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Pathogen inactivation with amotosalen plus UVA illumination minimally impacts microRNA expression in platelets during storage under standard blood banking conditions.

Author(s): Arnason NA, Johannson F, Landrö R, Hardarsson B, Irsch J, Gudmundsson S, Rolfsson O, Sigurjonsson OE

Publication: Transfusion, 2019, Vol. , Page

PubMed ID: 31674051 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared markers of platelet quality and activation and miRNA expression in untreated platelets stored in PAS, PI-treated platelets in PAS, and untreated platelets stored in plasma over the course of 7 days of storage. Blood from 24 healthy donors was centrifuged at 3578 × g for 11 min at 20°C. The buffy coats were then separated using an Optipress II. Specimens were then pooled to create three ABO matched pools (8 specimens/pool). The pools were mixed gently and then split and PAS (2 pools) or plasma (1 pool) was added. After 1 h at room temperature, platelet concentrates (PCs) were obtained from pools in PAS by centrifugation at 40 × g for 2 min followed by 463 × g for 6.5 min and from pools in plasma by centrifuging at 987 x g for 7 min. PCs were then leukocyte filtered and separated from buffy coat using an Optipress II. One of the PCs in PAS was then subjected to pathogen inactivation (PI) which included the addition of amotosalen solution, UVA light exposure (total 3.9 J/cm₂), and incubation with a compound absorption device and gentle agitation for 6 to 16 h at 22°C. PCs were stored for 7 days in a platelet incubator at 22°C with gentle agitation and sampled using a closed system on Days 1, 2, 4, and 7. Lactate, glucose, PO₂, PCO₂, and pH were analyzed immediately after sampling using an ABL90 blood gas autoanalyzer. Platelet counts, mean platelet value (MPV), and platelet distribution width (PDW) were determined using a Sysmex XN-1000 hematology auto-analyzer (Sysmex XN-1000) and Annexin V, glycoprotein IIb (GPIIb), GPIbα, P-selectin, and the CD63 were measured by flow cytometry. The platelets were subsequently pelleted by centrifugation at 1730 × g for 10 minutes and the pellet was white blood cell depleted using a magnet after incubation with CD45 Dynabeads for 15 min. The platelet supernatants were stored at -80°C until levels of soluble CD40 ligand (sCD40L), sCD62P/sP-Selectin, and CXCL4/platelet factor 4 (PF4) could be quantified by ELISA. RNA was isolated from platelets using the miRNeasy Mini Kit and stored at -80°C until quantification of miR-23a, miR-30c, miR-103, miR-142-3p, miR-451, and UniSp6 (spike-in control) by real-time RT-PCR.

   Summary of Findings:

   Platelet count decreased over storage with significantly smaller declines noted in untreated platelets in PAS than those in plasma (16.7% versus 19.8%, P=0.0132) or in untreated platelets in PAS (16.7% versus 20.4%, P=0.0165) but MPV and PDW were comparable between treatments and time-points. pH was higher on Day 4 than Day 2 or 7 but remained above the threshold of 6.4 in all treatments. Glucose and pCO₂ declined and lactate and pO₂ increased during storage with significantly higher levels of glucose, lactate, and pCO₂ in untreated platelets in plasma than platelets in PAS (P<0.05, all) and significantly lower pCO₂ in untreated platelets in PAS than PI-treated platelets in PAS on days Day 2 (P= 0.029) and Day 4 (P = 0.016). P-selectin and proportion of platelets with low GPIbα increased with storage and annexin V went up and down before increasing again. Untreated platelets in plasma had higher levels of P-selectin and Annexin V than untreated platelets in PAS at all time-points. Compared to untreated platelets in PAS, PI-treated platelets in PAS had higher P-selectin on Day 7 and higher proportion of platelets with low GPIbα on Days 4 and 7, indicating more platelet activation in PI-treated platelets but Annexin V binding was lower on Days 4 and 7. Supernatants from platelets stored in plasma had lower CD40L and higher sP-selectin than those stored in PAS on all days and higher PF4 on Day 4. Supernatants from PI-treated platelets in PAS had significantly higherCD40L concentration and higher sP-selectin and PF4 levels on Day 4 and higher levels of PF4 on Day 7 than supernatants from untreated platelets in PAS, confirming higher platelet activation following PI treatment. Untreated platelets stored in PAS had higher miR-326 on Day 7 (16.7%), lower miR146a on Day 2 (-10.8%), and lower lmiR-96-5p on Days 2 (-55.1%) and 4 (-51.4%) than platelets in plasma. Pathogen inactivated plasma in PAS had higher miR--17-3p on Day 2 (15%) and 4 (15.5%), higher miR-1260a on Day 7 (20.0%), lower miR-96-5p at all time-points (-51.4-61.9%), and lower miR-146a-5p on Day 2 (-7.9%). PCA of miRNA expression levels showed no clustering of specimens by treatment or sampling day.

   Biospecimens

   • Bodily Fluid - Buffy Coat

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Gas	Clinical chemistry/auto analyzer
   Carbohydrate	Clinical chemistry/auto analyzer
   Protein	Clinical chemistry/auto analyzer
   RNA	Real-time qRT-PCR
   Protein	ELISA
   Glycoprotein	Flow cytometry
   Cell count/volume	Flow cytometry
   Small molecule	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Short-term storage solution	Plasma PAS (SSP+)
   Biospecimen Aliquots and Components	Blood processing method	PI treated Not PI treated
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-23a miR-30c miR-103 miR-142-3p miR-451 UniSp6
   ELISA Specific	Targeted peptide/protein	soluble CD40 ligand sCD62P/sP-Selectin CXCL4/platelet factor 4
   Storage	Storage duration	1 day 2 days 4 days 7 days

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