nal von minden NADAL Troponin I Test Flow Chromatographic Immunoassay Instruction Manual

nal von minden NADAL Troponin I Test Flow Chromatographic Immunoassay

Intended Use

The NADAL® Troponin I Test is a lateral flow chromatographic immunoassay for the qualitative detection of cardiac troponin I in human whole blood, serum or plasma samples. The test provides a preliminary test result as an aid in the diagnosis of myocardial infarction (MI). The NADAL® Troponin I Test is designed for professional use only.
The preliminary result obtained with the NADAL® Troponin I Test should not be used for a diagnosis of exclusion of MI.

Introduction and Clinical Significance

Cardiac troponin I (cTnl) is a protein found in the cardiac muscle with a molecular weight of 22.5 kDa. Troponin I is a part of a troponin complex which also includes troponin T and troponin C. Along with tropomyosin, this protein complex forms the main component that regulates the calcium-sensitive ATPase activity of actomyosin in striated skeletal and cardiac muscles. Following a cardiac injury, cardiac troponin I is released into the blood 4-6 hours after the onset of pain. The release pattern of cTnl is similar to CK-MB, but whilst the CK-MB level returns to normal after 72 hours, troponin I remains elevated for 6-10 days, thus providing a longer time window for the detection of cardiac injury. The high specificity of cTnl measurements in the detection of myocardial damage has been demonstrated during surgical procedures, after marathon runs or in cases of a blunt chest trauma, amongst others. The release of cTnl has also been documented in heart conditions other than acute myocardial infarction (AMI), such as unstable angina, congestive heart failure and ischemic damage caused by coronary artery bypass surgery. Because of its high sensitivity and specificity for myocardial tissue, troponin I has recently become one of the most preferred biomarkers for myocardial infarction.

Test Principle

The NADAL® Troponin I Test is a lateral flow chromatographic immunoassay for the qualitative detection of cardiac Troponin I in human whole blood, serum or plasma samples.

The test enables the detection of cTnl through the visual interpretation of colour development on the internal test strip.
The detection of troponin I occurs with the aid of two specific anti-cTnI antibodies, of which one is colour-coded and the other mediates the binding to a capture reagent immobilised in the test region. The mixture migrates along the membrane by capillary action. If a sufficient amount of cTnl is present in the sample, a coloured line forms in the test line region (T) of the membrane. The presence of this coloured line indicates a positive result, whereas its absence indicates a negative result. The formation of a coloured line in the control line region (C) serves as a procedural control, indicating that the proper volume of sample has been added and membrane wicking has occurred.

Reagents and Materials Supplied

• 5/10/20 NADAL® Troponin I test cassettes (incl. disposable pipettes)
• 5/10/20 heparinised capillary tubes
• 1 buffer
• 1 package insert

Additional Materials Required

• Sample collection container
• Centrifuge (for serum or plasma samples only)
• Alcohol pad
• Lancets (for fingerstick whole blood samples only)
• Bulbs for capillary tubes (for fingerstick whole blood samples only)
• Timer

Storage & Stability

NADAL® Troponin I test kits should be stored at 2-30°C and used by the expiry date indicated on the packaging. Test cassettes should remain in sealed pouches until use. Do not freeze test kits. Do not use tests beyond the expiry date. Care should be taken to protect components of the test kit from contamination. Do not use the test if there is evidence of microbial contamination or precipitation. Biological contamination of dispensing equipment, containers or reagents can lead to false results.

Warnings and Precautions

• For professional in-vitro diagnostic use only.
• Carefully read through the test procedure prior to testing.
• Do not use the test beyond the expiry date indicated on the package.
• Do not use the test if the foil pouch is damaged.
• Do not reuse tests.
• Do not add samples to the reaction area (result area).
• In order to avoid contamination, do not touch the reaction area (result area).
• Avoid cross-contamination of samples by using a new sample collection container for each sample obtained.
• Do not substitute or mix components from different test kits.
• Do not eat, drink or smoke in the area where samples and test kits are handled.
• Wear protective clothing such as laboratory coats, disposable gloves and eye protection when samples are being assayed.
• Handle all samples as if they contain infectious agents. Observe established precautions for microbiological risks throughout all procedures and standard guidelines for the appropriate disposal of samples.
• The test kit contains products of animal origin. Certified knowledge of the origin and/or sanitary state of the animals does not completely guarantee the absence of transmissible pathogenic agents. It is therefore recommended that these products be treated as potentially infectious, and handled in accordance with usual safety precautions (e.g., do not ingest or inhale).
• Humidity and temperature can adversely affect test results.
• Used testing materials should be discarded according to local regulations.

Specimen Collection and Preparation

The NADAL® Troponin I Test can be performed using whole blood (from venipuncture or fingerstick), serum or plasma.
To collect fingerstick whole blood samples:

• Wash the patient’s hand with soap and warm water or clean it with an alcohol pad. Allow it to dry. • Massage the hand, without touching the puncture site, by rubbing along the hand towards the fingertip of the middle or ring finger.
  • Puncture the skin with a sterile lancet. Wipe away the first drop of blood.
  • Gently rub the hand from the wrist to the palm, and then to the finger to form a rounded drop of blood over the puncture site.

Fingerstick whole blood should be tested immediately.

Venipuncture whole blood samples
Containers containing anticoagulants, such as EDTA, citrate or heparin should be used for the preparation of venous whole blood or plasma samples.
Testing should be performed immediately after sample collection. Do not leave samples at room temperature for prolonged periods of time.
If the test is to be run within 2 days of sample collection, whole blood collected by venipuncture should be stored at 2-8°C.
Do not freeze whole blood samples.

Serum and plasma samples
Separate serum or plasma from blood as soon as possible to avoid haemolysis. Use only clear, non-haemolysed samples.
Testing should be performed immediately after sample collection. Do not leave samples at room temperature for prolonged periods of time. Serum and plasma samples can be stored at 2-8°C for up to 3 days. For long-term storage, samples should be kept at -20°C.
Bring samples to room temperature prior to testing. Frozen samples should be completely thawed and mixed well prior to testing. Samples should not be frozen and thawed repeatedly.
If samples are to be shipped, they should be packed in compliance with all applicable regulations for the transportation of etiologic agents.
Icteric, lipemic, haemolysed, heat-treated and contaminated samples may lead to inaccurate test results.

9. Test Procedure

Bring tests, samples, buffer and/or controls to room temperature (15-30°C) prior to testing.

1. Remove the test cassette from the foil pouch and use it as soon as possible. The best results will be obtained if the test is performed immediately after opening the foil pouch. Label the test cassette with the patient or control identification.

2. Place the test cassette on a clean and level surface.

3. For serum or plasma samples:
   Holding a pipette vertically, add 2 drops (approximately 50 μL) of a serum/ plasma sample to the sample well (S) of the test cassette.

   • For venipuncture whole blood samples:
     Holding a pipette vertically, add 3 drops (approximately 75 μL) of a whole blood sample to the sample well (S) of the test cassette, then add 1 drop of buffer to the sample well (S).

   • For fingerstick whole blood samples:

   • To use a capillary tube:
     Apply the end of the capillary tube to the blood until it is filled completely (approximately 100 μL). Avoid air bubbles forming. Dispense the entire whole blood sample into the sample well (S) of the test cassette, then add 1 drop of buffer to the sample well (S).

   • To use hanging drops:
     Position the patient’s finger so that a drop of blood is exactly above the sample well (S) of the test cassette. Allow 3 hanging drops of fingerstick whole blood to fall into the centre of the sample well (S) of the test cassette, then add 1 drop of buffer to the sample well (S).

4. Start the timer.

5. Wait for the coloured line(s) to appear. Read the test result after 10 minutes. Confirm negative test results after exactly 20 minutes. Do not interpret the result after more than 20 minutes.

Result Interpretation

Positive
Two coloured lines appear in the result area. One line appears in the control line region (C) and the other line appears in the test line region (T).

Note: The colour intensity in the test line region (T) may vary depending on the analyte concentration in the sample. Therefore, any shade of colour in the test line region should be considered positive, even if it is weak. Note that this is a qualitative test only and it cannot determine the analyte concentration in the sample.

Negative
Only one coloured line appears in the control line region (C). No coloured line appears in the test line region (T). Confirm negative test results after exactly 20 minutes.

Invalid
The control line (C) fails to appear. Results from any test which has not produced a control line at the specified reading time must be discarded. Please review the procedure and repeat the test with a new test cassette. If the problem persists, discontinue using the test kit immediately and contact your distributor.

Note: Insufficient sample volume, high sample viscosity, expired tests or incorrect operating procedure are the most likely reasons that the control line fails to appear.

Quality Control

An internal procedural control is included in the test cassette:
A coloured line appearing in the control line region (C) is considered an internal procedural control. It confirms sufficient sample volume, adequate membrane wicking and correct procedural technique.
Good laboratory practice (GLP) recommends the use of external control materials to ensure proper test kit performance.

Limitations

• The NADAL® Troponin I Test is for professional in-vitro diagnostic use only. This test should be used for the qualitative detection of cTnl in whole blood, serum or plasma samples only. Neither the quantitative value nor the rate of increase in cTnl concentration can be determined, using this qualitative test.
• The NADAL® Troponin I Test only indicates the presence of cTnl in the sample and should not be used as the sole criterion for the diagnosis of myocardial infarction.
• If the test result is negative and clinical symptoms persist, additional tests using other clinical methods should always be performed.
• Whole blood, serum or plasma specimens from patients who have been ingesting high levels of biotin in dietary supplements (multivitamins, biotin supplements them-selves, or other supplements for hair, skin and nail growth) may cause clinically significant inaccurate (false negative) test results. If the test result does not match the clinical presentation of a patient, biotin interference may be considered as a possible source of error.
• A negative result does not at any time exclude the presence cTnI in blood, as its concentration may be below the test’s detection limit (0.5 ng cTnI/mL). It should be noted that cTnI concentration usually increases a few hours after the onset of pain. If tested too early, the cTnI concentration might be too low to be reliably determined by the test. In case of slight damage to the cardiac muscle, only low cTnI concentrations may be released. For this reason, a negative result does not preclude at any time the possibility of myocardial infarction.
• Due to the heterogeneity of commercially available standard materials, the sensitivity of the test may vary with different standards/measurement methods. Concentrations below 0.5 ng/mL are no longer reliably detected by the test.
• As with all diagnostic tests, a definitive clinical diagnosis should only be made by a physician following the evaluation of all clinical and laboratory findings.
• Unusually high titers of heterophile antibodies or rheumatoid factors (RF) may affect results. Therefore, even if test results are positive, the results of further clinical examinations, therefore, should always be used to make a diagnosis.
• In rare cases, auto-antibodies in the blood of the patient prevent the antigen-antibody reaction in the test by blocking the binding sites. This may lead to false-negative test results. It should be noted that these problems may generally occur in all test methods in which protein detection occurs through an antibody reaction.
• Whole blood samples with a very high viscosity may lead to invalid test results. Using a new test cassette, repeat the test with a serum or plasma sample from the same patient. Whole blood samples which have been stored for more than 2 days may also cause migration problems.

Performance Characteristics

Analytical sensitivity
The detection limit of the test is 0.5 ng cTnI/mL sample.
The detection limit is checked regularly in the quality control procedure with commercially-available “Human Cardiac Troponin I-T-C Complex” from the company HyTest.

Diagnostic sensitivity and specificity
A correlation study was performed on 652 negative plasma samples (EIA confirmed) and 253 positive plasma samples (EIA confirmed). The results are presented in the following table:

| EIA
---|---

NADAL®

Troponin I Test

| +                         ̶                   Total
+

̶ Total

| 251| 4| 255
2| 648| 650
253| 652| 905

Relative sensitivity: 99.2 % (97.2%-99.9%) Relative specificity: 99.4% (98.4%-99.8%) Overall agreement: 99.3% (98.6%-99.8%) 95% Confidence interval

Inter- and intra-lot variability
The inter- and intra-lot variability of the test was determined by testing 3 independent test lots with the cTnI concentrations of 0 ng/mL, 0.5 ng/mL, 2 ng/mL, 5 ng/mL and 10 ng/mL in a 10-fold determination. The samples were correctly identified 99% of the time.

Cross-reactivity
Troponin I negative samples were tested with 10,000 ng/mL skeletal troponin I, 2,000 ng/mL of troponin T and 20,000 ng/mL cardiac tropomyosin. No cross- reactivity was observed. This demonstrates that the NADAL® Troponin I Test is highly specific for cTnI.
Samples with high concentrations of RF or heterophile antibodies may cause false positive results. 93% correct negative results were obtained with 100 RF positive samples. 96% correct negative results were obtained with 50 heterophile antibody positive samples.

Interfering substances
Specimens from patients who have been ingesting high levels of biotin in dietary supplements may exhibit false negative test results.
Drugs and substances listed below do not affect the generation of correct positive or negative test results obtained with the NADAL® Troponin I Test at the concentration of 50 μg/mL (unless otherwise indicated).

Acetaminophen| Captopril| Flunarizine hydrochloride| Oxazepam
---|---|---|---
Acetylsalicylic acid| Chloramphenicol| Furosemide| Pentoxifylline
Anisodamine| Chlordiazepoxide| Hydrochlorothiazide| Phenobarbital
Ascorbic acid| Cilazapril| Isosorbide Mononitrate| Quinine
Atenolol| Diclofenac| Labetalol| Ramipril
Atorvastatin Calcium| Digoxin| Metoprolol Tartrate| D,L-Tyrosine
C a f f e i n e| Erythromycin| Moracizine hydrochloride| Trimethoprim
Bisoprolol Fumarate| Felodipine| Nifedipine| Verapamil
Human albumin (110 mg/mL)| Bilirubin (6 mg/mL)| Haemoglobin (10 mg/mL)| Cholesterol (5 mg/mL)
Triglycerides (15 mg/mL)| | |

References

1. Adams, et al. Biochemical markers of myocardial injury, Immunoassay Circulation 88: 750-763 (1993).
2. Mehegan JP, Tobacman LS, Cooperative interaction between troponin molecules bound to the cardiac thin filament. J. Biol. Chem. 266:966 (1991)
3. Adams, et al. Diagnosis of Perioperative myocardial infarction with measurements of cardiac troponin I. N.Eng.J. Med 330:670 (1994).
4. Hossein-Nia M, et al. Cardiac troponin / release in heart transplantation. Ann.Thorac. Surg. 61 : 227 (1996).
5. Joint European Society of Cardiology/American College of Cardiology: J.Am. Coll. Cardio., 36(3), Myocardial Infarction Redefined, 2000.

References

• nal von minden GmbH - Spezialist für hochwertige in-vitro Diagnostik
• nal von minden GmbH - Spezialist für hochwertige in-vitro Diagnostik

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