Effect of Sample Transportation on the Proteome of Human Circulating Blood Extracellular Vesicles.
Author(s): Uldry AC, Maciel-Dominguez A, Jornod M, Buchs N, Braga-Lagache S, Brodard J, Jankovic J, Bonadies N, Heller M
Publication: Int J Mol Sci, 2022, Vol. 23, Page
PubMed ID: 35562906 PubMed Review Paper? No
Purpose of Paper
This paper compared extracellular vesicle size distribution and proteome profiles between case-matched platelet-poor plasma (PPP) and platelet-free plasma (PFP) that were separated from blood either transported by courier or by pneumatic tube system (PTS). The authors also investigated the potential influence of transportation metrics on the proteins identified.
Conclusion of Paper
PPP generally had fewer particles than PFP, but there no significant differences in particle number were attributed to transportation method. When blood was transported by PTS rather than courier, the PFP particles had larger median particle size, standard deviation in particle size, and particle size at maximum intensity, while PPP particles only had a larger standard deviation in particle size. Fewer proteins were detected in PFP than PPP from 11 of the 12 donors. A high percentage of the proteins unique to PPP (86.2%) were platelet-specific markers, reflecting greater platelet contamination in PPP than PFP. PPP also had a higher number of proteins classified as cell membrane, cell part, and cell surface proteins than PFP, but numbers of serum/plasma proteins (apolipoproteins, coagulation factors, complement factors and immunoglobulins) were similar. Markers enriched in PFP compared to PPP include those that are specific to erythrocytes, macrophages, endothelial cells, and exosomes. Conversely, markers enriched in PPP compared to PFP include those that are specific to platelets, and lymphocytes/monocytes. Importantly, levels of PFP- or PPP-specific markers did not differ significantly when blood transport methods were compared. Very few significant differences in PFP protein profile occurred when courier transport and PTS were compared; further the majority of the differences that were observed were limited to specimens from one or two patients.
As expected, differences in acceleration were observed between specimens transported by courier and those transported by PTS. The authors found that when blood was transported by courier, protein that correlated with the Teaker-Kaiser operator (TK), the root mean square (RMS), and the vibration dose value (VDV) overlapped with one another and were primarily involved in cytoskeleton regulation and cellular organization. In contrast, when blood specimens were transported by PTS, proteins formed two groups: proteins that were correlated with VDV were involved in mitochondrion-associated cellular respiration and proteins that were correlated with the TK and RMA were associated with organelles or involved in metabolism of proteins or nucleic acids. The authors identified 12 proteins that could classify specimens based on transport method.
Studies
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Study Purpose
This study compared extracellular vesicle size distribution and proteome profiles between case-matched platelet-poor plasma (PPP) and platelet-free plasma (PFP) that were separated from blood either transported by a courier or transported by pneumatic tube system (PTS). The authors also investigated the potential influence of transportation metrics on the proteins identified. Blood was collected from six healthy donors (3 males and 3 females, aged 39-56 years) and six patients diagnosed with myeloid malignancies (five males and one female, aged 31-82 years) into four S-Monovette 3.2% citrated tubes and one S-Monovette EDTA tube. Two of the citrate tubes were transported by PTS and the other two citrate tubes and the EDTA tube were transported by courier. Acceleration during transport was measured for all specimens using a Raspberry Pi Zero device with a SenseHat. Upon arrival, blood cell counts were quantified in the EDTA blood using a Sysmex XN-1000 instrument. Citrates plasma was separated from blood by centrifugation at 1500 g for 10 min at room temperature, and the PPP from the two tubes transported by the same method were combined. Aliquots of PPP were frozen at -80°C. The remaining PPP aliquots were then recentrifuged at 16,000 g for 2 min to obtain PFP. PFP was aliquoted and frozen at -80°C. Particle size, concentration, and volume were analyzed by nanoparticle tracking using a ZetaView. EVs were isolated from PPP and PFP by centrifugation 16,000 g for 40 min, were washed in PBS, and then pelleted by centrifugation at 16,000 g for 20 min three times. EVs were dissolved in a Tris HCl buffer (pH 8.0) containing Urea, DTT, and IAA and digested with trypsin and LysC. The proteins were profiled by untargeted label-free mass spectrometry.
Summary of Findings:
Compared to a previous study, the squared correlation of proteins found in PFP showed a modest correlation for cell surface proteins (R2=0.49), but strong correlations for serum/plasma proteins (R2=0.70), cellular proteins (R2=0.77), and cell membrane proteins (R2=0.75). Exclusion of three outliers increased the correlation for cell surface proteins between the two studies (R2=0.75). PPP generally had fewer particles than PFP. While there were no significant differences in particle number between the transportation methods evaluated, there was a trend toward higher particle number in PFP from blood transported by PTS compared to transport by courier. Particle size distribution did significantly differ between blood specimens transported by PTS and courier, with larger differences found in PFP than PPP. When blood was transported by PTS rather than courier, PFP particles had larger median particle size (P=6.6 x 10-7), standard deviation in particle size (P=5.1x10-9), and particle size at maximum intensity (P=2 x 10-4), while PPP particles had a larger standard deviation in particle size (P=0.049).
A total of 2144 proteins were detected in at least 2 technical replicates from at least one patient’s PPP or PFP. Fewer proteins were detected in PFP than PPP for 11 of the 12 donors. Overall, 456 proteins were unique to PPP specimens and 52 were unique to PFP. Of the 456 proteins unique to PPP, 393 (86.2%) were platelet-specific markers; only 12 of the 52 proteins (23.1%) unique to PFP were platelet-specific, reflecting higher platelet contamination in PPP than PFP. PPP had more proteins classified as cellular proteins (cell membrane, cell part, and cell surface) than PFP, but a comparable number of serum/plasma proteins (apolipoproteins, coagulation factors, complement factors and immunoglobulins). The markers enriched in PFP (compared to PPP) included those that are specific to erythrocytes (CD233), macrophages (CD14), endothelial cells (HSPG2), and exosomes (CD81); while those enriched in PPP (compared to PFP) included proteins that are specific to platelets (CD41, CD62P), and lymphocytes/monocytes (CD40, CD102). Importantly, levels of PFP- or PPP-specific markers did not differ significantly when blood transport methods were compared. Very few significant differences in were found between PFP protein profiles of blood transported by courier or PTS, an in most instances these differences were observed in specimens from one or two patients. However, PTS transported specimens were enriched for the erythrocyte-specific CD233 in half of the specimens examined. Platelet markers were correlated with coagulation factors and cell part, cell membrane, cell surface, and exosome protein abundances. Apolipoproteins were correlated with cell counts, with the exception of granulocyte and monocyte counts.
As expected, accelerometer data showed highly variable (>17 g) multidirectional acceleration of specimens transported by pneumatic tube system while those manually transported by courier showed regular gentle acceleration (>2.5 g). However, the median acceleration was similar between the two transport methods. The authors found that when blood was transported by courier, protein that correlated with the Teaker-Kaiser operator (TK), the root mean square (RMS), and the vibration dose value (VDV) overlapped with one another and were primarily involved in cytoskeleton regulation and cellular organization. In contrast, when blood specimens were transported by PTS, proteins formed two groups: proteins that were correlated with VDV were involved in mitochondrion-associated cellular respiration and proteins that were correlated with the TK and RMA were associated with organelles or involved in metabolism of proteins or nucleic acids. The authors identified 12 proteins that could classify specimens based on transport method.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Other
Platform:
Analyte Technology Platform Cell count/volume Light scattering Protein MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Within hospital transportation method Hand-delivered
Pneumatic tube system
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
Biospecimen Aliquots and Components Blood and blood products Platelet-poor plasma
Platelet free plasma