Craig Jenkins

A quality monitoring program for red blood cell components: in vitro quality indicators before and after implementation of semiautomated processing

Canadian Blood Services has been conducting quality monitoring of red blood cell (RBC) components since 2005, a period spanning the implementation of semiautomated component production. The aim was to compare the quality of RBC components produced before and after this production method change.

The effect of processing method on the in vitro characteristics of red blood cell products

While the clinical impact of differences in red blood cell (RBC) component processing methods is unknown, there are concerns they may be confounding variables in studies such as the ongoing ‘age of blood’ investigations. Here, we compare the in vitro characteristics of red cell concentrates (RCCs) produced by several different processing methods.

Bacterial survival and distribution during buffy coat platelet production.

At Canadian Blood Services, buffy coat (BC) platelet concentrates (BC‐PCs) show a generally lower bacterial contamination rate than apheresis PCs. This study investigated whether the PC production method contributes to this observation.

Stability of coagulation protein activities in single units or pools of cryoprecipitate during storage at 20–24°C for up to 24 h

Cryoprecipitate is a concentrated source of fibrinogen and other plasma proteins. Cryoprecipitate must be transfused within 4–6 h of thawing and storage at 20–24°C. We compared plasma protein activities in single or pooled cryoprecipitate units stored at 20–24°C for 0, 4 or 24 h.

Quality of frozen transfusable plasma prepared from whole blood donations in Canada: An update

BACKGROUNDnTransfusable plasma is obtained by processing whole blood donations, by apheresis, or as solvent/detergent plasma (SD plasma), a pooled pathogen-reduced plasma product. The quality of plasma is typically assessed by testing the activities of multiple coagulation-related plasma proteins, due to a lack of clinical trial data linking plasma composition to clinical endpoints. We sought to update previous quality surveys of Canadian frozen plasma (FP; manufactured from single donor whole blood donation and frozen within 24h of phlebotomy), to provide transfusionists with a more complete picture of its characteristics.nnnSTUDY DESIGN AND METHODSnFP units (n=131) were tested for: the activity of factors V, VII, VIII, X, and XI, protein S (PS), α2-antiplasmin (AP), and fibrinogen; and the activated partial thromboplastin (APTT) and prothrombin (PT) times. Comparisons were made to: previous Canadian FP surveys; and to studies of single-donor plasma and SD plasma from other nations.nnnRESULTSnMean FVIII, fibrinogen, or APTT values did not differ from the previous annual survey of Canadian FP; FV activity was increased and PT values decreased. FP produced with or without leukoreduction differed only in mean APTT. Canadian FP exhibited generally similar quality to that reported by other organizations in Europe and Asia for similarly manufactured single-donor plasma, but contained notably higher PS and AP (≈ four-fold) activities than did SD plasma.nnnCONCLUSIONnOur results indicate that Canadian FP is of similar quality to single-donor products produced in other jurisdictions. While it is of arguably superior in vitro quality to an SD plasma product recently licensed in Canada, these differences are highly unlikely to have clinical significance for most indications for plasma transfusion.

Relative safety of buffy coat platelet pools

1. Curtis BR. Genotyping for human platelet alloantigen polymorphisms: applications in the diagnosis of alloimmune platelet disorders. Semin Thromb Hemost 2009;34: 539-48. 2. Bertrand G, Bianchi F, Chenet C, Martageix C, Blanchet P, Baumler M, Kaplan C. New mutation in the platelet b3-integrin gene: implication for the diagnosis of fetomaternal alloimmunization. Transfusion 2006;46:2138-41. 3. Bertrand G, Kaplan C. The 262T>C silent mutation of the platelet b3-integrin gene is not restricted to a single family. Transfusion 2008;48:402. 4. Walchshofer S, Ghali D, Fink M, Panzer-Grümayer ER, Panzer S. A rare leucine/arginine polymorphism on platelet glycoprotein IIIa is linked to the human platelet antigen 1b. Vox Sang 1994;67:231-4.

Process improvement by eliminating mixing of whole blood units after an overnight hold prior to component production using the buffy coat method.

The elimination of a thorough manual mixing of whole blood (WB) which takes place following the overnight hold, but before the first centrifugation step, during buffy coat component production at Canadian Blood Services (CBS) was investigated. WB was pooled after donation and split. Pairs of platelet, red blood cell (RBC), and plasma components were produced, with half using the standard method and half using a method in which the mixing step was eliminated. Quality assessments included yield, pH, CD62P expression and morphology for platelets, hemoglobin, hematocrit, hemolysis, and supernatant K+ for RBCs, and volume and factor VIII activity levels for plasma. All components, produced using either method, met CBS quality control criteria. There were no significant differences in platelet yield between components produced with and without mixing. A significant difference was seen for RBC hemolysis at expiry (P = 0.03), but for both groups, levels met quality control requirements. Noninferiority of components produced without mixing was confirmed for all parameters. Manual mixing is laborious and has a risk of repetitive strain for production staff and its significance is unclear. Elimination of this step will improve process efficiencies without compromising quality.

Bacteria can proliferate in thawed cryoprecipitate stored at room temperature for longer than 4 h

Although key coagulation factor activities are maintained in thawed cryoprecipitate stored for up to 24 h at ambient temperature, several jurisdictions limit such storage to 4–6 h. Here, we separately spiked thawed cryoprecipitate units with four bacterial strains: Staphylococcus epidermidis, Serratia liquefaciens, Pseudomonas putida and Pseudomonas aeruginosa. No strains grew in the first 4 h of storage, but by 24 h, three of four exhibited up to 1000‐fold proliferation. Pathogen inactivation technologies could be explored to mitigate the safety risk posed by extending storage of thawed cryoprecipitate at room temperature.

Sporadic formation of cryoprecipitate-like particulate matter in thawed fresh-frozen plasma units

Fresh-frozen plasma (FFP) is a biologic solution with a high protein concentration, to which no excipients are added. Plasma protein precipitation is usually avoided when FFP is thawed, in preparation for transfusion, by traversing the solid/liquid-phase transition as rapidly as possible, typically by immersing FFP units in water bath– type thawing devices at temperatures from 30 to 37°C. Between September 2005 and February 2006, three apheresis FFP units were returned to Canadian Blood Services (CBS), Canada’s national blood transfusion agency, by an academic health sciences center in Eastern Canada, because of the presence of “clots.” In at least one instance, the particulate matter (PM) was not detected until after transfusion had been initiated, and an infusion pump alarm sounded due to blockage of the drip chamber. The returned units were shipped on ice to the CBS quality monitoring laboratory in Hamilton, Ontario, for investigation. PM was clearly visible (see Fig. 1) but was flocculent, rather than gel-like or fibrous, as was expected for a fibrin clot. Some PM dissolved on prolonged (5-hr) incubation at 37°C, and only an estimated 10% remained insoluble after 24 hours. Samples of all three units clarified by microcentrifugation exhibited fibrinogen levels (2.32.6 g/L) and activated partial thromboplastin times within normal ranges tested on an automated coagulation analyzer (STA-Compact, Abbott Laboratories, Abbott Park, IL); Factor (F)V levels were low-normal (0.46-0.49 IU/mL), likely reflecting some losses of this labile factor during storage and transit of the units at 1 to 6°C. Inspection of donor records revealed no unusual common features of the units, nor was there any commonality as to whether slow or fast approved freezing procedures were employed in their manufacture. The reversible nature of the PM suggested to us that they were either lipoprotein-enriched particles, previously suggested as one cause of white PM in blood components or cryoprecipitate. Aliquots of FFP units containing PM were microcentrifuged, and pellet and supernatant fractions were prepared and compared to a commercial normal plasma control by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis under reducing conditions. The enrichment of several bands in the PM pellets in the 45to 70-kDa mobility range was suggestive of increased fibrinogen. Inspection of gels and replicated immunoblots, loaded with samples normalized to contain equivalent amounts of human serum albumin (HSA), confirmed that the PM were enriched not only in fibrinogen, but also in von Willebrand factor (VWF) (Fig. 2). Fibrinogen and VWF are two of the proteins enriched in cryoprecipitate; others, for which we did not test, include FVIII and FXIII and fibronectin. Other abundant proteins such as HSA and immunoglobulins are also found in cryoprecipitate, but are not enriched over their concentration in the source plasma and are likely present as entrapped bystanders. Our data therefore suggested that an inadvertent and partial formation of cryoprecipitate-like PM had occurred during FFP thawing. Cryoprecipitate is produced by deliberate slow thawing of FFP at 1 to 6°C. Working with the hospital, CBS customer liaison staff determined that ThermoGenesis (Rancho Cordova, CA) plasma thawers were used to thaw up to 10 bags of FFP at a time, as permitted by the manufacturer’s instructions and that the temperature and water levels of the units were monitored appropriately. Two suggestions were made in hopes of minimizing zones of persistent ice/plasma mixtures that could promote cryoprecipitation, that the practice of bagging the FFP units in plastic be discontinued, and that chunks of partially thawed FFP be manually broken up during the thawing process. Following these steps, we are aware of no further PM-containing FFP units being returned to CBS in the ensuing 3 years. Even at the hospital in question, PM-containing FFP units were rare, representing three of the more than 1000 units thawed and transfused without incident during the 6 months when they were detected. We are uncertain if this problem is underreported and only came to the attention Fig. 1. Photograph of an apheresis FFP unit returned to CBS because of the presence of PM. The unit is shown from the unlabeled side, angled to maximize visibility of the PM, and inserted into a heat-sealable bag to guard against potential leakage during photography. White arrows highlight the position of some of the more visible PM.

Stability of Thawed Apheresis Fresh-Frozen Plasma Stored for up to 120 Hours at 1°C to 6°C

Regulations concerning the storage of transfusable plasma differ internationally. In Canada, plasma obtained from whole blood donations and frozen within 24 hours of phlebotomy (frozen plasma, FP) may be thawed and transfused within 120 hours of refrigerated storage. However, plasma frozen within 8 hours of phlebotomy following apheresis donation (FFPA) must be transfused within 24 hours of thawing and refrigeration. Our objectives were to measure coagulation factors (F) V, VII, and VIII, fibrinogen activities, and the prothrombin time (PT) in thawed refrigerated FFPA at 0, 24, and 120 hours of storage and to compare these values to those in thawed refrigerated FP. Fibrinogen activity remained unchanged over time, while mean factor levels in 28 FFPA units declined by 17% (FV), 19.7% (FVII), and 54.6% (FVIII) over 120 hours, while PT values rose to 7.6%. Factor activities were significantly higher in FFPA than FP after 120 hours of refrigerated storage. Residual FVIII activities in thawed FFPA met predefined noninferiority criteria compared to thawed FP after 120 hours. These results support a change in Canadian regulations to permit transfusion of thawed FFPA made in a closed system and refrigerated for up to 120 hours, one that could reduce wastage of transfusable plasma.