title: Stored packed red blood cells contain a procoagulant phospholipid reducible by leukodepletion filters and washing.
abstract: BACKGROUND
  Ageing RBC gradually increase the exposure of phosphatidylserine (PS) on their surface, due to loss of membrane asymmetry. PS expression on red cells is not normally a significant factor in the hemostatic process, because aged RBC are rapidly cleared from the circulation. We propose that the presence of many altered red cells during massive transfusion can lead to increased procoagulant activity similar to what is seen in disease states where it is known to play a pathophysiologic role in microvascular disease.
  
  
  METHODS
  Procoagulant activity of phospholipid generated during storage of PRBC was evaluated using PRBC's as the only source of phospholipid in the determination of modified Russell's viper venom times of 10 PRBC units in which half of each unit was left unfiltered and half of each unit filtered. Florescent labeled annexin V binding by PRBC was also assessed by flow cytometry over time in storage. The effect of washing and filtration on these parameters was also determined.
  
  
  RESULTS
  As time of storage increased, the Russell's viper venom time of both the unfiltered and filtered units shortened (p<0.01). There was a significant lengthening of the Russell's viper venom time at all time points measured when unfiltered units were compared to filtered units (p<0.01). In both unfiltered and filtered units, with increased length of storage, there was a gradual increase in the percentage of cells or particles binding annexin V (p<0.01). Filtration resulted in a significant reduction in the percentage of cells or particles binding annexin V at all time points measured (p<0.01). The effect of washing of PRBC units on the RVVT was assessed for unfiltered and filtered units on day 42. Washing resulted in a significant reduction of the RVVT in both unfiltered and filtered groups (p<0.01).
  
  
  CONCLUSIONS
  Levels of annexin V binding and procoagulant phospholipid activity similar to levels seen in disease states associated with significant vasoocclusive pathophysiology were found toward the end of the storage period of PRBC units. It was possible to reduce both of these parameters by leukodepletion at collection, and with washing of PRBC at the end of storage. Filtration at collection resulted in a 67% increase in RVVT over unfiltered units by day 42 of storage. On day 42 of storage, washing of filtered units resulted in a 21% increase in RVVT, and washing of unfiltered units resulted in a 34% increase in RVVT. The effects seen with filtration and washing were additive suggesting that in spite of filtration at collection, deterioration of cells continues based on age since further removal of phospholipid can be induced with washing of filtered units on day 42.
authors.affiliation: Department of Blood Research, Transfusion Medicine Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500, United States. lisa.cardo@us.army.mil
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Cardo
authors.forename: Peter
authors.initials: P
authors.lastname: Hmel
authors.forename: Donna
authors.initials: D
authors.lastname: Wilder
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D001779:Blood Coagulation Factors / Q000737:chemistry*; D001781:Blood Component Removal / Q000379:methods; D001793:Blood Preservation / Q000379:methods*; D017707:Erythrocyte Transfusion; D004912:Erythrocytes / Q000166:cytology* / Q000378:metabolism; D005374:Filtration; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000166:cytology; D010743:Phospholipids / Q000737:chemistry*; D011485:Protein Binding; D013048:Specimen Handling; D013997:Time Factors
chemical_list: D017304:Annexin A5; D001779:Blood Coagulation Factors; D005456:Fluorescent Dyes; D010743:Phospholipids