Cardiac Troponin I (cTnI) assay by immunochromatography method: Principle, Procedure, Interpretation and results

Rapid Assay for detection of Human Cardiac Troponin I (cTnl) in Serum/Plasma & Whole Blood by ImmunoChromatography

Fig. Troponin I kit showing Positive result

Principle:

This test utilizes the principle of immunochromatography, with a unique two-site sandwich immunoassay on a nitrocellulose membrane. The conjugate pad contains two components - monoclonal anti-cTnl conjugated to colloidal gold and rabbit IgG conjugated to colloidal gold. As the test sample flows through the membrane assembly of the device, the highly specific anti-cTnl antibody - colloidal gold conjugate complexes with cTnl in the sample and travels on the membrane due to capillary action along with rabbit IgG-colloidal gold conjugate. This sample moves further on the membrane to the test region (T) where it is immobilized by another specific anti-cTnl antibody coated on the membrane leading to the formation of a pink-purple band. A detectable colored band is formed if cTnl level is equal to or greater  than 0.1 ng/ml. The absence of this colored band in the test region indicates cTnl concentration < 0.1 ng/ml.

Interpreting and Correlating Abnormal Laboratory Values : Cardiac Function Tests

Diagnosis of Myocardial Infarction (MI) and Acute Coronary Syndrome

Fig. Getting MI attack

Since acute MI (AMI) requires rapid and accurate diagnosis, especially now that new treatment options with thrombolytic agents are available, the clinical laboratory has been called upon to provide serum diagnostic tests that can make this diagnosis at an early stage. Until recently, laboratory diagnosis was based on serial determinations of the MB fraction of creatine phosphokinase (CK-MB); confirmation of the diagnosis was provided by the so-called ‘flipped ratio’ of the isozymes of lactate dehydrogenase (LD) 24-36 hours after the initial acute event and/or by observation of the characteristic time courses for elevations of the three enzymes, CK, aspartate aminotransferase (AST) and LD.

Clinical utility of cardiac markers in monitoring reperfusion following thrombolytic therapy

When STEAMI occurs there is thrombus responsible for the occlusion. It can be lysed by administration of thrombolytic agents (urokinase, streptokinase, tissue plasminogen activator) or pushed downward by angioplasty. This therapy is given if the onset of chest pain is <12 hours. 

Unfortunately reperfusion can cause tissue damage a syndrome known as reperfusion-ischemic injury. Normally after thrombolytic therapy occlusion should be resolved within 90-120 minutes but sometimes upto 3 days is seen. Injury usually occurs due to sudden change in environment and mediated by increase in intracellular calcium, oxidative stress, pH, etc.

During monitoring markers during reperfusion following therapy, there must be washout of markers from the circulation if reperfusion is good. The rate of increase in cardiac biomarkers (cTnI, cTnT, Mb, CK-2) concentration after thrombolytic therapy can be assessed by frequent sampling. 

Reperfusion assessment should be done within 60-120 minute after therapy because at this time there is clot resolution and marker washout. To meet this testing of markers should be rapid. For this assessment markers should be measured prior to therapy and after therapy and then at later time usually at 60 and 90 minute.

Because of low molecular weight and its rapid turnover Mb is used to assess the myocardial perfusion after thrombolytic therapy but also other are measured. Increased CK-MB after 12 hours of therapy means failed reperfusion. Raised serum CK-MB >10 IU/L/hour over the first 2.5 hour of treatment indicates successful thrombolysis. Similarly rise in troponin in first hour after therapy indicates success of therapy. Increase in Mb 2 hr after therapy indicates success. 

Clinical utility of Myoglobin and LDH

Myoglobin:

It is known for its excellent clinical sensitivity early after MI (even before troponin and CK) but has lack of tissue specificity. Serum level rises 1 hr after MI, with peak at 2-12 hours. An attempt to improve clinical specificity of Mb is the measurement of carbonic anhydrase III (CA III). After an AMI, serum CA III remain unchanged, while both CK-2 and Mb increased. In patients with skeletal muscle trauma, Mb CK-2, CA III (skeletal muscle specific) were all elevated.

Lactate Dehydrogenase:

Due to its wide distribution, elevations occur in various clinical conditions like MI, hemolysis, liver, kidney, lung, muscle disorders. Hemolysis if severe produces the LD isoenzyme pattern similar to MI and also during megaloblastic anaemia where LD1 increases.

These are no longer measured. For historical perspective, for patients with AMI, serum LD values become elevated at 12-18 hr after the onset of symptoms, peaking at 48-72 hrs and normalizing after 6-10 days. LD-1 rises within 10-12 hours, peaks at 72-144 hours and normalizes in 10 days after AMI, paralleling total LD. Due to prolonged half-life LD-1 is a clinically sensitive marker for infarction when used more than 24 hours after occurrence. In the serum LD2 is more than LD1 but the appearance of more LD1 than LD2 is called flipped pattern typical of cardiac muscle damage but is non specific as it is seen during hemolysis, megaloblastic anaemia, chronic exercise, etc. This flipped ratio persists for 3 to 4 days after heart attack.

Clinical utility of CKMB

CK-MB measurement is an acceptable alternative to cardiac troponin in diagnosis of AMI. CK-2 rises in about 4-6 hours after onset of AMI and peaks at 24 hours. It returns to normal at 48-72 hours. It also has higher sensitivity after 3 hours (100%) and lower sensitivity at 0 hours in diagnosis of AMI. This suggest the traditional serial measurement of sample at 0, 12 and 24 hours might be replaced with four early serum CK-2 measurement at 0, 3, 6, 9 hours after presentation. 

(Source: Arneson's Clinical chemistry, 2nd edition)

 Healthy individual.                                          AMI.

Apart from heart disease CK activity is greatly elevated in all types of muscular disease, during old ages, myositis, etc. Damaged skeletal muscle may contain more CK-MB owing to phenomenon of fetal reversion, thus serum CK-MB isoenzyme may increase in such conditions. Hypothyroid subjects have elevated CK activity. In Duchene type muscular dystrophy value upto 50-100 times URL is seen.

Relative index (CK-MB mass assay/total CK x 100) may be used as an indicator of MI. A relative index >3% is indicative of AMI.

Clinical utility of Brain Natriuretic Peptide (BNP)

The general consensus about testing of BNP and proBNP is that, the testing should be performed to confirm the diagnosis of CHF in patients with a suspected diagnosis of CHF but presenting with ambiguous clinical features e.g. dyspnea which may be caused by COPD and cardiac failure. It is also used to monitor CHF patients following therapy.

It is also used to monitor CHF patients following therapy. BNP and NT-proBNP are used to identify patients with moderate to severe CHF and risk stratification of CHF and those with ACS. NPs increases in other conditions of volume overload – thus non specific. Used in risk stratification in CHF, ACS along with troponin measurement. Proposed for screening purpose considering risk factors

q  BNP usually >100 pg/mL in CHF.

The NT-proBNP (1-2 hr T_1/2) fragment is not cleared via receptor mediated process but 

predominantly by kidney. So, NT-proBNP is more sensitive to changes in renal function.

Since BNP is released by ventricles during stress condition, this can provide sensitive marker for changes in ventricular physiology. It is Proposed for screening purpose in high risk groups.

Clinical utility of Troponin

(Source: Tietz Clinical Chemistry 4th Edition)

Fig: Use of troponin to identify UA from MI

Increase above URL seen after 2-6 hours of onset of AMI and peaks at 48 hours. The initial increase in due to 3-6% cytoplasmic fraction of troponin (CK-MB is 100% cytoplasmic). 

Second, cTnI and cTnT can remain increased upto 4-14 days after AMI. This is due to release from 94% to 97% myofibril bound fraction. Third is very low to undetectable cardiac troponin values from patients without cardiac disease permit the use of lower discriminator concentrations compared with CK-MB for determination of myocardial injury.

Troponin is insufficient for effective early diagnosis as it shows low sensitivity and specificity upto 6 hr after onset of chest pain. But has high clinical sensitivity (>90%) above 8 hours after AMI.

Due to higher cytosolic content cTnT releases earlier than cTnI. In addition cTnT release is biphasic first cytosolic and second is muscle bound. cTnI release is monophasic due to lower free cTnI and also it exist and TIC, IC or free I.

72 hour troponin measurement correlates with infarct size. Since the second phase rise in cTnT occurs within 2-4 days, so if the infarct is severe then this phase is seen but if it is improving this phase is not seen.

Other causes of increase in troponins are hypothyroidism, renal failure, hypertension, rhabdomyolysis, sepsis, pulmonary embolism, etc.

CLINICAL UTILITY OF CARDIAC BIOMARKER MEASUREMENT

Measurement of cardiac specific proteins and providing their characteristics during myocardial injury can add in the diagnosis of disease. E.g as shown in figure;

(Source: Tietz Clinical Chemistry, 4th Edition)

According to European society of cardiology/American college of cardiology (ESC/ACS) the differential diagnosis of AMI from non-AMI can be done by biochemical cardiac biomarkers specially cTnI and cTnT. The following are biochemical indicators for detecting myocardial necrosis:

1.      Concentration of cTnI and cTnT exceeding 99^th percentile of the reference control group, on at least one occasion during the first 24 hour after the clinical event.

2.     CK-MB exceeding 99^th percentile the reference value on two successive samples or maximal value exceeding twice the upper reference limit during first hours after the clinical event (although CK-MB should rise or fall, a rising or falling pattern of CK-MB should be considered diagnostic; values that remain elevated without change are rarely caused by MI).

3.      In the absence of troponin or CK-MB assay, total CK greater than two times the URL may be used.

To date monitoring ACS patient to assist in clinical classification, cardiac troponin is the preferred biomarker. 

(Source: Bishop's Clinical chemistry, 6th edition)

Early diagnosis of MI (≤ 6 hr) – possible with two markers CK-MB isoforms and Mb

Characteristics of Biochemical indicators during myocardial necrosis:

(Source: Tietz Clinical Chemistry, 4th Edition)

Early release, long persistence and undetectable in patient without cardiac disease has made troponin a choice of test in MI diagnosis

(Source: Tietz Clinical Chemistry, 4th Edition)

This indicates high clinical sensitivity of cardiac biomarkers after 2-8 hours of clinical event

Several markers should no longer be used to evaluate cardiac disease like AST, total CK activity, LDH, LD isoenzymes with exception of hydroxyl butyrate dehydrogenase (HBD), which is used to estimate infarct size. HBD has sequence homology to H subunit of LDH, so can be considered as LD1.

In majority of patients’ blood should be obtained for testing at hospital admission (0 hours), at 6 to 9 hours, and again at 12 to 24 hours if the earlier specimens are normal and clinical event of suspicion is high. For patient in need of early diagnosis a rapidly appearing biomarker like myoglobin has been suggested to be added to serial cardiac troponin monitoring.

Cardiac biomarkers, measures of myocardial necrosis, were prioritized for use as follows:

 

Cardiac troponin > CK-MB mass > CK-MB activity > CK. An adequate biomarker should show a rising or falling pattern (at least one sample) when tested 6 hours apart (E.g. one at 0 hour and another at 6 hour) in the setting of clinical ischemia and absence of non cardiac cause of biomarker elevation. 

A positive biomarker was defined as exceeding the 99^th percentile or the lowest concentration at which a 10% CV can be demonstrated.

Other Cardiac biomarkers (CRP, BNP, OxLDL, Lp(a) etc)

C-REACTIVE PROTEIN:

It is an acute phase reactant produced during inflammation. Even a low concentration (so called hsCRP, assay detecting CRP <0.3 mg/L) can be a marker of atherosclerotic process which involves an inflammatory process. TNF, IL-1 which are produced during early inflammation stimulate IL-6 that then cause elaboration of CRP from liver.

For primary prevention, hsCRP values >3 mg/L are considered high risk. Value <1 mg/L is low risk; 1-3 mg/L is intermediate risk.

At present, it is recommended to take two measurements spaced 2 weeks apart, average them, and use the value to monitor risk for atherosclerosis inflammation. If the hs-CRP value is greater than 10 mg/L, the sample is invalid. High values are due to the presence of active infection or another inflammatory stimulus that has elevated the CRP.

The measurement of hs-CRP includes binding of hs-CRP with anti-CRP antibody and the light scatter by the precipitate is quantified.

BRAIN NATRIURETIC PEPTIDE: Marker of CHF

Brain (or B-type) natriuretic peptide (BNP) is a hormone stored mainly in the myocardium of cardiac left ventricles. Its level is increased in hypervolemic states like congestive heart failure and hypertension. So it is proposed to be a marker of CHF and edema due to heart failure.

Natriuretic peptide regulated fluid volume, blood pressure, and electrolyte balance. The main source of circulating BNP is the heart left ventricle, although isolated from porcine brain. BNP and ANP are released in response to atrial and/or ventricular stretch from volume overload e.g. during CHF.  NPs increases cardiac output by decreasing systemic and pulmonary vascular resistance, they reduce renin and aldosterone production by increasing renal blood flow, GFR and urine output. BNP (T_1/2 is 20-40 min) is formed from proBNP which produces NT-proBNP ((T_1/2 is 1-2 hr) which is a leader sequence and c-terminal BNP (physiologically active). BNP is degraded by neutral endopeptidase, by receptor mediated clearance, and bit by kidney.

Monoclonal antibody based ELISA test available although not standardized. POCT testing is approved by FDA.

Oxidized LDL:

It is a modified LDL and can enter cell via scavenger receptor especially in macrophage to form foam cells. It is formed in arterial wall where it is sequestered by proteoglycans and other extracellular matrix and protected from plasma antioxidants. This process is a free-radical driven lipi peroxidation chain reaction initiated by free radical attacking and double bond association with PUFA, leading to generation of MDA and 4-hydroxynonenal which then bind to apo B-100, giving it increased negative charge and rendering it unrecognizable by native LDL receptors.

Oxidized LDL (oxLDL) can be taken up by macrophage to form foam cells, it can be chemoattractant for circulating monocytes into macrophages, inhibition of motility of resident macrophages.  It is also cytotoxic and immunogenic since circulating anti-oxLDL antibodies were detected in serum.

ELISA method for determination of oxLDL is currently available. However, at present time the clinical relevance of oxLDL has not been established and so routine measurement is not recommended.

Lipoprotein (a):

Lp(a) is structurally related to LDL, but  has carbohydrate rich protein apo(a) covalently bound to apoB-100 through disulfide linkage. Apo(a) is homologous to plasminogen and  is serine protease. Lp(a) competitively inhibit plasminogen activity inhibiting clot lysis

ELISA based techniques using primary capture polyclonal antibodies against apo(a) and secondary detection monoclonal antibody against apoB-100. Then substrate added for color formation which is measured spectrophotometrically

>30 mg/dl – increased risk of CHD.

Other enzymatic cardiac markers (Myoglobin and LDH)

MYOGLOBIN:

It is a oxygen binding and storing protein. Its low molecular weight and cytoplasmic location accounts for its early release following muscle (heart or skeletal) injury. It cannot be used in differential diagnosis of muscle injury or cardiac injury.

Measured in serum by RIA, latex agglutination, and immunoassay based on monoclonal antibodies

LACTATE DEHYDROGENASE (EC 1.1.1.27) ISOENZYMES:

It is a cytoplasmic enzyme. This enzyme has mol weight of 134,000 and has four peptides chain of two types: M for muscle (or A) and H for heart (or B) each under separate genetic control as like M and B of CK. LD is inhibited by both pyruvate and lactate in excess although the effect of pyruvate is greater. Oxidation of –SH in enzyme and its inactivation is prevented by cysteine or glutathione.

Highest activities are found in skeletal muscle, liver, heart, kidney, and red blood cells. 

Isoenzymes                 % of LDH activity (Source)

q  LDH-1 (H₄)                 Mostly heart 60%, RBC 40%, kidney 28%

q  LDH-2 (H₃M)              Mostly kidney, myocardium, RBC, 30-34%

q  LDH-3 (H₂M₂)            Mostly spleen, lung, kidney, RBC

q  LDH-4 (HM₃)              Mostly Spleen, Lung, RBC, Kidney

 q  LDH-5 (M₄)                   Mostly liver 94%, skeletal muscle 76% 

A different sixth LD isoenzyme LD-X (also called LD_c) composed of four X (or C) subunits is present in post pubertal human testes. A seventh LD, called LD-6 has been identified in the sera of severely ill patients.

LDH catalyzes the reversible conversion of pyruvate to lactate. 

(Source: Tietz Clinical Chemistry, 4th Edition)

Analytical Measurement:

    

Enzymatic measurement: 

(Source: Tietz Clinical Chemistry, 4th Edition)

 

 

The decrease in absorbance at 340 nm followed. At pH 8.8 to 9.8 the reversible reaction is favored.

Reference range : 125-220 IU/L 

Isoenzyme analysis –

It can be done by electrophoresis or immunoinhibition assay where antibodies inhibit other isoenzymes except LD1. Formation of NBT formazan is the detection technique after electrophoretic separation. 

(Source: Harper's Illustrated biochemistry, 28th Edition)

CREATINE KINASE ISOENZYME AND ISOFORMS

Creatine kinase is a dimeric enzyme that will catalyze the reversible reaction of phosphorylation of creatine by ATP. 

(Source: Tietz Clinical Chemistry, 4th Edition)

The enzyme (CK) in serum is relatively unstable, activity being lost due to oxidation of active site sulfhydryl group of active site. This inactivation can be inhibited by incubating the enzyme with –SH compounds like N-acetylcysteine, dithiothreitol (Cleland reagent), and glutathione.

CK is dimer with 2 subunits (B and M) each with mol. Wt. of 40,000 KDa and it has 3 isoenzymes, CK-1, 2, 3. These are numbered according to their electrophoretic mobility with anodal form receiving the lowest number according to criteria of Commission on Biochemical Nomenclature.

CK exists as cytosolic isoenzyme (CK-1, 2, 3) and mitochondrial isoenzyme (CK-Mt). 

Isoenzymes                 % of CK activity (Source)

q  CK-1 (CK-BB)   -           >90% (brain, intestine).

q  CK-2 (CK-MB) –           1% (Skeletal muscle) 25-30% (Myocardial muscle)

q  CK-3 (CK-MM) –          98% (Skeletal muscle), 77% (myocardium)

q    CK-MM – 3 isoforms

 q  CK-MB – 4 isoforms

CKMM and CKMB isoforms are formed by post-translational modification (cleavage of C-terminal lysine by carboxypeptidase)

Analytical Measurement:

Isoenzymes of CK are measured by using monoclonal anti-CK2 antibodies and rely in immunoinhibition method which will inhibit the M subunit of CK-MB and measures the B subunits. Isoforms measurements are also done by advanced techniques. ELISA methods are also used to measure CK-MB using solid phase anti-MB antibody and using anti-B antibody conjugated with enzyme as detection antibody.

Total CK activity can be measured by enzymatic kinetic method: Also after inhibition of CK-M the CK-B is also assayed by the same process. 

(Source: Tietz Textbook of Clinical chemistry, 4th Edition)

 

 

There is increase in absorbance at 340 nm followed every minutes for 3 minutes and CK-MB activity obtained by multiplying the CK-B activity by 2. N-acetyl-L-cysteine is added as an activator to maintain a supply of reduced sulfhydryl groups necessary for the complete activation of CK. Adenylyl kinase present in serum can produce ATP from ADP producing positive interference so, this enzyme is inhibited by adding diadenosine pentaphosphate or by running sample blank using AK instead of CK.

The reference range at 37°C for total CK is 46-174 U/L for adult men and 96-140 U/L for adult women. The reference range for CK-MB activity is <6% of total CK and CK-MB mass is 0-5 ng/L.

Addition of EDTA will also stabilize the enzyme by chelating with calcium.

CK isoenzymes can also be quantified by electrophoresis: 

CK-MB activity assay have been replaced by CK-MB mass assay. This assay can detect an increased amount of CK-MB about 1 hour earlier than activity based assays. These assay based on sandwich technique where solid phase capture antibody directed to M subunit and detection antibody directed to B subunit. This detects only CK-MB. In addition, calculation of a relative index (CK-MB mass assay/total CK x 100) may be used as an indicator of MI. A relative index >3% is indicative of AMI.

Mechanism of cardiac injury:

Mechanism of cardiac injury:

(Source: Harper's Biochemistry 28th Edition)

CARDIAC BIOMARKERS: Characteristics and Types

CARDIAC BIOMARKERS:

The characteristic features of cardiac markers are:

a.      Should be tissue specific and rapidly release to circulation after tissue injury.

b.      Should be sensitive enough to distinguish minor damage in tissue.

c.     There must availability of rapid, accurate and standardized analytical method for measurement.

d.      Should persist in circulation to provide late diagnosis.

e.      Should be cheap and easily available

f.        Should be absent or not detectable in patients without myocardial damage.

  TYPES OF CARDIAC BIOMARKERS: 

Enzymatic	Non enzymatic
CK (CK-MB) – marker of injury	Troponins (T, I) – Injury marker
LDH (LDH1) – marker of injury	Myoglobin – Injury marker
AST– marker of injury	BNP (NT-proBNP) – marker of CHF
Myeloperoxidase – inflammatory marker	Ischemia modified albumin – marker of ischaemia
Matrix metalloproteinase – degrades collagen and ECM.	Cytokines (TNF, IL-1, 68, 12, 18) – inflammatory markers
	Unbound FFA – markers of ischaemia
	CRP – inflammation marker
	Oxidized LDL – atherosclerotic marker, inflammatory marker
	Nourin – released by stressed monocytes and inflammatory marker

CARDIAC TROPONIN I AND T:

These are the regulatory contractile proteins. These are the complex of 3 protein subunits, troponin C (the calcium-binding component), troponin I (the inhibitory component that inhibit myosin ATPase) and troponin T (the tropomyosin binding component). Although present in muscle, unique isoforms of troponin are present in heart called cTnT and cTnI. cTnI has additional 31 amino acids as compared to skeletal muscle making it heart specific. Similarly cardiac specific cTnT has additional 11 amino acids as compared to skeletal muscle. Different forms of troponins I exists like TIC, IC binary complex, and free I. Different modified form of troponins are also available, phosphorylated, oxidized, reduced, etc. 3-6% is cytoplasmic and remaining is present in myofibrils.

Analytical Measurement

Monoclonal antibody based ELISA test to measure cTnI and cTnT are approved by FDA which do not cross react with skeletal muscle troponins. Increase in troponins above 99^th percentile should be followed up with serial samples over 6 to 12 hour period after presentation.

The Reaction Principle

cTnT (cTnI) reacts with the monoclonal cTnT (cTnI) antibody to form a complex. This complex is linked to a dye, enzyme, or chemiluminescent reagent that is linked to a second antibody to allow for quantitation of the cTnT (cTnI) present in the patient’s sample. Chemiluminesence is the most sensitive test at this time for cTnT or cTnI

•      Reference range <0.010 µg/L

•      Reference range <0.050 µg/L

Lateral flow method: 

The test principle employs mouse monoclonal anti-cTnI antibodies, dye conjugated, anti-cTnI antibodies (goat) and polyclonal anti-mouse IgG antibodies (goat) in control line. As sample migrates through the absorbent pad human TnI reacts with anti-cTnI antibodies to form a sandwich between capture and detection antibodies this binds to the test line and produces a red violet test line. Excess conjugate reacts in the control line with the anti-mouse IgG forms a second violet line which is a control line. The test cannot detect less than 0.5 ng/ml of cTnI.

Here troponin in the sample is sandwiched between gold coated antibody and enzyme linked antibody. This complex will migrate and produce colored band in test area. Control area will also produce band and serves as quality check.