Background
Leukoreduced whole blood (LR‐WB) has received renewed attention as alternative to component‐based transfusion in trauma. According to the manufacturer's instructions, leukoreduction should ...be carried out within 8 h after collection. This study assessed impact of (1) WB collection bag, (2) LR filtration, and (3) timing of filtration on in vitro quality.
Study Design and Methods
WB collected into different vendor bags was held at room temperature for <8 h or >16 h but <24 h prior to LR. In vitro quality was assessed before and after filtration, and throughout 3 weeks of storage at 4°C. Cell count and hemoglobin levels were determined by hematology analyzer, platelet activation, and responsiveness to ADP by surface expression of P‐selectin by flow cytometry, hemolysis by HemoCue, and metabolic parameters by blood gas analyzer. Hemostatic properties were assessed by rotational thromboelastometry. Plasma protein activities and clotting times were determined by automated coagulation analyzer or quantitative immunoblotting.
Results
Bag type had no impact on WB in vitro quality. LR by filtration had some impact, but is aligned with data in the literature. The time between donation and filtration resulted in some statistically significant differences in metabolic activity, platelet yield, platelet activation, and factor protein activity initially; however, these differences in in vitro quality attributes decreased throughout 21‐day cold storage.
Conclusion
WB hold time showed only a minor impact on WB in vitro quality, so it may be possible for blood processing facilities to explore extended hold times prior to filtration in order to provide greater operational flexibility.
Background
Pathogen inactivation (PI) accelerates the platelet (PLT) storage lesion, including apoptotic‐like changes. Proteomic studies have shown that phosphorylation levels of several kinases ...increase in PLTs after riboflavin and UV light (RF‐PI) treatment. Inhibition of p38MAPK improved in vitro PLT quality, but the biochemical basis of this kinase's contribution to PLT damage requires further analysis.
Study Design and Methods
In a pool‐and‐split design, apheresis PLT concentrates were either treated or kept untreated with or without selected kinase inhibitors. Samples were analyzed throughout 7 days of storage, monitoring in vitro quality variables including phosphatidylserine exposure, degranulation, and glucose metabolism. Changes in the protein expression of Bax, Bak, and Bcl‐xL and the activities of caspase‐3 and ‐9 were determined by immunoblot analysis and flow cytometry, respectively.
Results
The expression levels of the proapoptotic proteins Bax and Bak, but not the antiapoptotic protein Bcl‐xL, were significantly increased after the RF‐PI treatment. This trend was reversed in the presence of p38MAPK inhibitor SB203580. As a result of increasing proapoptotic protein levels, caspase‐3 and ‐9 activities were significantly increased in RF‐PI treatment during storage compared with control (p < 0.05). Similarly, p38MAPK inhibition significantly reduced these caspase activities compared with vehicle control after RF‐PI treatment (p < 0.05).
Conclusion
These findings revealed that p38MAPK is involved in signaling leading to apoptosis triggered by RF‐PI. Elucidation of the biochemical processes influenced by PI is a necessary step in the development of strategies to improve the PLT quality and ameliorate the negative effects of PI treatment.
In this study, we show how defocused spatially offset Raman spectroscopy (SORS) can be employed to recover chemical information from media of biomedical significance within sealed plastic transfusion ...and culture bags using a commercial SORS instrument. We demonstrate a simple approach to recover subsurface spectral information through a transparent barrier by optimizing the spatial offset of the defocused beam. The efficiency of the measurements is assessed in terms of the SORS ratio and signal-to-noise ratio (S/N) through a simple manual approach and an ordinary least squares model. By comparing the results for three different biological samples (red blood cell concentrate, pooled red cell supernatant and a suspension of Jurkat cells), we show that there is an optimum value of the offset parameter which yields the maximum S/N depending on the barrier material and optical properties of the ensemble contents. The approach was developed in the context of biomedical applications but is generally applicable to any three-layer system consisting of turbid content between transparent thin plastic barriers (i.e., front and back bag surfaces), particularly where the analyte of interest is dilute or not a strong scatterer.
Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, but the underlying mechanisms remain unknown. We used a dedicated ...N-terminomics technique, iTRAQ terminal amine isotopic labeling of substrates (TAILS), to characterize the human platelet N-terminome, proteome, and posttranslational modifications throughout platelet storage over 9 days under blood-banking conditions. From the identified 2938 proteins and 7503 unique peptides, we characterized N-terminal methionine excision, co- and posttranslational Nα acetylation, protein maturation, and proteolytic processing of proteins in human platelets. We also identified for the first time 10 proteins previously classified by the Human Proteome Organization as “missing” in the human proteome. Most N termini (77%) were internal neo-N termini (105 were novel potential alternative translation start sites, and 2180 represented stable proteolytic products), thus highlighting a prominent yet previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metalloproteinase and other proteinase substrates and their respective cleavage sites suggests novel mechanisms of the effect of proteases on protein activity and platelet function during storage. All data sets and metadata are available through ProteomeXchange with the data set identifier PXD000906.
•TAILS proteomics identified 2938 human platelet proteins, pervasive proteolytic processing, and precise proteolytic cleavage sites in stored platelets.•During storage, metalloproteinases were predominantly involved in proteolytic processing, while other proteinases were mainly involved in degradation.
BACKGROUND
Biochemical analyses of mechanisms triggered in platelets (PLTs) upon pathogen inactivation (PI) are crucial to further understand the impact of PI on PLT functionality and, subsequently, ...quality.
STUDY DESIGN AND METHODS
PLT concentrates (PCs) were split into four small illumination bags: 1) untreated control, 2) treated with riboflavin and ultraviolet light (RF/UV), and spiked with 3) solvent control dimethyl sulfoxide and 4) p38 mitogen‐activated protein kinase (MAPK) inhibitor SB203580 before RF/UV treatment. Flow cytometry was used to monitor PLT mitochondrial potential (ΔΨm); generation of intracellular reactive oxygen species (ROS); and release of microvesicles (MVs), mitochondria (MT), and MVs containing MT (MVs/MT). Quantitative polymerase chain reaction (qPCR) was used to quantify extracellular mitochondrial DNA (mtDNA). Translocation of selected mitochondrial proteins was analyzed in subcellular fractions by immunoblot.
RESULTS
RF/UV treatment triggered an increased mitochondrial translocation of both Bax and Bid (p < 0.05, Day 7) and cytochrome c release (p < 0.01, Day 7), loss of ΔΨm (p < 0.05, Day 5 and Day 7), and ROS generation (p < 0.01, Day 5 and Day 7) in PCs compared to the untreated control during storage. These PI‐triggered changes were inhibited by SB203580 (p < 0.05). The release of MVs, MT, and MVs/MT was increased upon the RF/UV treatment during storage (p < 0.05) and, with the exception of MT, the release was decreased by the inhibitor (p < 0.05). qPCR analysis showed that RF/UV does not trigger mtDNA release during storage.
CONCLUSION
These findings further our understanding of mechanisms in PLTs initiated by the RF/UV treatment, demonstrating that this treatment induces p38 MAPK‐dependent mitochondrial signaling and MV release in apheresis PCs.
Background
Transfusion medicine standards in Canada state that adult recipients can be transfused with cryoprecipitate of any ABO group, however, not all hospitals follow this guideline. There is a ...paucity of data on cryoprecipitate anti‐A/B levels to reinforce standards.
Study Design and Methods
Manual tube antibody titration was performed on 7 units of group O plasma and the corresponding cryosupernatant plasma and cryoprecipitate. IgG/IgM levels were determined by nephelometry. Additionally, 10 cryoprecipitate each from groups A, B, and O were similarly assessed. From the antibody titer distribution among these samples, the probability of making a pool of cryoprecipitate with a titer ≥1:100 was calculated using bootstrap analysis.
Results
Anti‐A/B titers in cryoprecipitate were equivalent to those in corresponding plasma; partitioning of anti‐A/B activity into cryoprecipitate was not observed. Average IgM concentration was higher in cryoprecipitate than in plasma (P < .01). However, no correlation between IgM levels and anti‐A/B titers was established. Among 30 cryoprecipitates from routine blood bank inventory, the median antibody titer and mode were 1:32 and 1:16, respectively. Of the samples tested, 4 of 30 and 9 of 30 had titers above 1:100 and 1:50, respectively. The probability of transfusing an adult dose of cryoprecipitate (pool of 10 cryoprecipitate) with a titer higher than 1:100 was calculated to be less than 1 in 3 million.
Conclusions
This study provides strong evidence to support current Canadian transfusion medicine standards on the safety of transfusion of cryoprecipitate without the need for blood group matching in adult recipients.
See editorial on page 1–4, in this issue
50 years of Vox Sanguinis International Forums Lozano, Miquel; Dunbar, Nancy M.; Devine, Dana V.
Vox sanguinis,
August 2020, 2020-Aug, 2020-08-00, 20200801, Volume:
115, Issue:
6
Journal Article
Keywords: blood center operations; FFP transfusion; transfusion practices (adult) Byline: Claudia S. Cohn, Lise Estcourt, Brenda J. Grossman, Monica B. Pagano, Elizabeth S. Allen, Evan M. Bloch, ...Arturo Casadevall, Dana V. Devine, Nancy M. Dunbar, Farid Foroutan, Thomas J. Gniadek, Ruchika Goel, Jed Gorlin, Michael J. Joyner, Ryan A. Metcalf, Jay S. Raval, Todd W. Rice, Beth H. Shaz, Ralph R. Vassallo, Jeffrey L. Winters, Gregory Beaudoin, Aaron A. R. Tobian
BACKGROUND
In neonate transfusion, the use of a dedicated red blood cell (RBC) unit decreases donor exposure. A separate safety measure involves gamma irradiation of the RBCs to abrogate the ...possibility of transfusion‐associated graft‐versus‐host disease. However, in combination, storage of gamma‐irradiated RBCs leads to accumulation of potentially harmful substances in the supernatant.
STUDY DESIGN AND METHODS
For this study, RBCs were pooled and split into three study arms. Centrifugation or gravity was used to pack RBCs of matched units thereby reducing the amount of supernatant that would be present in neonate transfusion aliquots; these were compared to matched control units. Supernatant measurements of potassium, hemoglobin (Hb), RBC microvesicle (RMV) content, and mannitol were made in aliquots prepared weekly up to 21 days after gamma irradiation. RBC morphology and osmotic fragility were also assessed to determine if supernatant reduction methods affected the storage lesion.
RESULTS
Potassium and mannitol were significantly decreased in transfusion aliquots prepared with either of the supernatant reduction methods. On Day 21, potassium levels from supernatant‐reduced aliquots were below those of Day 7 control aliquots. A decrease in free Hb was only detected on Day 21 in centrifuged aliquots. RMVs were significantly reduced in centrifuged aliquots and significantly increased in gravity‐settled aliquots. The only measurable effect on storage lesion was a small increase in osmotic fragility of the RBCs subjected to supernatant reduction.
CONCLUSION
Supernatant reduction by centrifugation effectively reduces potassium, mannitol, and RMVs in aliquots from gamma‐irradiated RBCs stored up to 21 days.
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