Troponin is a protein found in skeletal and cardiac muscle fibers that regulates muscular contraction. Cardiac troponin I and T are specific to the heart and are used as diagnostic biomarkers to detect heart injury. Troponin levels in the blood rise when the heart muscle has been damaged, such as during a heart attack. High sensitivity troponin assays can detect very low levels of troponin in the blood and are used to rapidly diagnose heart attacks.
Opening Summary
The color of the tube used to collect blood samples for high sensitivity troponin I (hs-cTnI) assays can impact test results. Most hospitals and labs recommend using gold or gold/black top tubes containing a clot activator and gel separator for hs-cTnI testing. The gold top tubes help to completely separate the serum from the blood cells through the gel barrier. This provides the cleanest serum sample for analyzing the very low levels of troponin detected by high sensitivity assays. Other tube additives like heparin or EDTA in green or purple top tubes can interfere with the highly sensitive troponin antibody immunoassays. For accurate hs-cTnI results, specially designed gold top tubes are required.
Troponin Function and Structure
Troponin plays a vital role in striated muscle contraction. It is comprised of three regulatory proteins – troponin C, troponin I, and troponin T – that are bound to tropomyosin on muscle thin filaments. In response to calcium ion signals, conformational changes in troponin alter tropomyosin positioning, unblocking actin-myosin binding sites and initiating muscle contraction through ATP-dependent cross-bridge cycling. Troponin I inhibits actin-myosin interactions at low calcium levels, while troponin C contains calcium binding sites to sense intracellular calcium fluctuations that trigger contraction. Troponin T anchors the troponin complex to tropomyosin on the thin filament.
While all three troponin subunits are found in skeletal muscle, cardiac troponin I and T isoforms are unique to heart muscle. After injury to the myocardium, such as during a myocardial infarction, cardiomyocyte necrosis releases cTnI and cTnT into the circulation. Detection of elevated cardiac troponin in the blood provides a very specific and sensitive biomarker of myocardial damage.
High Sensitivity Troponin Assays
Standard troponin assays can detect levels above the 99th percentile value in healthy patients, which is approximately 0.04 ng/mL for cTnI. However, even minor elevations in troponin below this cut-off are indicative of myocardial stress and damage. High sensitivity troponin assays use advanced antibody technology to improve the lower limit of detection 10- to 100-fold compared to standard assays.
Levels of cTnI in healthy individuals are normally undetectable by hs-cTnI assays. Detectable concentrations above the assay-specific 99th percentile reference limit signals myocardial injury has occurred. This allows rapid diagnosis of myocardial infarction in patients with chest pain or other symptoms suggestive of acute coronary syndrome. High sensitivity troponin testing also aids in risk stratification and provides prognostic information for cardiovascular disease patients.
Sample Collection Tubes
Because hs-cTnI assays can detect troponin at very low picogram/mL or nanogram/mL concentrations, the blood collection tube used is critical. Sample contamination and troponin complex instability can adversely affect test results if improper tubes are used.
For hs-cTnI testing, most laboratories recommend using serum separator tubes with a gold top or speckled gold/black tops. These tubes contain clot activators, typically silica particles, to accelerate clot formation. They also contain a gel separator barrier that isolates the serum from the clotted blood cells after centrifugation. The gel forms a physical barrier to prevent fragments of red blood cells, platelets, or fibrin from contaminating the serum layer where hs-cTnI levels will be measured.
Recommended Tubes
| Tube Additive | Tube Color |
|---|---|
| Clot activator and gel separator | Gold top |
| Clot activator, silica particles, gel separator | Speckled gold/black top |
This provides the highest quality serum sample for sensitive troponin analysis. Other non-gel tube types may not completely separate serum from blood cells. Trace amounts of cellular components in the serum could interfere with the immunoassay antibody reactions used to measure troponin levels. The gel barrier offers a cleaner, cell-free serum for cTnI detection.
Avoiding Interfering Substances
Certain chemicals present in blood collection tubes can also disrupt hs-cTnI analysis if they sequester or denature troponin. Tubes with liquid lithium heparin or sodium heparin anticoagulants typically have green or light green tops. These heparin salts bind proteins like troponin, preventing them from interacting properly with detection antibodies used in the immunoassays. The negatively charged heparin molecules can additionally interfere with antibody antigen binding through electrostatic interactions. Using lithium or sodium heparin tubes is not recommended for hs-cTnI testing.
EDTAtubes with lavender tops that contain ethylenediaminetetraacetic acid salt as an anticoagulant can similarly interfere in the troponin assay. EDTA chelates metal ions like calcium and magnesium, which are essential cofactors for the function of many immunoassay antibodies and enzymes. The metal chelation can reduce troponin detection accuracy. Lavender top EDTA tubes should also be avoided for high sensitivity troponin analysis.
Tubes to Avoid for hs-cTnI
| Tube Additive | Tube Color |
|---|---|
| Liquid lithium heparin anticoagulant | Light green top |
| Sodium heparin | Green top |
| EDTA anticoagulant | Lavender top |
Plasma from these tubes may contain substances that interfere with antibody binding or troponin structure. This could reduce the accuracy of detecting very low troponin concentrations present in healthy patients using high sensitivity assays.
Serum Separator Tubes
The gold or gold/black speckled top tubes specifically designed for serum separation are the best option for collecting blood for hs-cTnI analysis. The gel effectively partitions the serum from cellular components of the blood after centrifugation. This removes sources of contamination like lipids, proteins, cytosolic contents, and particulate matter from damaged blood cells that could affect troponin assay results.
The exact composition of the separator gel does vary between tube manufacturers. But most contain a thixotropic gel made from a formulated silicone. This water-insoluble gel has a density between that of blood cells and serum. Upon centrifugation, it migrates to form a distinct barrier layer between the serum and cellular fractions. The gel may also be optimized to have minimal chemical reactivity with assay components to avoid any technique interference.
Overall, gold top serum separator tubes provide the cleanest serum specimen with the fewest interfering substances. This allows for the most accurate measurement of very low troponin levels using high sensitivity immunoassay methods. The gold standard tubes are specially designed to ensure optimum sample integrity for hs-cTnI testing.
Sample Handling
In addition to using proper tube types, attention must be paid to sample handling procedures. Once blood is collected in gold top tubes for hs-cTnI analysis, the tubes should be gently inverted 5-10 times to distribute clot activators. The tubes should then be transported vertically and centrifuged for 10-15 minutes at 1100-1300xg within 2 hours of collection. After centrifugation, the serum should be transferred to a plastic transport tube for analysis or refrigerated at 2-8°C up to 24 hours. If the serum cannot be analyzed within 24 hours, it should be stored frozen at -70°C and avoid freeze-thaw cycles. With careful handling using gold separator tubes, hemolyzed, lipemic, or icteric samples should not pose a problem for analysis.
Conclusion
In summary, gold or gold/black top serum separator tubes containing clot activators and a gel barrier are strongly recommended for collection of blood samples for high sensitivity troponin testing. The tubes are specially designed to provide cell-free serum with minimal interfering substances that could impact very low troponin detection by immunoassays. Green, purple, or other tube types may sequester or alter troponin in ways that reduce assay accuracy. With proper handling, the gold standard separator tubes yield a clean serum specimen optimal for precise hs-cTnI measurement to rapidly diagnose myocardial infarction.
