Vox Sanguinis (2014) © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12154


Quality and safety of red blood cells stored in two additive solutions subjected to multiple room temperature exposures  Ducas,1 M. Girard,1 M. Methot,1,2 M. Brien1,2 & L. Thibault1,2 M. J. de Grandmont,1 E. 1

He ma-Que bec, Operational Research, R&D, Quebec, QC, Canada Laval University, Department of Biochemistry, Microbiology and Bioinformatics, Quebec, QC, Canada


Background and Objectives Many international standards state that red blood cell (RBC) products should be discarded if left out of controlled temperature storage for longer than 30 min to reduce the risk of bacterial growth and RBC loss of viability. This study aimed to verify whether repeated short-time exposures to room temperature (RT) influence RBCs quality and bacterial proliferation. Materials and Methods Saline–adenine–glucose–mannitol (SAGM) and AS-3 RBC units were split and exposed to RT for 30 or 60 min on day 2, 7, 14, 21, and 42 of storage while reference units remained stored at 1–6°C. Red blood cell in vitro quality parameters were evaluated after each exposure. In a second experiment, SAGM and AS-3 RBC units were split and inoculated with Staphylococcus epidermidis (5 CFU/ml), Serratia marcescens (1 CFU/ml), and Serratia liquefaciens (1 CFU/ml). Reference units remained in storage while test units were exposed as described previously. Bacterial concentrations were investigated after each exposure. Results No differences were noticed between reference and test units in any of the in vitro parameters investigated. S. epidermidis did not grow in either reference or exposed RBCs. While S. marcescens did not grow in AS-3, bacterial growth was observed in RT-exposed SAGM RBCs on day 42. Similar growth was obtained for S. liquefaciens in the two additive solutions for both reference and test units.

Received: 22 September 2013, revised 13 March 2014, accepted 20 March 2014

Conclusion Short-time exposures to RT do not affect RBC quality and do not significantly influence bacterial growth. An expansion of the ‘30-minute’ rule to 60 min should be considered by regulatory agencies. Key words: 30-minute rule, bacterial growth in RBCs, red blood cell quality, storage lesions, temperature deviations.

Introduction To preserve red blood cells (RBCs) quality and to limit the risk of bacterial proliferation, handling of blood units should be performed under strict temperature controls. In Canada, the Canadian Standards Association (CSA) requires that RBC units must be stored at a temperature between 1 and 6°C for up to 42 days [1]. Moreover, the CSA and the Canadian Society for Transfusion Medicine Correspondence: Louis Thibault, Hema-Quebec, Operational Research, R&D, 1070, avenue des Sciences-de-la-Vie, Quebec, G1V 5C3 QC, Canada E-mail: [email protected]

state that RBC units that have been left out of the controlled temperature storage for more than 30 min cannot return to the inventory and must be discarded if their temperature reaches ≥10°C [1,2]. This ‘30-minute rule’, which has been implemented by many regulatory agencies, appears to origin from a study conducted by Pick and Fabijanic who demonstrated, in the early 1970s, that whole blood (WB) units, left at room temperature (RT), reached a core temperature of 10°C within 45–60 min [3]. It is interesting to note that this study does not present any data regarding the RBC quality or bacterial growth. Regarded as a universal standard, the 30-min rule has never been officially validated even though its application leads to significant logistical constraints for blood


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banks operations as well as substantial blood product wastage [4]. There is currently little evidence in the literature that the limit of 10°C displayed by several regulatory standards to limit excursions of RBC units outside controlled temperature storage is an acceptable threshold to maintain the quality and safety of packed RBC units. As a result, this standard has been considerably challenged. It has been demonstrated that RBC units stored at 10°C for 28 days presented no more indication of cell damage than did units stored at 4°C [5]. Recent studies on RBC warming have shown that RBC in vitro quality is not affected by single or multiple exposures to higher storage temperature [6–9]. Wagner et al. recently reported the acceptable in vitro quality of RBC units warmed up to 13°C for a maximum of 24 h, once a week during the 42 days of storage [10]. Thomas et al. have studied the in vitro parameters of RBCs exposed to 30°C for three periods of 30 or 60 min. The authors did not observe any significant increase in the rate of haemolysis in these units [11]. In addition, Ramirez-Arcos et al. did not report any additional risks of bacterial proliferation by exposing RBCs contaminated with mesophilic or psychrophilic microorganisms to RT for multiple periods of 30 and 60 min, suggesting the feasibility of an extension to 60 min [8]. There is a need to revise and adapt the ‘30-minute rule’ to modern blood processing, while ensuring the quality of blood products and the safety of patients [4]. There is a lack of published data concerning the quality of RBCs stored in AS-3 in the context of temperature deviation studies. More information about bacterial growth in RBCs subjected to multiple RT excursions is also required to ensure that blood product safety is not compromised if an extension of the ‘30-minute’ rule is considered. In this work, we investigated the impact of weekly RT exposures of 30 and 60 min of RBC units stored in AS-3 and SAGM (saline–adenine–glucose–mannitol) solutions on their in vitro parameters and bacterial proliferation risk.

Materials and methods Blood collection Informed consent was obtained from 24 healthy blood donors according to the standards state by our research ethics committee. Whole blood (WB) (450 ml) was collected either with 63 ml of citrate–phosphate–doubledextrose (CP2D) in Leukotrap WB system (Haemonetics, Braintree, VT, USA) or with 63 ml of citrate–phosphate– dextrose (CPD) in Atreus blood collection system (TerumoBCT, Zaventem, Belgium). Both blood collection systems were used according to manufacturer’s instructions.

Blood storage and component preparation To prepare RBCs stored in AS-3 additive solution, WB was stored overnight at 1–6°C. After leucoreduction performed with the collection set in-line filter, WB was centrifuged at 5147 g for 5 min. Following plasma extraction, 100 ml of AS-3 additive solution was added to RBCs. For SAGM RBCs, WB was rapidly chilled to 22 – 2°C after collection. Following overnight storage, WB was processed into blood components using the Atreus instrument (TerumoBCT). Red blood cells were suspended in 100 ml of SAGM and leucoreduced as per the manufacturer’s instructions. All RBC units were stored at 1–6°C.

Study 1 – Assessing the quality of warmed RBCs To evaluate the impact of short-term RT exposures of RBCs on their in vitro parameters, freshly processed AS-3 and SAGM RBC units were split into three portions (A–C) into 150-ml polyvinyl chloride (PVC) transfer bags (Baxter, Mississauga, ON, Canada) using a sterile connection device. Each portion of about 100 ml was next inserted between two 600-ml PVC bags filled, either side, with 50 ml of saline. This design was validated to reproduce the core temperature pattern of a 310-ml RBC unit exposed to RT (Fig. 1). On days 2, 7, 14, 21, and 42, bags A and B were exposed to RT for 30 or 60 min respectively, while bag C remained at 1–6°C (reference unit). At the end of each warming period, samples were taken from bags A–C to perform in vitro analysis and bags were returned to 1–6°C. This experiment was repeated six independent times for RBCs stored in both additive solutions. The design of the insulation device was validated with mock bags containing an identical volume of saline probed with a temperature sensor. The core temperature was monitored during RT exposures of 30 and 60 min using temperature logger devices (HOBO, Pacasset, MA, USA). Temperature profiles were compared to reference bags filled with 310 ml of saline. Saline solution has been used instead of blood to simplify the experimental design and because both fluids have similar heat capacities [12– 14]. In this work, the minor difference between WB and saline heat capacities results in a slightly faster heating rate for WB units. This difference has little impact on the heating time in our experimental setup, since it is within the range of the experimental error associated with temperature measurements.

Biochemistry and haematology analysis Glucose, lactate, sodium, potassium, pO2 and pCO2 were determined using a GEM 3000 analyzer (Instrumentation Laboratory, Bedfort, MA, USA). Complete blood counts © 2014 International Society of Blood Transfusion Vox Sanguinis (2014)

RBC multiple exposures to room temperature 3




Fig. 1 Insulation device containing a paediatric red blood cell unit. Front view (a) and view by the top (b). Core temperature was monitored using a temperature logger device (c).

were analysed with an ACP 5 diff AL haematologic analyzer (Beckman-Coulter, Miami, FL, USA). pH measurements were performed with a pH metre (Model Φ360; Beckman-Coulter, Fullerton, CA, USA), and plasma haemoglobin levels were measured with a HemoCue Plasma/ Low HB photometer (HemoCue, Angelholm, Sweden). The ATP and the 2,3–DPG levels were determined with commercial kits according to manufacturers’ instructions (Perkin Elmer, Waltham, MA, USA for ATP and ROCHE Diagnostics, Indianapolis, IN, USA, for 2,3-DPG). Red blood cell mechanical fragility was assessed according to Raval et al. [15].

Study 2 – Assessing bacterial proliferation Bacterial strains Staphylococcus epidermidis (ATCC49134), Serratia marcescens (ATCC43862), and Serratia liquefaciens (ATCC35551) were obtained from the American type and culture collection (ATCC, Manassas, VA, USA). These micro-organisms were selected because of their involvement in adverse transfusion reactions implying contaminated RBCs [16,17]. The two psychrophilic bacteria, S. marcescens and S. liquefaciens, grow well under refrigerated conditions, but could be impacted by warming. Conversely, S. epidermidis, a mesophilic bacterium, does not proliferate well in refrigerated blood products and exposures to RT could favour its proliferation. Although not isolated from contaminated blood samples, these three bacterial strains grow well in blood products and have been used by others in similar experiments [18,19]. © 2014 International Society of Blood Transfusion Vox Sanguinis (2014)

Study design To evaluate the impact of multiple warmings of RBCs on bacterial proliferation, units were split into three equal portions. Each of these portions was inoculated with 1–5 colony-forming units (CFU)/ml of either S. epidermidis, S. marcescens, or S. liquefaciens. Prior to inoculation, overnight bacterial cultures were serially diluted in either AS-3 or SAGM additive solution. An aliquot of the final dilution was used for spiking, and samples from spiked RBC units were plated in triplicate on blood agar plates to verify bacterial viability and concentration of the inoculum. The use of 1 or 5 CFU/ml inoculum can be questioned, but it refers to the World Health Organization guidelines which recommend to use a concentration as small as 003–03 CFU/ml for blood spiking experiments [20]. Immediately after inoculation, each bag was subdivided into three portions of about 34 ml (A, B, and C) into 150-ml transfer bags. These bags were placed between two PVC bags filled on either side with 110 ml of saline and stored at 1–6°C. On days 2, 7, 14, 21, and 42, bags A and B were warmed at RT for a period of 30 or 60 min, respectively. Bag C remained at 1–6°C. After each exposure, a sample was aseptically taken from bags A, B, and C, which were immediately returned to 1–6°C. Bacterial CFUs were determined by plating 100 ll or 500 ll of RBC samples in duplicate on blood agar plates followed by incubation at 37 – 2°C for S. epidermidis and S. marcescens or 26 – 2°C for S. liquefaciens. Colony counting was performed after 18–24 h of incubation. This experiment was repeated on six independent times for RBCs stored in both additive solutions.

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Results Temperature pattern of RBCs during ambient temperature exposures Figure 2 depicts the typical temperature pattern of mock saline bags that was used to validate the experimental system of this work. This device mimics the core temperature profile of routine RBC units exposed to RT. Reference bags filled with 310 ml of saline reached a temperature of 110 – 04°C and 148 – 06°C when exposed to ambient temperature for a period of 30 or 60 min, respectively. According to thermodynamic principles, a 310-ml whole blood unit should have reached theoretical temperatures of 113°C and 149°C when exposed to RT for a period of 30 or 60 min, respectively, which is very similar to temperatures reached by saline bags. Using 100-ml bags in Study 1, average temperatures of 118 – 05°C and 151 – 02°C were obtained after RT exposures of 30 and 60 min, respectively. With the design used in Study 2, bags filled with 34 ml of saline reached a temperature of 118 – 05°C and 151 – 04°C after the 30-min and 60-min RT exposures, respectively. No statistically significant differences were observed between the three temperature profiles (P < 012), indicating that our experimental design reproduces well the core temperature variation of a routine 310–ml product.

RBC quality is not affected by multiple RT exposures Some results of the in vitro assays of Study 1 are presented in Fig. 3 for AS-3 RBCs and in Fig. 4 for SAGM RBCs. Glucose, ATP, sodium, 2,3-DPG concentrations, and RBC mechanical fragility decreased with storage time. Levels of lactate, potassium, and haemolysis increased gradually during storage. End-of-storage haemolysis was slightly greater in SAGM compared to AS-3 RBCs, but remained below the acceptable limit of 08% in all products. Levels of 2,3-DPG are greater in AS-3 than in SAGM on day 2. Notwithstanding those observations, no

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(b) 18 Temperature (°C)

Values are presented as averages – standard deviations. Generalized estimating equations (GEE) were used to analyse the temperature pattern. To compare each storage condition for the level of in vitro parameters and bacterial concentrations, statistical analyses were carried out using a two-way analysis of variance (ANOVA). All analyses were performed using the computer software SAS Enterprise guide 4.1.3 (SAS Institute, Cary, IL, USA). Values were considered significantly different if P values were

Quality and safety of red blood cells stored in two additive solutions subjected to multiple room temperature exposures.

Many international standards state that red blood cell (RBC) products should be discarded if left out of controlled temperature storage for longer tha...
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