Differences in Cardiac Troponin T Composition in Myocardial Infarction and End-Stage Renal Disease Patients: A Blood Tube Effect?
Author(s): Vroemen WHM, Denessen EJS, van Doorn WPTM, Pelzer KEJM, Hackeng TM, Litjens EJR, Henskens YMC, van der Sande FM, Wodzig WKWH, Kooman JP, Bekers O, de Boer D, Mingels AMA
Publication: J Appl Lab Med, 2024, Vol. , Page
PubMed ID: 38816928 PubMed Review Paper? No
Purpose of Paper
This paper compared levels and relative proteoform abundance of cardiac troponin T (cTnT) in serum, lithium heparin plasma, and EDTA plasma in leftover specimens as well as specimens from patients with a myocardial infarction or end-stage renal disease. Additionally, effects of serum/plasma storage at 37°C on proteoform abundance was examined using blood from one volunteer that was spiked with 40 kDa cTnT.
Conclusion of Paper
Overall, cTnT levels did not differ significantly between case-matched serum and lithium heparin or EDTA plasma nor did they differ between lithium heparin and EDTA plasma, regardless of patient diagnosis or in the storage duration (at 37°C for up to 48 h) of spiked specimens. When 40 kDa cTnT was spiked into blood specimens from a healthy volunteer, the relative abundance of cTnT fragments differed among matched serum, lithium heparin, and EDTA plasma specimens based on the duration of plasma/serum storage (37°C). In the spiked serum specimen, the 40kDa fragment degraded immediately (0% detectable in immediately frozen serum) into a 29 kDa fragment, which was further degraded during storage of serum at 37°C (100% at 0 h; 90-91% at 2, 4 and 8 h; 56% at 24 h; and 0% at 48h) into 15-18 kDa fragments (0% at 0 h; 9-10% at 2, 4, and 8 h; 44% at 24 h; and 100% at 48h). In lithium heparin plasma, 97-98% of cTnT was 40 kDa at the 2-24 h timepoints and 90% was 40 kDa at the 48 h timepoint, with the remaining 2-10% being the 29 kDa fragment. In EDTA plasma, the 40kDa fragment degraded faster than in the lithium heparin plasma but much slower than in serum (96% at 0 h, 87% at 2 h, 68% at 4 h, 21% at 8 h, 1 % at 24 h and 0% at 48 h) which first corresponded with an increase in the 29 kDa fragment (4% at 0 h, 10% at 2 h, 27% at 4 h, 43% at 8 h, 24% at 24 h and 5% at 48 h) and a subsequent increase in the 15-18 kDa fragments (0% at 0 h, 3% at 2 h, 5% at 4 h, 35% at 8 h, 75% at 24 h and 95% at 48 h). The cTnT composition differed among specimens from patients with myocardial infarction (generally, the 40 kDa fragment was prevalent in both plasma types and the 29 kDa fragment was prevalent in serum), but 15-18 kDa fragments were predominant in all specimens from patients with end-stage renal disease, regardless of tube type.
Studies
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Study Purpose
This study compared levels and relative proteoform abundance of cardiac troponin T (cTnT) in serum, lithium heparin plasma, and EDTA plasma in leftover specimens as well as specimens from patients with a myocardial infarction or end-stage renal disease. Additionally, effects of serum/plasma storage at 37°C on proteoform abundance was examined using blood from one volunteer that was spiked with 40 kDa cTnT. To investigate the potential effects of collection tube type on cTnT levels, cTnT was quantified using a Cobas 6000 automated analyzer in residual lithium heparin (31 specimens) and/or EDTA plasma (73 specimens) that were collected from the same venipuncture as serum (82 specimens) from patients undergoing cTnT assessment. To study the stability of cTnT, 40 kDa cTnT was spiked into serum, lithium heparin and K2 EDTA tubes containing the blood of a healthy volunteer. The spiked specimens were kept at room temperature for 30 min before separation of serum/plasma by centrifugation at 18885 g for 10 min. The matched serum/plasma aliquots were then frozen at -80°C either immediately (control) or after 0, 2, 4 8, 24 or 48 h at 37°C, and the cTnT proteoform composition was analyzed by gel-filtration chromatography. To investigate the effect of tube type on patient diagnosis, blood was collected from 11 patients with myocardial infarction (initial cTnT concentration >400 ng/L) and 10 patients with end-stage renal disease (cTnT > 50 ng/L) into serum, lithium heparin and EDTA tubes and cTnT was quantified by immunoprecipitation followed by Western blotting.
Summary of Findings:
cTnT levels were not significantly different between case-matched serum and lithium heparin or EDTA plasma nor were they different between lithium heparin and EDTA plasma. Further, cTnT levels were very strongly correlated between serum and lithium heparin (ρ=0.99) or EDTA (ρ=1.00) plasma and between lithium heparin and EDTA plasma (ρ=1.00). When 40 kDa cTnT was spiked into a blood specimen of a healthy volunteer, the relative abundance of cTnT fragments differed among matched serum, lithium heparin, and EDTA plasma specimens based on the duration of specimen storage (37°C), but the overall concentration of cTnT was not affected by storage of serum, lithium heparin plasma or EDTA plasma at 37°C for 48 h. In the spiked serum specimen, the 40kDa fragment degraded immediately (0% detectable in immediately frozen serum) into a 29 kDa fragment, which then further degraded during storage of serum at 37°C (100% at 0 h; 90-91% at 2, 4 and 8 h; 56% at 24 h; and 0% at 48h) into 15-18 kDa fragments (0% at 0 h; 9-10% at 2, 4, and 8 h; 44% at 24 h; and 100% at 48h). In contrast, the 40 kDa cTnT degraded more slowly in lithium heparin and EDTA plasma. In lithium heparin plasma, 97-98% of cTnT was 40 kDa at the 2-24 h timepoints and 90% was 40 kDa at the 48 h timepoint with the remaining 2-10% being the 29 kDa fragment. In EDTA plasma, the 40 kDa fragment degraded faster than in the lithium heparin plasma but much slower than in serum (96% at 0 h, 87% at 2 h, 68% at 4 h, 21% at 8 h, 1 % at 24 h and 0% at 48 h) into the 29 kDa fragment (4% at 0 h, 10% at 2 h, 27% at 4 h, 43% at 8 h, 24% at 24 h and 5% at 48 h) and then into 15-18 kDa fragments (0% at 0 h, 3% at 2 h, 5% at 4 h, 35% at 8 h, 75% at 24 h and 95% at 48 h). No overall effect of tube type was found on cTnT levels in the blood of patients with myocardial infarction or end-stage renal disease; however, the cTnT composition displayed diagnosis-dependent effects, with the 40 kDa form predominant in both plasma types from myocardial infarct patients, the 29 kDa form being predominant in serum from myocardial infarct patients, but the 15-18 kDa fragments being predominant in all specimens from patients with end-stage renal disease, regardless of tube type.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Cardiovascular Disease
- Normal
- Other diagnoses
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Serum tube
Lithium heparin tube
K2EDTA tube
Biospecimen Acquisition Anticoagulant EDTA
Lithium heparin
None
Storage Storage duration 0 h
2 h
4 h
8 h
24 h
48 h
Preaquisition Diagnosis/ patient condition Myocardial infarction
End stage renal disease