Impact of clinical sample handling and processing on ultra-low level measurements of plasma cytokines.
Author(s): Cohen L, Keegan A, Melanson SEF, Walt DR
Publication: Clin Biochem, 2019, Vol. 65, Page 38-44
PubMed ID: 30633878 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of storage of blood at room temperature and 4˚C, delayed analysis of plasma, sample dropping, pneumatic tube system (PTS) transport, and freeze-thaw cycling of plasma on levels of interleukin (IL) -2, IL-6, and IL-10 and interferon (IFN) γ in plasma.
Conclusion of Paper
Pre-centrifugation storage of blood at room temperature resulted in significant declines in plasma levels of IL-6, IL-10, IFNγ, and IL-2, but levels were not significantly lower when blood was stored at 4˚C for 10 h compared to 2 h. Plasma levels of IL-6, IL-10, IFNγ, and IL-2 were unaffected by plasma storage at room temperature for 6 h, after one versus three3 freeze-thaw cycles, by dropping the blood specimen repeatedly, or by pneumatic tube transport.
Studies
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Study Purpose
This study investigated the effects of pre-centrifugation storage of EDTA blood at room temperature and 4˚C on levels of IL-6, IL-10, IFNγ, and IL-2 in plasma. Blood was collected from healthy volunteers into 2.0 mL EDTA tubes and aliquoted after 2 h at room temperature (20 specimens) or 4˚C (9 specimens). Plasma was obtained after an additional 4 h (6 h total) or 8 h (10 h total) at room temperature or 8 h (10 h total) at 4˚C by centrifugation at 2000 x g for 15 min and then aliquoted and frozen at -80˚C. To further investigate the effects of delayed processing, blood from four healthy individuals was centrifuged immediately or after 4 h at room temperature. Cytokines levels were determined using the Simoa ultra-sensitive immunoassays.
Summary of Findings:
Pre-centrifugation storage of whole blood for 6 or 10 h rather than 2 h resulted in significantly lower plasma levels of IL-6 (15%, P<0.0001 and 18%, P<0.0001; respectively) IL-10 (2%, P=0.0400 and 5%, P=0.0064; respectively) and IFNγ (7%, P=0.0250 and 11%, P=0.0250; respectively). Similarly, storage at room temperature for 4 h rather than 0 h resulted in an average decrease of 25% in IL-6, an average decrease of 8% in IL-10, and large decreases in IFNγ and IL-2 in one specimen, but significance was not determined. IL-2 levels were lower when whole blood was stored for 6 or 10 h rather than 2 h (11% and 15%, respectively), but only the decline at 10 h was significant (P=0.0420). While levels of IL-6, IL-10, IFNγ, and IL-2 were 3-5% lower in plasma from blood stored for 10 h than 6 h, only the difference in IL-6 was significant (P=0.0258). In contrast, pre-centrifugation storage of blood at 4˚C for 10 h rather than 4 h did not significantly affect levels of any of the tested cytokines.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Immunoassay Glycoprotein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 4˚C
Room temperature
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Time at room temperature 0 h
4 h
Storage Storage duration 2 h
6 h
10 h
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Study Purpose
This study investigated the effects of plasma storage and freeze-thaw cycling on cytokine levels. To test the effects of plasma storage, blood was collected from ten healthy volunteers into 2.0 mL EDTA tubes and centrifugation at 2000 x g for 15 min within 2 h of collection. Plasma was aliquoted and frozen at -80˚C immediately (2 h from draw) or after an additional 4 h (6h from draw). To test the effects of freeze-thaw cycling, plasma was obtained from 10 excess blood specimens from the clinical hematology laboratory by centrifugation at 2000 x g for 15 min then aliquoted and frozen at -80˚C. Specimens were thawed for 1 h at room temperature and refrozen daily for 1 or 3 cycles. IL-6, IL-10, IFNγ, and IL-2 levels were determined using the Simoa ultra-sensitive immunoassays.
Summary of Findings:
Plasma levels of IL-6, IL-10, IFNγ, and IL-2 were not significantly different in specimens stored for 2 or 6 h, but decreases in IL-6 were observed in two specimens. There were no differences in levels of IL-6, IL-10, IFNγ, or IL-2 between plasma thawed once and plasma subjected to three freeze-thaw cycles.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
- Normal
Platform:
Analyte Technology Platform Glycoprotein Immunoassay Protein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Time at room temperature 2 h
6 h
Storage Freeze/thaw cycling 1 cycle
3 cycles
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Study Purpose
The purpose of this study was to determine the effects of repeatedly dropping the specimen and pneumatic tube transport on plasma levels of IL-6, IL-10, IFNγ, and IL-2. Ten EDTA blood specimens were divided between tubes and subjected to a five-foot drop or normal handling and five pneumatic tube transports or not transported. Plasma was obtained by centrifugation at 2000 x g for 15 min and then aliquoted and frozen at -80˚C. IL-6, IL-10, IFNγ, and IL-2 levels were determined using the Simoa ultra-sensitive immunoassays.
Summary of Findings:
Although specimens dropped 5 times displayed visible hemolysis, plasma levels of IL-6, IL-10, IFNγ, and IL-2 were comparable to those in specimens that were not dropped. There were no effects of transport of blood by pneumatic tube system on levels of IL-6, IL-10, IFNγ, and IL-2 in plasma.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Glycoprotein Immunoassay Protein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Hemolysis Shaking-induced
Not induced
Storage Within hospital transportation method Pneumatic tube system
Not transported