

N O

O H

O H

+Janovsky Complex

max = 490-500 nm

Max Eduard Jaffe (1841-1911), German physiologic chemist


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Modifications of the Jaffe method

Fullers Earth (aluminum silicate, Lloyds reagent)

adsorbs creatinine to eliminate protein interference Acid blanking

after color development; dissociates Janovsky

complex

Pre-oxidation

addition of ferricyanide oxidizes bilirubin

Kinetic methods


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Kinetic Jaffe method

Absorbance(=

5

20nm)

Time (sec) 0 8020

Fast-reacting

(pyru

vate,glucose,asc

orbate)

Slow-reacting

(protein)

t

A

rate

t

A

creatinine (and -keto acids)


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Enzymatic creatinine methods

Creatininase

creatininecreatineCKADPPKLD Creatinase

creatininecreatinesarcosinesarcosineoxidaseperoxideperoxidase reaction

Creatinine deaminase (iminohydrolase)

most common


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Creatinine deaminase method

Creatinine

Creatinine

iminohydrolase

+ H2ON-Methylhydantoin

ATP

ADP

NMH amidohydrolase

N-Carbamoylsarcosine

H2O

PeroxidaseOxygen receptor Colored product

Sarcosine

NCS

amidohydrolase

- NH3, CO2

+ O2

Sarcosine oxidase

H2O H2O2

Formaldehyde + glycine


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Measurement of urine protein

Specimen

Timed 24-h is best

Urine protein/creatinine ratio can be used withrandom specimen

Normal protein excretion is


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Dipstick method for urine protein

Method is based on protein association with pH

indicator

Test pad contains dye tetrabromphenol blue atpH=3

If protein binds to the pH indicator, H+ is displaced

and the color changes from yellow to green (or

blue) Most sensitive to albumin (poor method for

detecting tubular proteinuria)


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What causes excess urinary protein?

Overload proteinuria

Bence-Jones (multiple myeloma)

Myoglobin (crush injury, rhabdomyolysis)

Hemoglobin

Tubular proteinuria

Mostly low MW proteins (not albumin)

Fanconis, Wilsons, pyelonephritis, cystinosis Glomerular proteinuria

Mostly albumin at first, but larger proteins appear

as glomerular membrane selectivity is lost.


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Classification of proteinuria: Minimal


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Classification of proteinuria: Moderate

1.0 - 4.0 grams of protein per day

Usually associated with glomerular disease Overflow proteinuria from multiple myeloma

Toxic nephropathies


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Classification of proteinuria: Severe

>4 grams of protein per day

Nephrotic syndrome ( GBM permeability)

Sx: edema, proteinuria, hypoalbuminemia,

hyperlipidemia

In adults, usually 2 to systemic disease (SLE,diabetes)

In children, cause is usually primary renal disease

Minimal Change Disease (Lipoid Nephrosis)

Most common cause of NS in children

Relatively benign (cause unknown, not autoimmune)


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Proteinuria due to glomerulonephritis

Acute, rapidly progressive, or chronic GN can

result in severe proteinuria

Often the result of immune reaction (Circulating

Immune-Complex Nephritis)

Antigen can be endogenous (SLE) or exogeneous

Glomerular damage is mostly complement-mediated

If antigen is continuously presented, GN can

become chronic


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How do red blood cells get in urine?

Hematuria can result from bleeding anywhere in

the kidneys or urinary tract Disease, trauma, toxicity

Hemoglobinuria can result from intravascular

hemolysis

Disease, trauma, toxicity


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Dipstick method for hemoglobin

Ascorbic acid inhibits the reaction, causing a false

negative test

Depends on RBC lysis (may not occur in urine

with high specific gravity)

Detection limit approximately 10 RBC/ L

H2O2 + chromogen* Oxidized chromogen + H2OHeme

Peroxidase

*tetramethylbenzidine; oxidized form is green


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Microscopic examination of urine sediment


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Significance of RBC casts in urine

Indicative of blood crossing the GBM

Casts form in the distal tubules Stasis produces brown, granular casts

RBC casts almost always reflect glomerular disease


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Brights Disease (acute glomerulonephritis)

Characterized by oliguria, proteinuria, and

hematuria Most common cause is immune-related

Richard Bright(1789-1858)


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Primary Glomerulonephritis

Proliferative GN

Acute Post-infectious GN Idiopathic or Crescentic GN

-GBM disease

Membranoproliferative GN

Focal GN

IgA nephropathy


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Primary Glomerulonephritis, cont.

Idiopathic membranous GN

Histological diagnosis, probably immune complex

Chronic GN

Clinical Dx; many potential causes

Lipoid Nephrosis

Histological findings normal; Nephrosis

Focal Glomerular Sclerosis

Probably immune (IgM) related


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Secondary Glomerulonephritis

Systemic Lupus Erythematosus

Renal failure accounts for 50% of SLE deaths

Polyarteritis (inflammatory vasculitis) Wegeners Granulomatosis (lung and URT)

Henoch-Schnlein Syndrome

Lacks edema assoc. with post-streptococcal GN

Goodpastures Syndrome (pulmonary hemorrhage)

Hemolytic-Uremic Syndrome

Progressive Systemic Sclerosis (blood vessels)


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44

Case 3: Chest Pain


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Case 3: Chest Pain

A 63 year old male was brought to the emergency department

after complaining of severe chest pain that had lasted for two

hours. He had been mowing his lawn when the pain developed,

and he became concerned when the pain did not subside afterhe stopped the activity. He had no previous history of heart

disease. On presentation he was moderately overweight, dia-

phoretic, and in obvious discomfort. He described his chest

pain as beginning in the center of my chest, then my arms,

neck, and jaw began to ache too.

Diagnostic procedures were performed.


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Questions

What is the most important consideration in the

triage of this patient?

What tests should be ordered?


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Chest pain

One of the most common reasons for seeking

medical attention

Characteristics of cardiogenic chest pain (angina)

induced by exercise

described as pressure

radiates to extremities

MI notrelieved by rest or vasodilatory drugs (NG) Only 25% of patients presenting with chest pain as

the primary complaint will ultimately be diagnosed

as MI (specificity=25%; sensitivity=80%)


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The Heart

Aorta

Superior vena cava

RA LA

RV

LV

Pulmonary arteries


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The Heart (posterior view)

Aorta

Superior vena cava

Inferior vena cava

Pulmonary veins

Pulmonary arteries


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Cardiac physiology


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Cardiac conduction system

Sinoatrial (SA) node

Atrioventral (AV) node

His bundle

Right bundle branch

Left bundle branch


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Normal Electrocardiogram

PQ

R

S

T

U


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Myocardial infarction

Right coronary artery

Left coronary artery

Anterior left ventricle


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ECG changes in myocardial infarction

S

P

R

T

Q

S-T elevation


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Diagnostic value of ECG

ECG changes depend on the location and severity of

myocardial necrosis

Virtually 100% of patients with characteristic Q-wave and S-T segment changes are diagnosed with

myocardial infarction (100% specificity)

However, as many as 50% of myocardial infarctions

do not produce characteristic ECG changes(sensitivity 50%)

ECG may be insensitive for detecting prognostically

significant ischemia


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History of cardiac markers

1975: Galen describes the use of CK, LD, and

isoenzymes in the diagnosis of myocardial infarction.

1980: Automated methods for CK-MB (activity) andLD-1 become available.

1985: CK-MB isoforms are introduced.

1989: Heterogeneous immunoassays for CK-MB

(mass) become available.

1991: Troponin T immunoassay is introduced.

1992: Troponin I immunoassay is introduced.


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Enzyme markers

Aspartate transaminase (AST; SGOT)

2-Hydroxybutyrate dehydrogenase

Lactate dehydrogenase Five isoenzymes, composed of combinations of H

(heart) and M (muscle) subunits

Creatine kinase

Three isoenzymes, composed of combinations ofM (muscle) and B (brain) subunits


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Lactate dehydrogenase (LD)

Pyruvate

LD activity is measured by monitoring absorbance

at = 340 nm (NADH)

Methods can be P L or L PBut. . .reference range is different

Total LD activity has poor specificity

LactateLD

NAD+NADH


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Tissue specificity of LD isoenzymes

LD isoenzyme Tissues

LD-1 Heart (60%), RBC, Kidney

LD-2 Heart (30%), RBC, Kidney

LD-3 Brain, Kidney

LD-4 Liver, Skeletal muscle, Brain, Kidney

LD-5 Liver, Skeletal muscle, Kidney


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LD isoenzyme electrophoresis (normal)

LD-1

LD-2

LD-3LD-4

LD-5

LD-2 > LD-1 > LD-3 > LD-4 > LD-5

Cathode (-) Anode (+)


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LD isoenzyme electrophoresis (abnormal)

LD-1

LD-2

LD-3

LD-4 LD-5

LD-1 > LD-2

Cathode (-) Anode (+)


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Direct measurement of LD-1

Electrophoresis is time-consuming and only semi-

quantitative Antibodies to the M subunit can be used to

precipitate LD-2, 3, 5, and 5, leaving only LD-1

Method can be automated

Normal LD-1/LDtotal ratio is less than 40%


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Sensitivity and specificity of LD-1

Sensitivity and specificity of the LD 1:2 flip, or

LD-1 > 40% of total, are 90+% within 24 hours of

MI,but. . . May be normal for 12 or more hours after

symptoms appear (peak in 72-144 hours)

May not detect minor infarctions

Elevations persist for up to 10 days

Even slight hemolysis can cause non-diagnostic

elevations in LD-1


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Creatine Kinase (CK)

Phosphocreatine

ADP

HKGlucose Glucose-6-phosphate

NADPH

=340 nm

NADP+

GPD

6-Phosphogluconate

Oliver and Rosalki method (1967)

CreatineCK

ADP ATP

Mg++


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Tissue specificities of CK isoenzymes

Tissue

CK-1

(BB)

CK-2

(MB)

CK-3

(MM)

Skeletal muscle 0% 1% 99%

Cardiac muscle 1% 20% 79%

Brain 97% 3% 0%


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Measurement of CK isoenzymes

Electrophoresis (not used anymore)

Immunoinhibition/precipitation

Antibody to M subunit

Multiply results by 2

Interference from CK-1 (BB)

Most modern methods use two-site (sandwich)

heterogeneous immunoassay

Measures CK-MB mass, rather than activity

Gives rise to a pseudo-percentage, often called the

CK-MB index


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Sensitivity/specificity of CK-MB

Sensitivity and specificity of CK-MB for

myocardial infarction are >90% within 7-18 hours;

peak concentrations occur within 24 hours CK is a relatively small enzyme (MW = 86K), so it

is filtered and cleared by the kidneys; levels return

to normal after 2-3 days

Sensitivity is poor when total CK is very high, andspecificity is poor when total CK is low

Presence of macro-CK results in false elevations


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CK isoforms

C-terminal lysine is removed from the M subunit--

therefore, there are three isoforms of CK-3 (MM)

t: CK-MB1 > CK-MB2

Ratio of CK-MB2 to CK-MB1 exceeds 1.5 within six

hours of the onset of symptoms

Only method currently available is electrophoresis

CK-MB2 (tissue) CK-MB1 (circulating)

C-terminal lysine

Plasma carboxypeptidase


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Myoglobin

O2-binding cytosolic protein found in all muscle

tissue (functional and structural analog of

hemoglobin)

Low molecular weight (17,800 daltons)

Elevations detected within 1-4 hours after

symptoms; returns to normal after 12 hours

Nonspecific but sensitive marker--primarily used

for negative predictive value

Usually measured by sandwich, nephelometric,

turbidimetric, or fluorescence immunoassay


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Temporal changes in myoglobin and CK-MB

0

200

400

600

800

0 8 16 24 32 40 48

Time after symptoms

Myoglobin(ug/L)

0

10

20

30

4050

60

CK-MB(ug/L

)

Myoglobin CK-MB


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Troponin

Thick Filament

Myosin

Tropomyosin Actin

TnC

TnT (42 Kd)

TnI (23 Kd)


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Tissue specificity of Troponin subunits

Troponin C is the same in all muscle tissue

Troponins I and T have cardiac-specific forms,

cTnI and cTnT Circulating concentrations of cTnI and cTnT are

very low

cTnI and cTnT remain elevated for several days

Hence, Troponins would seem to have thespecificity of CK-MB (or better),andthe long-term

sensitivity of LD-1


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Is cTnI more sensitive than CK/CK-MB?

-1

-0.5

0

0.5

1

1 8 40 66

Hours since presentation

logXnormal

CK

CK-MB

CK-MB Index

cTnI

79 y/o female with Hx of HTN, CHF, CRI, Type II diabetes


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Measurement of cTnI and cTnT

All methods are immunochemical (ELISA, MEIA,

CIA, ECIA)

Roche Diagnostics (formerly BMC) is the sole

manufacturer of cTnT assays

First generation assay may have had some cross-

reactivity with skeletal muscle TnT

Second generation assay is cTnT-specific

Also available in qualitative POC method

Many diagnostics companies have cTnI methods


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W.H.O. has a Myocardial Infarction?

A clinical history of ischemic-type chest discomfort

Changes on serially obtained ECG tracings

A rise and fall in serum cardiac markers

A patient presenting with any two of the following:

SourceJACC28;1996:1328-428


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Sensitivity/Specificity of WHO Criteria

0%

20%

40%

60%

80%

100%

Chest Pain ECG changes Serum

markers

Sensitivity

Specificity


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What Cardiac Markers do Labs Offer?

0500

1000

1500

2000

25003000

3500

#o

flabsreporting

CK-MB

(ng/mL)

CK-MB

(IU/L)

cTnI cTnT

1997

1998

cardiac markers and renal function

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