Laboratory assessment of the quality of adult and neonatal red cell concentrates manufactured from whole blood, exchange transfusion or intrauterine transfusion red cell units.

BACKGROUND: We evaluated the quality of red cell components in additive solution over 42 days of storage when re-manufactured from neonatal exchange transfusion (ExTx) or intrauterine transfusion (IUT) units on day 7 for issue to adults, neonates or infants. MATERIALS AND METHODS: Red cell concentrates (RCC) manufactured from WB were compared to RCC re-manufactured from ExTx or IUT on day 7, and red cell splits (RCS) manufactured from WB were compared to RCS re-manufactured from ExTx or IUT on day 7. All components were stored at 2-6°C and tested throughout storage until day 42 for in vitro parameters of red cell quality. One RCS manufactured from each of WB, ExTx or IUT, was irradiated on day 14 and tested on day 28 along with a non-irradiated RCS from the same unit. RESULTS: All the re-manufactured arms had no worse haemolysis, red cell microvesicle (RCMV) release or ATP over storage compared to controls. All arms complied with the 0·8% haemolysis UK specification, except for re-manufactured RCS from the IUT arm irradiated on day 14 and tested on day 28. Re-manufactured units had significantly decreased potassium levels compared to control over storage (P < 0·001 all). CONCLUSION: RCC or RCS re-manufactured from ExTx or IUT units on day 7 are suitable for transfusion up to the standard day 35 of storage. Re-manufactured RCS from ExTx units (but not IUT), may be irradiated up to day 14 and stored for 14 days post-irradiation.

In vitro quality of apheresis platelets divided into paediatric-sized units and stored in PVC bags plasticised with TOTM, BTHC or DINCH.

BACKGROUND AND OBJECTIVES: Two of the predictive factors of the quality of small volumes of platelets suitable for paediatric use are bag size and material. This study evaluated the storage properties of paediatric platelet aliquots in TOTM-, BTHC- or DINCH-PVC bags. METHODS/MATERIALS: (i) Three apheresis platelet concentrates (PC) were pooled and split into three units. One was retained as an adult unit (control; polyolefin bag). The second and third units were split into four MacoPharma TOTM-PVC and BTHC-PVC paediatric bags, respectively. (ii) Two apheresis PC were pooled and split into two units. One PC was retained as an adult unit, and the other was split into four Fresenius DINCH-PVC paediatric bags. Testing was performed on storage for pH, blood gases, hypotonic shock response, soluble CD62P, LDH, glucose and lactate, ATP, CD62P, CD63, platelet-derived microparticles and annexin V. RESULTS: The volumes, platelet yields and pH of all paediatric units met local specifications. The TOTM-PVC bag showed no worse quality than the adult bag up to day 7 for all parameters studied, and it maintained pH higher than BTHC-PVC and DINCH-PVC over storage. The BTHC-PVC bag was shown to be the most gas permeable; however, it had the highest glucose consumption rates and the highest platelet activation. CONCLUSION: All bags showed an acceptable in vitro quality. Overall, the TOTM-PVC paediatric bag showed better platelet quality compared to the other storage bags, whereas storage in the BTHC-PVC bag resulted in poorer platelet quality.

Randomized trial of red cell washing for the prevention of transfusion-associated organ injury in cardiac surgery.

BACKGROUND: Experimental studies suggest that mechanical cell washing to remove pro-inflammatory components that accumulate in the supernatant of stored donor red blood cells (RBCs) might reduce inflammation and organ injury in transfused patients. METHODS: Cardiac surgery patients at increased risk of large-volume RBC transfusion were eligible. Participants were randomized to receive either mechanically washed allogenic RBCs or standard care RBCs. The primary outcome was serum interleukin-8 measured at baseline and at four postsurgery time points. A mechanism substudy evaluated the effects of washing on stored RBCs in vitro and on markers of platelet, leucocyte, and endothelial activation in trial subjects. RESULTS: Sixty adult cardiac surgery patients at three UK cardiac centres were enrolled between September 2013 and March 2015. Subjects received a median of 3.5 (interquartile range 2-5.5) RBC units, stored for a mean of 21 ( sd 5.2) days, within 48 h of surgery. Mechanical washing reduced concentrations of RBC-derived microvesicles but increased cell-free haemoglobin concentrations in RBC supernatant relative to standard care RBC supernatant. There was no difference between groups with respect to perioperative serum interleukin-8 values [adjusted mean difference 0.239 (95% confidence intervals -0.231, 0.709), P =0.318] or concentrations of plasma RBC microvesicles, platelet and leucocyte activation, plasma cell-free haemoglobin, endothelial activation, or biomarkers of heart, lung, or kidney injury. CONCLUSIONS: These results do not support a hypothesis that allogenic red blood cell washing has clinical benefits in cardiac surgery. CLINICAL TRIAL REGISTRATION: ISRCTN 27076315.

Quality control of apheresis platelets: a multicentre study to evaluate factors that can influence pH measurement.

BACKGROUND AND OBJECTIVES: Blood operators routinely monitor the pH of apheresis platelets as a marker of the so-called storage lesion, which can result from manufacturing problems. It is also suspected that some donor characteristics can increase the risk of poor platelet storage. To explore this hypothesis, we analysed a large, multinational data set of quality control (QC) pH test results on apheresis platelets. MATERIALS AND METHODS: For the period between September 2011 and August 2014, seven blood operators in Canada, the USA, the Netherlands, the United Kingdom, France and Australia provided pH QC test results and donor characteristics on a total of 21,671 apheresis platelets. RESULTS: Some variations in pH distribution between blood operators were in part explained by differences in collection, processing and testing methods. Younger age and female gender were significantly associated with a pH value below the 10th percentile. Among donors who had two or more pH measurements (n = 3672), there was a strong correlation between pH results (r = 0·726; P < 0·0001). CONCLUSION: The strong intradonor correlation of pH measurements and the association between donor characteristics and pH results suggest that donor factors play a role in the quality of platelets.

Platelet responses to agonists in a cohort of highly characterised platelet donors are consistent over time.

BACKGROUND AND OBJECTIVES: Platelet function shows significant inheritance that is at least partially genetically controlled. There is also evidence that the platelet response is stable over time, but there are few studies that have assessed consistency of platelet function over months and years. We aimed to measure platelet function in platelet donors over time in individuals selected from a cohort of 956 donors whose platelet function had been previously characterised. MATERIALS AND METHODS: Platelet function was assessed by flow cytometry, measuring fibrinogen binding and P-selectin expression after stimulation with either cross-linked collagen-related peptide or adenosine 5'-diphosphate. Eighty-nine donors from the Cambridge Platelet Function Cohort whose platelet responses were initially within the lower or upper decile of reactivity were retested between 4 months and five and a half years later. RESULTS: There was moderate-to-high correlation between the initial and repeat platelet function results for all assays (P ≤ 0·007, r2 0·2961-0·7625); furthermore, the range of results observed in the initial low and high responder groups remained significantly different at the time of the second test (P ≤ 0·0005). CONCLUSION: Platelet function remains consistent over time. This implies that this potential influence on quality of donated platelet concentrates will remain essentially constant for a given donor.

Quality of red cells after combination of prion reduction and treatment with the intercept system for pathogen inactivation.

BACKGROUND AND OBJECTIVES: The pathogen inactivation (PI) INTERCEPT Blood System for Red Blood Cells utilises amustaline (S-303) to inactivate a broad range of pathogens in red cell concentrates (RCC). The aim of this study was to investigate the effect on red cell quality of INTERCEPT treatment with and without prion reduction. METHODS/MATERIALS: Five pools of five RCC each were prepared. These were split and treated as follows: (i) stored at 2-6 °C for 18 h, (ii) stored at 18-24 °C for 18 h, (iii) PI-treated, (iv) PI-treated then prion reduced and (v) prion reduced then PI-treated. Prior to storage, PI-treated RCC underwent an exchange step to remove S-303 and other breakdown products. Components were tested throughout 35 days of storage for in vitro parameters of red cell quality. RESULTS: All RCC met specification for volume and haemoglobin content. Haemolysis, microvesicle formation, supernatant potassium and deformability were lower and ATP levels higher in PI-treated units when compared with control units. The effect of prion reduction in addition to PI treatment was minimal in all parameters tested except haemolysis, which was increased in units prion-reduced after being PI-treated. CONCLUSION: The PI-treatment process did not increase red cell haemolysis or decrease ATP levels over storage. The lower haemolysis and supernatant potassium levels in treated RCC compared with control RCC were attributed to the exchange step. The effects of combining PI treatment and prion reduction were not more than additive when prion reduction precedes PI treatment.

Paired comparison of methylene blue- and amotosalen-treated plasma and cryoprecipitate.

BACKGROUND AND OBJECTIVES: Cryoprecipitate is used in the treatment of patients with acquired hypofibrinogenaemia. Studies have not directly compared cryoprecipitate produced following pathogen inactivation (PI) of fresh-frozen plasma (FFP) using different systems. The effects of methylene blue (MB) and amotosalen (AS) PI systems on the quality of FFP and cryoprecipitate were investigated in a paired study. MATERIALS AND METHODS: Seven group A and 7 group O pools of plasma were prepared and split into individual units and rapidly frozen to produce FFP. Units of FFP were thawed and either PI treated with MB or amotosalen, or left untreated (control). Samples of FFP along with the corresponding cryoprecipitate were tested for a range of coagulation factors, thrombin generation (TGT) and rotational thromboelastometry (ROTEM). RESULTS: AS-FFP showed a smaller decrease following treatment for most coagulation factors analysed than MB-FFP, except fibrinogen (antigen) and factor VII, partly due to lower volume losses. There was no significant difference between treatment methods for fibrinogen content of cryoprecipitate with treated units meeting current UK specification, or TGT and ROTEM parameters studied. CONCLUSIONS: MB-cryo contained a significantly higher content of FVIII and lower content of FXIII when compared to AS-cryo, with no difference in fibrinogen activity.

Storage of washed or irradiated red cells in AS-7 improves their in vitro characteristics.

BACKGROUND: AS-7 is a new alkaline hypotonic red cell additive solution (AS) shown to improve red cell quality during storage compared with AS-1. We sought to compare red cells stored in AS-7 with those stored in SAGM using RCC that were either untreated, or washed or irradiated on day 14 of storage. STUDY DESIGN AND METHODS: A pooled and split study design was used to produce seven identical RCC (four in SAGM and three in AS-7). At day 14 following donation, two RCC (one in SAGM and one in AS-7) were gamma irradiated and three RCC (two in SAGM and one in AS-7) were washed and resuspended in either SAGM or AS-7. RCC were sampled for analysis throughout storage and at end of shelf life: day 28 for washed or irradiated and day 35 for untreated RCC. RESULTS: For untreated, washed or irradiated RCC, those stored in AS-7 had lower haemolysis, red cell microvesicles and supernatant potassium content than RCC in SAGM. In addition, ATP levels and pH were better maintained in AS-7 RCC than in SAGM RCC. CONCLUSION: These data suggest that the quality of these components may be improved by storage in AS-7 compared with SAGM.

Thawed and liquid plasma--what do we know?

There is increasing interest in the use of liquid or frozen plasma thawed and stored for extended periods (>24 h) to reduce wastage and to improve rapid availability of plasma in massive transfusion protocols advocating the early use of plasma in trauma by some centres. There is now a body of studies that have assessed individual coagulation factors during storage of thawed plasma. These show that factor VIII (FVIII) is the worst affected factor and that its activity is mainly lost during the first 24 h following thawing. However, for most factors studied, there is a continual decline during further storage. The few studies that have assessed thrombin generation in thawed plasma have shown variable results. Extended storage of plasma is associated with an increase in levels of DEHP in the component and could theoretically increase the risk of bacterial contamination, although the latter does not appear to have been an issue in countries that have adopted the use of thawed plasma. There are no clinical studies relating to the efficacy of extended-thawed plasma, and therefore, the potential reduction in its efficacy must be balanced with the clinical need for the component.

Thrombin generation and coagulation factor content of thawed plasma and platelet concentrates.

BACKGROUND: We assessed the haemostatic capacity of thawed plasma produced after ambient storage of whole blood for 24 h (RTFP24), and the supernatant of buffy-coat derived platelet concentrates (PC). METHODS: Platelet concentrates (n = 20) were tested on days 1, 5 and 7 of storage at 22°C and RTFP24 (n = 10) immediately following thawing and after 4 and 6 days storage at 4°C. Coagulation factor activity, thrombin generation ± an activator of protein C (PROTAC) and rotational thromboelastometry (ROTEM) were assessed. RESULTS: In plasma and buffy-coat derived PC, there was a < 10% loss of factors II, IX and FX, but much higher loss of factors FV, FVII and FVIII. In plasma, the total or peak amount of thrombin generated was unaffected by storage for 6 days, with or without Protac, but there was an increase in lag time and decreased rate of clot formation by ROTEM. In PC, but not plasma, there was a 16% increase in FXII activity and increase in resistance to activated protein C, co-incidental to 30% loss of free protein S. CONCLUSIONS: These data suggest thrombin generation is relatively unaltered when RTFP24 is thawed and stored for 6 days, and that the supernatant of PC has significant haemostatic capacity.

Modern banking, collection, compatibility testing and storage of blood and blood components.

The clinical practice of blood transfusion has changed considerably over the last few decades. The potential risk of transfusion transmissible diseases has directed efforts towards the production of safe and high quality blood. All transfusion services now operate in an environment of ever-increasing regulatory controls encompassing all aspects of blood collection, processing and storage. Stringent donor selection, identification of pathogens that can be transmitted through blood, and development of technologies that can enhance the quality of blood, have all led to a substantial reduction in potential risks and complications associated with blood transfusion. In this article, we will discuss the current standards required for the manufacture of blood, starting from blood collection, through processing and on to storage.

The development and evaluation of options for improving future U.K. blood component labelling--outcome of the 2013 U.K. hospital survey.

BACKGROUND/OBJECTIVES: U.K. blood component labels have evolved to accommodate a plethora of information. Concern has, however, been expressed that current U.K. labelling is too 'cluttered', detracting from the clarity of critical information. This prompted a holistic review of labelling and available information technology (IT) with the aim of improving the situation. METHODS/MATERIALS: A survey was circulated requiring U.K. hospital participants to rank each item of information on the label according to its 'criticality' and assess three novel 'future' and one 'transition' prototype labels. Prototypes were based on applicable regulatory standards, best practice guidance, international benchmark data and U.K. expert input. The prototypes support steps towards 'full face' label printing and utilise 2D and quick response (QR) barcodes. RESULTS: Two-hundred eleven completed surveys were received identifying 110 contributing hospitals with 41% from clinical staff, 37% from transfusion laboratory staff and 22% from transfusion practitioners. There was excellent agreement between the three groups on the critical information, i.e., blood group, expiry date, blood component name, unique donation identification number (DIN) and blood component volume but far less on the other information, especially the various warning messages. Of the 'future' labels, option 3 (closest to the current 'quadrant model') was most popular. Option 1, with its additional inverted section replicating critical information was least popular and prompted significant safety concerns. CONCLUSION: The prototype labels correctly identified the critical items of information and extensive comments confirmed that this was more prominently and clearly displayed. Laboratory staff commented that the transition label was essential to enable IT systems to be adapted.

Ensuring that blood transfusion sets administer an effective dose of functional blood components.

BACKGROUND: Proposed changes to ISO 1135-4 will require that blood transfusion administration sets are demonstrated by the manufacturers to be suitable for the range of cellular and plasma blood components for which they are designated. AIMS: To design a test protocol to asses the depletion of the blood components by transfusion sets and damage and activation of blood components during their passage through the set. METHODS: Transfusion giving sets (CareFusion Ref no. 60895 180311 and Fresenius Ref no. 2900032) were assessed by comparing samples of the blood component taken prior to and after passage through the transfusion set in strict accordance with the manufacturer's instructions. As well as depletion of red cells, platelets and FVIIIc, the following markers of damage/activation were assessed: red cells-supernatant haemolysis and potassium; FFP-prothrombin fragments 1 and 2 and fibrinopeptide A and platelets-pH, CD62P, CD63 and sP-selectin. RESULTS: The CareFusion and the Fresenius transfusion sets gave less than 5% depletion of blood components and caused negligible and clinically insignificant effects on red cells, platelet concentrates and FFP. CONCLUSION: A practical test protocol has been established to assess the depletion, damage to and activation of the key constituents of commonly requested blood components. This protocol would provide a valuable addition to ISO 1135-4 in assuring the suitability of transfusion sets.

Lipaemic plasma induces haemolysis in resuspended red cell concentrate.

INTRODUCTION: We investigated whether haemolysis in red cells suspended in plasma was affected by the lipid content and/or methylene blue (MB) treatment of fresh-frozen plasma (FFP). We also investigated whether haemolysis was affected by the conditions under which lipaemic plasma was stored. METHODS: Study 1: Visibly lipaemic (n = 22) or nonlipaemic FFP (n = 24) units were thawed, pooled and split into identical pairs, one of which was MB treated. These units were used to resuspend red cell concentrates (RCC) and tested for haemolysis immediately and after 24 and 48 h of storage at 2-6°C. Study 2: Fresh plasma was aliquoted into 15-ml tubes and stored in one of four ways as follows: room temperature; 2-6°C; frozen and thawed; or twice frozen and thawed. A sample of RCC was resuspended in each of these plasmas and haemolysis measured after 2 h. Study 3: Plasma was divided into 15-ml tubes and stored as in study 2 followed by storage left standing upright in a refrigerator (2-6°C) for 24 h (with the exception of the room temperature sample). Plasma was separated into top, middle and bottom fractions and used to resuspend RCC that were assessed for haemolysis after 2 h. RESULTS: The levels of haemolysis in RCC were immediately greater when suspended in lipaemic plasma (0·70 ± 0·53% v 0·05 ± 0·06% for nonlipaemic plasma), which increased further on subsequent storage for 48 h (1·22 ± 0·40% v 0·15 ± 0·14% for nonlipaemic plasma). This was irrespective of whether plasma was MB treated. Lipaemic plasma stored frozen and then thawed resulted in the greatest haemolysis. In lipaemic plasma stored at 2-6°C, the chylomicron-rich top fraction caused the highest level of haemolysis. CONCLUSION: Haemolysis in red cells is increased in those suspended in lipaemic plasma and is dependent upon the storage conditions of that plasma prior to suspension. These data are relevant to the choice of plasma used to suspend red cells for neonatal exchange transfusion.

Repeated short-term warming of red blood cell concentrates has minimal effect on their quality.

BACKGROUND AND OBJECTIVES: Blood components must be stored under controlled temperature conditions, for reasons of component quality and safety. However, there are occasions when components may be exposed to conditions outwith the defined limits. This study aimed to generate prospective data on the effect of red cell exposure to extremes of temperature. MATERIALS AND METHODS: Study 1: red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAGM), made after ambient overnight hold of whole blood, were exposed to either +22°C or -2°C for up to three periods of 3 h on days 3, 8 and 15 of storage, followed by a 5 h exposure on day 29. Study 2: RCC in SAGM were exposed to 25°C for 24 or 48 h from day 2. In vitro markers of cell quality were measured during storage to 43 days, and compared with control units that had been stored at 2-6°C. RESULTS: Multiple short-term exposures to +22°C or -2°C did not cause any significant changes to pH, haemolysis, supernatant potassium, cellular ATP, 2,3-DPG, or deformability, when compared to control units. Exposure of RCC to 25°C for 24 or 48 h caused a significant fall in pH, ATP, and deformability. CONCLUSION: Red cells may be damaged by prolonged exposure to warm temperatures, but repeated short-term exposure to 22°C or -2°C does not appear to affect the in vitro quality of RCC. It is important to note that no bacterial growth studies were performed during this study.

Red cell concentrate storage and transport temperature.

BACKGROUND AND OBJECTIVES: This study investigated the current U.K. guidelines for storage and transport of red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAGM). The guidelines stipulate storage at 2-6 °C but allow exposure to between 1-10 °C core temperature in a single occurrence of less than 5 h and a surface temperature of 2-10 °C for no more than 12 h during transportation. METHODS AND MATERIALS: Twenty RCC units in SAGM were selected on the day of blood collection (day 0) and in vitro quality was tested pre- and post-temperature deviation at 10 °C and up to day 42 of storage. Each group of 10 RCC units was incubated for either 12 h or for both 5 and 12 h. RESULTS: Haemolysis was below the 0·8% U.K. limit at day 42 in all units, although there was an unexpected trend towards lower haemolysis in packs incubated for 5 and 12 h rather than just 12 h alone. Supernatant potassium was significantly higher than reference data on day 35 (P < 0·05) with a maximum of 58 mmol L(-1) and day 42 (P < 0·001). All units incubated at 10 °C had comparable levels of adenosine triphosphate and, 2,3-diphosphoglycerate to reference data from previous studies, throughout storage. CONCLUSION: These results suggest that exposure to 10 °C for 12 h or for 5 and 12 h did not adversely affect in vitro red cell quality for the remainder of the components shelf life.

Comparison of platelet activation in platelet concentrates measured by flow cytometry or ADVIA 2120.

BACKGROUND AND OBJECTIVES: The ADVIA 2120 Haematology Analyser is capable of measuring parameters that can be used as markers of platelet activation, mean platelet component (MPC), platelet component distribution width (PCDW) and mean platelet mass (MPM). This study investigated the degree of correlation of these measures of platelet granularity with CD62P measurement of platelet activation by flow cytometry in platelet concentrates. MATERIALS AND METHODS: Pooled platelets in plasma/citrate phosphate dextrose (CPD) anticoagulant or apheresis platelets in plasma/acid citrate dextrose formula A (ACD-A) anticoagulant were evaluated. Pooled platelets were tested during 13 day storage, and apheresis platelets within 24 h of venepuncture. These were assessed for platelet activation using CD62P and the ADVIA, with or without extra EDTA anticoagulant. RESULTS: In pooled platelets, PCDW correlated strongly with CD62P, both with and without the addition of extra EDTA anticoagulant. There was a good correlation between MPC and CD62P with additional EDTA, but a weaker correlation without extra EDTA. There was no correlation between CD62P and MPM. In apheresis platelets the correlation between PCDW and CD62P was poor, whereas MPC correlated strongly with CD62P if EDTA anticoagulant was added. CONCLUSION: The usefulness of ADVIA platelet granularity measures to predict the degree of platelet activation depends upon the anticoagulant present in the platelet concentrate, and whether extra EDTA is added to the sample. Although ADVIA MPC and PCDW measurement could not replace CD62P or other gold standard methods of assessing platelet activation, these ADVIA 2120 parameters may provide a quick check of platelet concentrate quality.

Effect of X-irradiation on the quality of red cell concentrates.

INTRODUCTION: The irradiation of blood components is used to inactivate T-lymphocytes to prevent transfusion-associated graft-versus-host (GVH) disease in susceptible recipients. X-irradiation can be used as an alternative to gamma irradiation and does not involve the use of a radioactive source. This study investigated the effect of X-irradiation on the quality of various red cell concentrates (RCC). METHODS: Whole blood units were processed into RCC in additive solution (SAG-M) or RCC in plasma suitable for intra-uterine (IUT) or neonatal exchange transfusion. RCC-SAG-M were irradiated at day 14 and sampled before and 0, 7 and 14 days following irradiation. RCC-IUT and RCC-Exchange were irradiated on day 4 and sampled before and 0 and 24 h following irradiation. Extracellular potassium levels and free plasma haemoglobin (haemolysis) were compared in X or gamma-irradiated units during storage. RESULTS: X-irradiation of RCC in SAG-M, or RCC for IUT or Exchange resulted in haemolysis comparable with gamma irradiation and levels were below the current UK/CE limit of 0.8% at the end of shelf life. X-irradiation of RCC-SAG-M or RCC-IUT with haematocrit of 75% and 85% resulted in potassium leakage comparable with gamma-irradiation. However, X-irradiation of RCC for neonatal exchange transfusion showed statistically significantly higher supernatant potassium levels after 24 h than gamma irradiation, although this difference is considered to be clinically insignificant. CONCLUSION: X-irradiation of RCC in SAG-M or IUT or RCC in plasma suitable for neonatal exchange transfusion resulted in acceptable levels of haemolysis and potassium leakage compared with the current process of gamma irradiation.