Tuesday, May 24, 2016

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.

The rabbit IgG-colloidal gold conjugate and unbound complex, if any, moves further to the reference region (R) that contains pre-calibrated anti rabbit IgG antibodies, corresponding to 1 ng/ml cTnl, immobilized on the membrane. The intensity of the pink purple colored band at the reference region (R) corresponds to a cTnI concentration of 1 ng/ml. The reference band would form even in a negative specimen. Semi-quantitative information about the concentration of cTnl can be deduced by comparing the intensity of the test band against the reference band. If the intensity of test band is less than the reference band, cardiac Troponin I (cTnI) concentration is equal to or above 0.1 ng/ml and less than 1 ng/ml. If the intensity of the test band is equal to or greater than reference band, cardiac Troponin I (cTnl) concentration is equal to or greater than 1 ng/ml. The unreacted conjugate along with unbound complex if any, move further on the membrane and are subsequently immobilized by the anti-rabbit antibodies coated on the membrane at the control region (C), forming a pink-purple colored band. This control band acts as a procedural control and serves to validate test results.

Specimens: Serum

Materials (All available in kit)

A. Individual pouches each containing­
  1. Test device: Membrane assembly predispensed with monoclonal anti-cTnI colloidal gold conjugate, rabbit IgG colloidal gold conjugate, monoclonal anti- cTnI antibody and anti-rabbit antiserum coated at the respective regions.
  2. Desiccant pouch.
  3. Sample dropper.
B. Sample Running buffer in a dropper bottle.

C. Package insert.      

PROCEDURE & INTERPRETATION OF RESULTS:

1. Bring the Core Troponin I- kit components to room temperature before testing.
2. Open the pouch by tearing along the notch.
3. Retrieve the device, sample dropper and desiccant. Check the color of the desiccant. It should be blue. If it has turned colorless or pink, discard the device and use another device.
4. Once opened the device must be used immediately.
5. Tighten the vial cap of the sample running buffer provided with the kit in clockwise direction to pierce the pierce the dropper bottle nozzle.
6. Label the device with specimen identity.
7. Place the testing device on a flat horizontal surface.
8. Holding the sample dropper vertically, carefully dispense four (4) drops of serum/plasma/whole blood into the sample port 'A'
9. Add four (4) drops of sample running buffer in buffer port 'B'. 10. At the end of 15 minutes read result as the diagram given in the kit protocol.

Discussion:

Discovered by Ebashi, Troponins are regulatory proteins in cardiac muscle that modulate the interaction between actin and myosin, during the calcium-mediated contraction of cardiac muscle. Three distinct tissue specific isoforms of Troponin I have been identified, two in skeletal muscle and one in cardiac muscle. The cardiac isoform of Troponin I (cTnl) has an additional sequence of 31 amino acids at the N terminal end that accounts for cardiac specificity, with a molecular weight of 22.5 kDa. This absolute specificity of Troponin I for cardiac tissue makes it an ideal biomarker for myocardial injury.

Clinical study results have demonstrated that elevated serum levels of cardiac Troponin I (cTnl) are detectable within 4 to 6 hours after the onset of chest pain, reach peak concentration in approximately 12 hours and remain elevated for 3-10 days following acute myocardial infarction. Thus cardiac Troponin I (cTnI) meets the entire criterion laid down by National Academy of Clinical Biochemistry (NACB) for an ideal cardiac biomarker in early identification and risk stratification of patients with chest pain suggestive of ischemia and identification of patients that present after infarction.

Immediately after a cardiac event, the damaged myocardial cells start releasing cardiac Troponin I (cTnl) in circulation and their level rises in a time specific manner. Since patients present at varying times-for testing following the onset of chest pain in a cardiac event, it is necessary to perform sequential testing for optimal diagnostic accuracy.


A protocol for measuring cardiac Troponin I (cTnl) levels requires testing at admission or 3 hours after onset of chest pain and at 6 and 9 hours. Modification may be necessary depending upon specific clinical situation. Hence sequential testing of cardiac Troponin I (cTnl), together with ECG results and patient history and symptoms are necessary for differential diagnosis between acute myocardial infarction and unstable angina pectoris. The positive and negative likelihood ratios correspond to the clinical concepts of ruling in and ruling out disease. Thus, a higher positive likelihood ratio means that a test result is better for ruling in disease when positive, and a lower negative likelihood ratio means that a test result is better for ruling out disease when negative. Examination of likelihood ratios reveals that levels of cardiac Troponin I (cTnl) are very useful at ruling out AMI when the value is negative at 10 or more hours from the onset of chest pain. However, a negative test value early in the course of episode of chest pain does very little to reduce the likelihood of AMI. A positive cardiac Troponin I (cTnl) value after 6 or more hours after the onset of chest pain appears to be very useful at ruling in AMI. Thus a negative cardiac Troponin I (cTnl) level identifies patient at low risk for adverse cardiac events.

(Source: Information from Kit and clinical chemistry textbook)
Related Posts Plugin for WordPress, Blogger...