Relevance of pre-analytical blood management on the emerging cardiovascular protein biomarkers TWEAK and HMGB1 and on miRNA serum and plasma profiling.
Author(s): Basso D, Padoan A, Laufer T, Aneloni V, Moz S, Schroers H, Pelloso M, Saiz A, Krapp M, Fogar P, Cornoldi P, Zambon CF, Rossi E, La Malfa M, Marotti A, Brefort T, Weis TM, Katus HA, Plebani M
Publication: Clin Biochem, 2017, Vol. 50, Page 186-193
PubMed ID: 27847340 PubMed Review Paper? No
Purpose of Paper
This paper compared levels of cardiovascular protein, MALDI-TOF MS spectra, and microRNA (miRNA) yield and profiles in EDTA, sodium citrate, lithium heparin, and acid citrate dextrose (ACD) plasma and serum subjected to precentrifugation refrigerated or room temperature storage and one or two centrifugation steps.
Conclusion of Paper
Serum had higher levels of IL-8 and TWEAK and lower levels of HMGB1 than plasma, regardless of anticoagulant type. Precentrifugation storage at room temperature significantly affected IL-8 levels, the MS spectra, and the miRNA profile of serum specimens. The number of centrifugation steps had little to no effect on the miRNA profile or levels of IL-8, TWEAK, and HMGB1 in serum or plasma (regardless of anticoagulant). Delayed centrifugation affected IL-8 levels in lithium heparin plasma, MS spectra and miRNA yield from EDTA plasma, and miRNA yield from ACD plasma but significance depended on storage temperature. The second centrifugation step eliminated the tRNA peaks but also reduced miRNA yield.
Studies
-
Study Purpose
This study compared levels of IL-8, TWEAK, and HMGB-1 in K2EDTA, sodium citrate, lithium heparin, and ACD plasma and serum subjected to precentrifugation refrigerated or room temperature storage and one or two centrifugation steps. Blood was collected from nine patients with hemochromatosis, six patients with polyglobulia, and one patient each with essential thrombocytosis and porphyria cutanea tarda using a 21-gauge needle into SSTMII advance (serum), K2EDTA, sodium citrate, lithium heparin, and acid-citrate-dextrose tubes. Half the tubes were stored at room temperature and half in a refrigerated box for 30 min, 3 h, 6 h (9 patients only), or 9 h (9 patients only) before centrifugation at 1200 x g for 10 min at room temperature or refrigerated. Half of the resultant serum or plasma was frozen at -80˚C while the other half was centrifuged again at 2500 x g for 15 min before freezing. Specimens were thawed immediately before quantification of TWEAK and HMGB1 by Elisa and IL-8 by an immunoassay.
Summary of Findings:
Serum levels of IL-8 were low when centrifuged twice within 30 min of blood collection, but IL-8 levels in plasma were undetectable, regardless of anticoagulant type. TWEAK levels were significantly higher in serum and lower in lithium heparin plasma than in other plasma types (P<0.0001 and P<0.05, respectively). In contrast, HMGB1 levels were significantly lower in serum than in plasma, regardless of anticoagulant (P<0.0001). IL-8 levels in serum and lithium heparin plasma increased with room temperature storage (P=0.023), but there was no effect on IL-8 levels in ACD, K2EDTA, or sodium citrate plasma nor was there an effect of refrigerated storage. TWEAK and HMGB1 were unaffected by precentrifugation storage temperature or duration. IL-8, TWEAK, and HMGB1 levels were unaffected by the number of serum or plasma centrifugation steps.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Other diagnoses
- Hemochromatosis
Platform:
Analyte Technology Platform Protein Immunoassay Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Lithium heparin
Potassium EDTA
Acid-citrate-dextrose
Sodium citrate
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Different number of centrifugation steps compared
Storage Storage temperature Room temperature
4˚C
Storage Storage duration 30 min
3 h
6 h
9 h
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
-
Study Purpose
This study compared the MALDI-TOF spectra of serum, EDTA plasma, and heparin plasma and investigated the effects of precentrifugation storage temperature and duration and number of centrifugation steps. Blood was collected from two patients using a 21-gauge needle into SSTMII advance (serum), K2EDTA, and lithium heparin tubes. The tubes were stored at room temperature for 30 min or 9 h before centrifugation at 1200 x g for 10 min at room temperature or refrigerated. Half of the resultant serum or plasma was frozen at -80˚C while the other half was centrifuged again at 2500 x g for 15 min before freezing. Specimens were thawed immediately before MALD-TOF analysis.
Summary of Findings:
The MS spectra differed among the three specimen types. In serum, two of the four features selected for analysis increased in intensity with precentrifugation storage at room temperature while the other two decreased, regardless of centrifugation protocol. Refrigerated storage of serum did not significantly affect peak intensity. In EDTA plasma, smaller changes in peak intensity were noted during room temperature storage, but effects of cold storage were limited to storage for more than 6 h. The authors report no effect of storage on the spectra of lithium heparin plasma.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Other diagnoses
- Hemochromatosis
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Protein MALDI-TOF MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Lithium heparin
Potassium EDTA
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Different number of centrifugation steps compared
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Storage Storage duration 30 min
3 h
6 h
9 h
Storage Storage temperature Room temperature
4˚C
-
Study Purpose
This study compared the miRNA yield and profile in serum and K2EDTA, sodium citrate, lithium heparin, and ACD plasma subjected to precentrifugation refrigerated or room temperature storage and one or two centrifugation steps. Blood was collected from three patients with hemochromatosis, polyglobulia, essential thrombocytosis, or porphyria cutanea tarda using a 21-gauge needle into SSTMII advance (serum), K2EDTA, sodium citrate, lithium heparin, and acid-citrate-dextrose tubes. Half the tubes were stored at room temperature and the other half in a refrigerated box for 30 min, 3 h, 6 h (9 patients only), or 9 h (9 patients only) before centrifugation at 1200 x g for 10 min at room temperature or refrigerated. Half of the resultant serum or plasma was frozen at -80˚C while the other half was centrifuged again at 2500 x g for 15 min before freezing. RNA was extracted using the miRNeasy Serum/Plasma Kit and quality and quantity were evaluated by bioanalyzer. miRNAs were quantified using custom microarrays.
Summary of Findings:
A second centrifugation eliminated the tRNA peaks (70-90 bp) for EDTA, citrate, and ACD plasma but also reduced miRNA yield (P=0.0319, P=0.0194, and P=0.0033). Pre-centrifugation storage significantly increased miRNA yield from EDTA (P=0.0208) and ACD plasma (P=0.026), but not citrate plasma. In contrast, there was no effect of delayed centrifugation or number of centrifugation steps for serum, but storage temperature affected the miRNA profile. Heparin plasma interfered with miRNA quality assessment so effects of storage and centrifugation were not investigated.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Hemochromatosis
- Other diagnoses
Platform:
Analyte Technology Platform RNA DNA microarray RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Lithium heparin
Potassium EDTA
Acid-citrate-dextrose
Sodium citrate
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Different number of centrifugation steps compared
Storage Storage temperature Room temperature
4˚C
Storage Storage duration 30 min
3 h
6 h
9 h
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum