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The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray.

Author(s): Bæk R, Søndergaard EK, Varming K, Jørgensen MM

Publication: J Immunol Methods, 2016, Vol. 438, Page 11-20

PubMed ID: 27568281 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to compare the signal intensity of EV markers (combined CD9, CD63 and/or CD81) in serum, and CPDA, EDTA, and lithium heparin plasma after room temperature processing delays of up to 24 h, after storage on a shaker, and isolation using a one- or two-step centrifugation protocol. The effects of plasma/serum storage temperature, freeze-thaw cycling and exosome isolation on EV signal intensity were also examined. The authors further investigated the effects of delayed centrifugation, storage on a shaker, and EV isolation on levels of 15 EV markers in select specimens using a phenotyping array. Blood was collected from five healthy donors into CPDA (citrate phosphate dextrose adenine), EDTA (K3EDTA), lithium heparin and serum clot activator tubes. Unless otherwise specified, plasma/serum were obtained by centrifugation at 1800 g for 6 min immediately after blood collection and stored at -40°C for a few days before analysis. To test the effects of delayed centrifugation, one tube of each type was centrifuged after 0 h (serum was excluded), 1, 8 and 24 h at room temperature. To test the effects of centrifugation method, case-matched specimens were centrifuged once at 1800 g for 6 min or twice at 2000 g for 30 min. To test the effects of agitation during transport, one tube of each type was subjected to storage on an orbital shaker for 1 h before centrifugation. To test the effects of post-thaw centrifugation, thawed aliquots of EDTA plasma were centrifuged once at 14,000 g for 2 min or twice at 2500 g for 15 min before immediate analysis. To test the effects of plasma/serum storage temperature, aliquots of each type of plasma/ serum were stored at room temperature, 4°C, −20°C, −40°C, −80°C, and −160°C for days (number not specified) or 3 months. To test the effects of freeze-thaw cycling of serum/plasma, aliquots of each type stored at -40°C were subjected to one, two, three or four freeze thaw cycles (thawing method not specified).  To test the effects of ExoQuick EV isolation, EVs were isolated from plasma/serum aliquots before or after storage at -40°C using ExoQuick and compared to specimens for which no EV isolation was performed. Proteins were detected in isolated EVs, plasma and serum using two different protein microarrays. EV signal intensity was evaluated using an array with a mixture of antibodies for CD9, CD63, and CD81. Further characterization of some samples was performed using an array with fifteen antibodies allowing for EV phenotyping.

   Summary of Findings:

   Relative intensity of EV signals were not significantly altered by delayed centrifugation in heparin plasma or serum, although significant effects were observed in CPDA and EDTA plasma after processing delays.  In CPDA plasma, EV signal intensities were lower in specimens processed after 1 or 8 h compared to immediately processed specimens (P<0.001 and P<0.005, respectively), but after an increase toward baseline, were significantly higher after a 24 h delay compared to a 1 h delay (P<0.001). Further investigation showed that only CD9 signal intensity differed significantly between CPDA plasma processed after 1 h and 24 h (P<0.005). EV signal intensities were significantly higher in EDTA plasma subjected to an 8 or 12 h delay than those subjected to a 1 h delay (P<0.005 for both), but levels were not significantly different from immediately centrifuged specimens at any timepoint and significance was not reached for any marker individually. A significant increase in EV signal intensity was observed when EDTA blood was stored on a shaker for an hour compared to tubes stored on the benchtop (P<0.0001) and further analysis showed significantly higher signals from Annexin V, CD19, TNF RL, and CD106 (P<0.05, all). In contrast, blood agitation via a shaker did not significantly affect the signal intensity of EVs from CPDA plasma, heparin plasma, or serum. Use of a two-step rather than a one-step centrifugation procedure resulted in higher EV signal intensities and lower background noise for CPDA and EDTA plasma but did not affect the signal intensity or background in serum or heparin plasma. For EDTA plasma, post-thaw centrifugation also reduced background, but there was no clear difference between one- and two-step centrifugation. EV signal intensities were significantly affected by the temperature of plasma/serum short-term (days) storage. Significant differences in EV signal intensity were observed among case-matched EDTA or heparin plasma stored at all temperatures examined  relative to room temperature storage; CPDA plasma stored at -40 or -80°C relative to room temperature storage, at -40°C, -80°C and -160°C relative to 4°C and at -40°C, and -160°C relative to -80°C; Serum stored at -80°C, and -160°C relative to -40°C When serum or plasma were stored for months instead of days, significant differences in EV signal intensities were found between heparin plasma stored at room temperature and 4°C versus each of the other temperatures, between serum stored at room temperature and each of the other temperatures, but in CPDA plasma significant differences were limited to between storage at room temperature and 4°C and in EDTA plasma between specimens stored at 4°C and those stored at room temperature, -40°C or -80°C. Relative EV signal intensities differed between heparin plasma or serum that was freeze-thaw cycled once compared to twice, and EDTA plasma that was freeze-thaw cycled 3 times compared to four times. When an exosome isolation step was included before or after freezing, the relative intensity of EV markers was higher in EDTA plasma and lower in heparin plasma compared to when no isolation was performed. However, including an exosome isolation step did not significantly alter the EV signal intensity in serum or CPDA plasma. The authors state the increase in EV signal intensities in EDTA plasma following exosome isolation were mainly due to a decrease in background intensity.  The intensity of annexin V, CD9, TNFR1, CD142, CD42a, and ICAM-1 were lower when exosomes were isolated from heparin plasma before freezing than when no isolation was performed, indicating isolation removes platelet contamination, microparticles and immune related markers.. The authors concluded that heparin plasma demonstrated the greatest EV stability, but its use may be inadvisable due to platelet activation and EV uptake.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma
   • Bodily Fluid - Serum

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Antibody microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	None Potassium EDTA Lithium heparin Citrate phosphate dextrose adenine 1
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Different number of centrifugation steps compared Multiple speeds compared
   Biospecimen Acquisition	Type of collection container/solution	Serum clot activator tubes K3EDTA tube Lithium heparin tube CPDA tube
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Plasma exosomes Serum
   Storage	Storage conditions	On a shaker On the bench
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles 4 cycles
   Storage	Storage temperature	Room temperature 4°C -20°C -40°C -80°C -160°C
   Storage	Time at room temperature	0 h 1 h 8 h 24 h
   Analyte Extraction and Purification	Analyte isolation method	Exosomes isolated No isolation performed
   Storage	Storage duration	Days 3 months

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