Storage of whole blood for up to 24 hours at ambient temperature before component preparation: implementation in the Netherlands.

The aim of this article is to reflect on the historical background on what urged the authors in the 1980s to investigate the prolonged storage of whole blood at ambient temperature before component preparation, the routine implementation of the method in the Netherlands, and the effect on logistics and plasma procurement.

Pooled platelet concentrates: an alternative to single donor apheresis platelets?

Three types of platelet concentrates (PC) are compared: PC either processed with the platelet-rich plasma (PRP) or the Buffy coat (BC) method from whole blood units and PC obtained by apheresis. Leuko-reduction (LR) pre-storage is advocated to improve quality of the PC during storage and reduce adverse reactions in recipients. Standardization of methods allow preparation of PC with comparable yields of approximately 400 x 10(9) platelets in pooled non-LR-PRP, approximately 370 x 10(9) in pooled LR-BC-PC and in LR apheresis PC the number of platelets can be targeted on 350 x 10(9) or more with devices of various manufacturers. While viral transmission can be prevented by outstanding laboratory tests, the risk of bacterial contamination should be reduced by improved arm disinfection, deviation of the first 20-30 ml of blood and culture or rapid detection assays of the PC pre-issue. In a large prospective multicenter trial no significant difference was observed between cultures of apheresis PC (n = 15,198): 0.09% confirmed positive units versus 0.06% in pooled BC-PC (n = 37,045), respectively. Though platelet activation as measured by CD62 expression may differ in vitro in PC obtained with various apheresis equipment, and also between PC processed with the two whole blood methods there is scarce literature about the clinical impact of these findings. In conclusion the final products of LR-PC derived from whole blood or obtained by apheresis can be comparable, provided the critical steps of the processing method are identified and covered and the process is in control.

The effect of plastic overwraps on storage measures of red cell concentrates.

BACKGROUND: For hygienic purposes, plastic overwraps can be used for storage of leucoreduced red cell concentrates (LR-RCC). However, the effect of the use of such overwrapping on in vitro parameters during 42 days of storage was unknown. METHODS: In paired experiments, LR-RCCs in SAGM (saline, adenine, glucose, mannitol) were packed in two types of polyethylene overwrap or in a polypropylene overwrap; no overwrap served as reference (n = 12 paired experiments). Units were stored at 2-6 degrees C for 42 days and sampled at regular intervals for in vitro analysis. RESULTS: No significant effect was found for any of the overwraps investigated. All units contained > 2.7 micromol adenosine triphosphate per gram haemoglobin and had a haemolysis rate well below 0.8% on Day 42. CONCLUSION: The use of plastic overwraps does not affect red cell quality markers in vitro.

Metabolic energy reduction by glucose deprivation and low gas exchange preserves platelet function after 48 h storage at 4 degrees C.

INTRODUCTION: We showed earlier that metabolically suppressed platelets (MSP) prepared by incubation in glucose-free, antimycin A medium at 37 degrees C better sustained storage at 4 degrees C than untreated controls at 22 degrees C. However, the use of the mitochondrial inhibitor antimycin A is incompatible with platelet transfusion. OBJECTIVES: The aim of this study was to investigate how energy-reduced (ER) platelets could be prepared in the absence of antimycin A. STUDY DESIGN AND METHODS: Platelets in gas-impermeable bags in glucose-free medium were kept at 22 degrees C for 4 h to reduce energy stores and thereafter stored at 4 degrees C (ER22-4). Controls were energy-reduced platelets without prior incubation at 22 degrees C (ER4), and MSPs in test tubes and untreated platelets in gas-permeable bags with glucose and stored at 22 degrees C (C22) and 4 degrees C (C4). RESULTS: After 48 h storage, ER22-4 were superior to C22 with respect to pH preservation (6 x 4 +/- 0 x 4 vs. 5 x 0 +/- 0 x 4, n= 4), platelet count (800 +/- 225 vs. 650 +/- 150 x 10(9)), thrombin receptor-activating peptide-induced aggregation (50 +/- 15 vs. 10 +/- 5%) and glycoprotein (GP)Ib alpha expression (60 +/- 15% vs. 28 +/- 15). GPIb alpha expression was higher in ER22-4 than in ER4, indicating that energy suppression preserved GPIb alpha during cold storage. CONCLUSION: Metabolic suppression without the use of antimycin A could be mimicked by storage of platelets in glucose-free medium in gas-impermeable bags. Energy suppression preserved GPIb alpha expression during storage at 4 degrees C.

Guidelines for the transfusion of platelets.

Recommendations aiming at standardising and rationalising clinical indications for the transfusion of platelets in Belgium were drawn up by a working group of the Superior Health Council. To this end the Superior Health Council organised an expert meeting devoted to "Guidelines for the transfusion of platelets" in collaboration with the Belgian Hematological Society. The experts discussed the indications for platelet transfusions, the ideal platelet concentrate and the optimal platelet transfusion therapy. The recommendations prepared by the experts were validated by the working group with the purpose of harmonising platelet transfusion in Belgian hospitals.

Sterilization method of platelet storage containers affects in vitro parameters.

BACKGROUND AND AIM: Four butyryl trihexyl phthalate plasticized polyvinyl chloride (BTHC-PVC) containers were compared for storage of leukoreduced platelet concentrates (LR-PC): three ethylene oxide (EtO) sterilized (Gambro, Haemonetics, Fresenius), and one steam-sterilized (Fresenius). METHODS: LR-PCs were made from 5 buffy coats and 300 ml Composol additive solution, and leukoreduced by filtration. Four LR-PCs were pooled and subsequently divided over the 4 BTHC-PVC bags to prevent donor-dependent differences, and sampled for in vitro analysis on day 1, 2, 5, 7 and 9. RESULTS: The pH values on day 9 were (mean +/- SD, n = 10): 7.12 +/- 0.03 (Gambro), 7.12 +/- 0.04 (Haemonetics), and 7.07 +/- 0.09 (Fresenius, EtO-sterilized) (not significantly different), vs. 6.91 +/- 0.12 (Fresenius, steam-sterilized; P < 0.001 vs. all EtO-sterilized bags). LR-PCs stored in the steam-sterilized bag exhibited significantly higher glucose consumption and lactate production (P < 0.001 vs. all EtO-sterilized bags). CONCLUSION: All BTHC-PVC containers allow storage of LR-PCs for up to 9 days with good in vitro parameters. However, the method of sterilization affects the storage conditions of the LR-PCs in these bags.

An evaluation of automated blood collection mixers.

BACKGROUND AND OBJECTIVES: We investigated the mixing capacity of two whole blood (WB) collection mixers. MATERIALS AND METHODS: WB was simulated by using a 25% glycerol solution warmed to 35 degrees C. Citrate-phosphate-dextrose (CPD) anticoagulant of a collection system was stained with toluidine blue, and simulated WB was added at 30, 60 or 90 ml/min, respectively (n = 3 per flow speed). The optical density (OD) of 10-ml fractions was measured, and results are expressed as percentage of a well-mixed '100%-sample'. RESULTS: CompoGuard showed adequate mixing at all three flow speeds (average ODs 96-103%). HemoLight showed good mixing at 60 and 90 ml/min (ODs from 97 to 101%). At 30 ml/min, mixing appeared suboptimal, but still conformed to our requirements with ODs from 96% to 104%. CONCLUSION: Both mixers give sufficient mixing of whole blood with anticoagulant.

Optimal sampling time after preparation of platelet concentrates for detection of bacterial contamination by quantitative real-time polymerase chain reaction.

BACKGROUND AND OBJECTIVES: A universal quantitative real-time polymerase chain reaction (PCR), based on bacterial 16S rDNA, to detect bacterial contamination of platelet concentrates (PCs), was developed previously and compared with automated culturing. In the present study, this real-time PCR method was evaluated to determine the optimal sampling time for screening of bacterial contamination in PCs. MATERIALS AND METHODS: Routinely prepared PCs were spiked with suspensions of Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Propionibacterium acnes to 1, 10 and 100 colony-forming units (CFU)/ml and stored at room temperature for 7 days. The presence of bacteria in these PCs was monitored by quantitative real-time PCR. As a reference method (additional control), BacT/Alert automated culturing was used. For PCR, 1-ml aliquots were drawn from all (spiked) PCs on days 0, 1, 2, 3, 6 and 7 of storage. As a control, triplicate samples (10 ml) were inoculated into aerobic and anaerobic BacT/Alert culture bottles immediately after spiking (day 0) and after storage for 1, 2, 3, 6 or 7 days. RESULTS: With quantitative real-time PCR, all bacterial species tested were reproducibly detected on day 1 after spiking at original concentrations of 10 and 100 CFU/ml. Bacteria were also detected on day 1 from PCs spiked with an initial concentration of 1 CFU/ml, except for E. coli, which was detected in only one of the three samples and P. aeruginosa, for which analysis was not performed on day 1. With the reference method, bacteria were detected in culture bottles (inoculated on day 0) within a mean time of 20.1 h, with the exception of P. acnes which was detected at a mean time of 102.3 and 49.3 h (for original spiking concentrations of 10 and 100 CFU/ml respectively). CONCLUSIONS: PCR enables the rapid detection of low initial numbers of bacteria in PCs. For reliable detection, our results support that sampling of PCs for real-time PCR screening should not be carried out earlier than 1 day after preparation (48 h after blood collection). Importantly, the real-time PCR approach has the potential to be used before the release of PCs from the blood centre or shortly before they are transfused in the hospital.

Amplified-fragment length polymorphism analysis of Propionibacterium isolates implicated in contamination of blood products.

Propionibacterium acnes is implicated in most cases of bacterial contamination of platelet concentrates (PCs). To determine the source of contamination, amplified-fragment length polymorphism (AFLP) analysis was applied. This DNA fingerprinting technique was used to study the molecular relationship of 44 isolates derived from 22 PCs and 22 corresponding red blood cells concentrates (RBCs) from the same whole blood donations. The AFLP results together with sequencing analysis of the 1,200 bp of the 16S ribosomal RNA gene revealed the existence of three main groups: two groups (groups 2 and 3) (55%) consisted of isolates that did not originate from skin flora and another group (group 1) (45%) comprised bacteria belonging to the skin flora. This latter group showed complete homology with reference strains of P. acnes. Therefore these isolates can be considered as P. acnes strains. In contrast, contaminants from groups 2 and 3 were shown to be molecularly unrelated to the P. acnes found on the skin surface. The AFLP is reproducible and gave invaluable information about the nature of Propionibacteria contaminating PCs. To gain more insights into the source of contamination, this technique could be exploited in further studies to determine the molecular relationship of different bacteria commonly found in blood products.

Gamma irradiation does not affect 7-day storage of platelet concentrates.

BACKGROUND AND OBJECTIVES: Extension of storage of leucocyte-reduced platelet concentrates (LR-PCs) to 7 days has recently been introduced, but the effect of gamma irradiation on in vitro parameters was unknown. MATERIALS AND METHODS: A paired experiment was carried out, with gamma irradiation on day 1 or day 5 with 25 Gy, and no irradiation served as a reference. RESULTS: Measurements during storage up to 8 days after blood collection showed no significant differences of irradiated LR-PCs vs. the control. CONCLUSIONS: Irradiation, either on day 1 or day 5, had no effect on platelet quality during a 7-day storage period.

White blood cell fragments in platelet concentrates prepared by the platelet-rich plasma or buffy-coat methods.

BACKGROUND AND OBJECTIVES: White blood cell (WBC) fragments in platelet concentrates (PCs) may induce allo-immunization in the recipient. MATERIALS AND METHODS: As the level of WBC fragments can differ between PCs produced using different methods, we compared PCs prepared by using the buffy-coat method (BC-PCs) in plasma or platelet additive solution (Composol) and PCs prepared using the platelet-rich plasma method (PRP-PCs). RESULTS: Post-filtration results revealed identical levels of WBC, but significantly higher CD62p expression and a significantly lower amount of total DNA, cell-free DNA and number of WBC fragments (0.6 vs. 4.2 WBC equivalents/microl) in PRP-PCs than in BC-PCs in either plasma or Composol. CONCLUSIONS: The number of WBC fragments is significantly higher in BC-PCs than in PRP-PCs.

Development of white blood cell fragments, during the preparation and storage of platelet concentrates, as measured by using real-time polymerase chain reaction.

BACKGROUND AND OBJECTIVES: White blood cell (WBC) fragments may cause human leucocyte antigen (HLA) immunization in recipients. We investigated the occurrence and production of WBC fragments in platelet concentrates (PCs) and plasma units, during storage and filtration, by using real-time polymerase chain reaction (PCR) and flow cytometry. MATERIALS AND METHODS: To study the occurrence of WBC fragments, 'male' WBCs were spiked into double-filtered 'female' PCs in a concentration series of 0.03-100 WBCs/microl (n = 4 per level). To study the production of WBC fragments, 'male' WBCs were spiked into 'female' plasma units to 4 x 10(9) WBCs/l and stored at room temperature prior to filtration (n = 4 per storage time; t = 0, 24 or 48 h). DNA was measured by both albumin real-time PCR and Y real-time PCR. Intact WBCs were counted by using flow cytometry. The number of WBC fragments was calculated by subtracting cell-free DNA (real-time PCR on supernatant) and intact WBCs (flow cytometry) from the total DNA amount (real-time PCR). RESULTS: Spiking of 'male' WBCs into 'female' PCs showed that the Y real-time PCR is linear and has a reproducible quantitative range down to 0.03 WBC/microl, but that the albumin-PCR, in unspiked samples, revealed a total of 6-10 WBC equivalents/microl (eq/microl). After centrifugation, half of this was observed as cell-free DNA in the supernatant, suggesting that the remaining DNA is derived from WBC fragments. The number of intact WBCs, amount of cell-free DNA and number of WBC fragments after filtration increased significantly when filtration was delayed for up to 48 h, from 0.1 WBC/microl, 1.3 WBC eq/microl and 0.6 WBC eq/microl at t = 0 h to 25 WBC/microl, 38 WBC eq/microl and 57 WBC eq/microl at t = 48 h, respectively. CONCLUSIONS: WBC fragments occur in WBC-reduced PCs and increase when products are stored, prior to filtration, up to levels that are equivalent to the amounts of intact WBCs that induce HLA immunization (i.e. > 5 x 10(6)/unit).

Correlation between the extent of platelet activation in platelet concentrates and in vitro and in vivo parameters.

BACKGROUND AND OBJECTIVES: Platelet activation, which is necessary to stop bleeding, also occurs in vitro during the storage of platelet concentrates (PCs). However, it is unknown whether in vitro-activated platelets are able to reduce blood loss in the patient. We studied correlations between platelet activation in PCs and in vitro parameters (pH, platelet count, swirling effect, storage time). In addition, we studied the correlation between platelet activation and in vivo parameters [the volume of thorax drain fluid as a measure of blood loss, platelet count, international normalized ratio (INR), and activated partial thrombin time (APTT)] in a clinical pilot study. MATERIALS AND METHODS: White blood cell-reduced PCs prepared from five buffy coats and one plasma unit (n = 55; storage time: median, 5 days; range, 2-12 days) were sampled. Platelet activation (CD62p, CD63, CD42b), as measured by flow cytometry, pH, platelet count, swirling effect and storage time, was determined. For the in vivo pilot study, PCs (n = 21) stored for 2-7 days were also checked for the above parameters and transfused into patients (n = 21) immediately after coronary artery bypass graft surgery. The volume of thorax drain fluid was measured for up to 12 h after surgery, and the platelet count, INR and APTT were measured < 1 h and 16-24 h postsurgery. RESULTS: A good correlation (r2 > 0.5) was observed between CD62p and CD63, between CD62p and CD42b, between CD62p or CD63 and pH and between CD62p or CD63 and swirling effect. Also, a significant increase in platelet activation was observed for PCs stored for > or = 8 days (mean +/- standard deviation: CD62p, 41.6 +/- 30.7; CD63, 23.8 +/- 18.6), compared to PCs stored for 2-7 days (mean +/- standard deviation: CD62p, 12.3 +/- 4.8; CD63, 10.4 +/- 3.6). No correlation (r2 < or = 0.1) was observed between platelet activation and the in vivo parameters. CONCLUSIONS: Although a correlation between platelet activation and in vitro parameters was observed, no correlation was found between platelet activation and in vivo parameters. Possible explanations for this are a too low variance in platelet activation in transfused PCs, and too small a number of patients.

In vivo PLT increments after transfusions of WBC-reduced PLT concentrates stored for up to 7 days.

BACKGROUND: Bacterial screening and improvement of storage conditions of leukoreduced PLT concentrates (LR-PCs) allows extension of their storage period from 5 to 7 days. STUDY DESIGN AND METHODS: For in vitro studies, 40 LR-PCs made from five buffy coats and plasma were studied for 8 days. For in vivo studies, routinely produced LR-PCs stored for 2 to 7 days after blood collection were administered to clinically stable thrombocytopenic patients. CI1 h was calculated after 353 transfusions (67 patients), and CCI1 h, after 195 transfusions (55 patients), with pretransfusion PLT counts of not greater than 20 x 10(9) per mL. RESULTS: Storage experiments showed that the pH of LR-PCs remained greater than 6.8 for 8 days, provided that the PLT concentration was less than 1.3 x 10(9) per mL. Routinely produced LR-PCs had a volume of 282 +/- 15 mL (n = 10,193) and contained 329 x 10(9)+/- 40 x 10(9) PLTs (n = 3467). For 7-day-old LR-PCs, 76 of 78 (97%) of the transfusions resulted in a CI1 h of at least 10 and 37 of 39 (95%) in a CCI1 h of at least 7.5, which indicated levels for successfulness. Mean +/- SE values of CI1 h and CCI1 h of 7-day-old LR-PCs were 28.7 +/- 2.3 (n = 78) and 19.0 +/- 2.0 (n = 39), respectively. No significant differences were observed between 5- and 7-day-old LR-PCs transfused with respect to CI1 h and CCI1 h values. CONCLUSION: In vitro and in vivo studies showed that LR-PCs can be stored for up to 7 days with excellent clinical results, provided that they are routinely screened for bacterial contamination.

Influence of cell-free DNA in plasma on real-time polymerase chain reaction for determination of residual leucocytes in platelet concentrates.

BACKGROUND AND OBJECTIVES: Real-time quantitative (RQ) polymerase chain reaction (PCR) can be used to determine the number of residual leucocytes in leucocyte-reduced platelet concentrates (LR-PCs), which should contain < 3.3 leucocytes/ micro l. In this study we investigated the extent to which cell-free DNA, known to be present in plasma, might interfere with this determination. In this study, RQ-PCR was employed to determine the following: the influence of filtration of platelet concentrates (PCs) on the amount of cell-free DNA; the variation in concentration of cell-free DNA between the buffy coats (BCs) of different donors; and the amount of cell-free DNA during storage and processing of whole blood. MATERIALS AND METHODS: PCs were sampled before and after filtration (n = 5), BCs were sampled (n = 100) and whole blood units were sampled < 2 h and 16-20 h after collection, and the BCs were also sampled after processing the whole blood (n = 10). Samples were centrifuged to obtain cell-free plasma in which the amount of cell-free DNA was determined using an RQ-PCR for the albumin gene. RESULTS: The amount of cell-free DNA was not influenced by filtration of the PCs [1.7 +/- 0.8 vs. 1.5 +/- 0.8 leucocyte-equivalents (eq)/ micro l]. However, the amount of cell-free DNA in plasma of the BCs varied considerably, from 0.1 to 18.2 leucocyte-eq/ micro l (median = 1.5 leucocyte-eq/ micro l; mean +/- SD: 2.2 +/- 2.4 leucocyte-eq/ micro l). In 18% of the BCs the amount cell-free DNA was > 3.3 leucocyte-eq/ micro l. The amount of cell-free DNA increased during storage, from 0.3 +/- 0.3 leucocyte-eq/ micro l (< 2 h after collection) to 0.9 +/- 0.6 leucocyte-eq/ micro l (16-20 h after collection) and, after processing the whole blood, to 2.0 +/- 2.0 leucocyte-eq/ micro l. CONCLUSIONS: Variable amounts of cell-free DNA in plasma will interfere if RQ-PCR is applied to estimate leucocyte numbers in leucocyte-reduced PCs.

Comparison of various dimethylsulphoxide-containing solutions for cryopreservation of leucoreduced platelet concentrates.

BACKGROUND AND OBJECTIVES: Leucoreduced platelet concentrates (LR-PCs) can be stored at 20-24 degrees C for 5-7 days. When LR-PCs are cryopreserved they can be stored for several years. For cryopreservation to become applicable in blood-bank practice, an off-the-shelf cryoprotectant is needed that can be added to the LR-PC in a sterile manner. For this, we varied the composition of the cryopreservation medium and studied various parameters of cryopreserved LR-PCs for up to 24 h after thawing at room temperature. MATERIALS AND METHODS: LR-PCs in plasma or Composol were concentrated and divided into 2 units. To each unit, an equal part of 10% dimethylsulfoxide (DMSO) in plasma, Composol with or without 5% albumin, or GPO (pasteurized plasma-protein solution) was added. Freezing occurred at 1 degrees C/min and LR-PCs were placed in the vapour phase of nitrogen. LR-PCs were thawed at 37 degrees C and stored at room temperature. LR-PCs were tested for morphology, platelet recovery, swirling effect, and activation antigens at various time-points thereafter. RESULTS: LR-PCs in 100%, 65% and 50% plasma supplemented with Composol showed good morphology scores (>250), limited CD62P expression (<35%), low CD63 expression (<20%) and a swirling effect of about 2, at 24 h after thawing. At the same time-point, platelet recovery was >80% under all conditions and CD42b expression varied between 70 and 85%. Results of LR-PCs in 15% plasma and Composol, with or without plasma substitutes, were not acceptable at 24 h after thawing, i.e. the morphology score was <200 and the CD62P expression was >40%. CONCLUSIONS: A minimum of 50% plasma in the cryopreserved LR-PC is necessary to maintain an acceptable in vitro quality of platelets up to 24 h after thawing. Composol is a good candidate for using to prepare an off-the-shelf cryoprotectant.