Influence of prestorage leukoreduction and subsequent irradiation on in vitro red blood cell (RBC) storage variables of RBCs in additive solution saline-adenine-glucose-mannitol

BACKGROUND: There exists only very few data on in vitro and in vivo effects of gamma irradiation of red blood cells (RBCs) that have been leukoreduced by filtration before a subsequent irradiation. Reported studies reflect neither the current Food and Drug Administration (FDA) nor the European recommendations on timing of irradiation and subsequent storage.
STUDY DESIGN AND METHODS: We studied 40 RBC units that were prepared from inline filtered whole blood and 40 RBC units that were filtered after component separation. All RBCs were stored in the additive solution saline-adenine-glucose-mannitol and leukoreduced on the collection day. In both groups, 20 components were irradiated on Day +14 with 30 Gy, and 20 served as nonirradiated controls. In vitro evaluation of both irradiated and nonirradiated RBC units was performed before and after irradiation on Days +1, +7, +14, +21, +28, +35, and +42 from the collection day.
RESULTS: Gamma irradiation induced enhanced leakage of potassium ions and lactate dehydrogenase and an enhanced in vitro hemolysis rate in the irradiated components. However, in vitro hemolysis rate of both nonirradiated and irradiated components was remarkably lower than 0.8 percent, and the preservation of adenosine triphosphate over 42 days was satisfying.
CONCLUSIONS: This study reflects the current FDA and European recommendations on timing of irradiation and subsequent storage. Our findings together with recent results of other investigations on the effect of gamma irradiation on leukoreduced RBCs allow the proposal that a storage time up to 28 days after irradiation is allowable.     

Anaerobic storage of red blood cells in a novel additive solution improves in vivo recovery

BACKGROUND: In preliminary studies, anaerobic red blood cell (RBC) storage reduced oxidative damage and phosphatidylserine exposure while maintaining adenosine triphosphate levels. The purpose of this study was to compare the 24-hour recovery and life span of autologous RBCs stored 6 and 9 weeks using OFAS3 additive solution in an anaerobic environment, compared to control RBCs aerobically stored in AS-3 for 6 weeks.
STUDY DESIGN AND METHODS: Eight subjects were entered into a randomized, crossover study. Whole blood was collected from each subject twice separated by 12 weeks or more into CP2D and leukoreduced. Controls were stored in AS-3. Test units in OFAS3 were oxygen depleted with argon then stored 9 weeks in an anaerobic chamber at 1 to 6°C. At the end of each storage period, RBCs were labeled with ⁵¹Cr and ^99mTc and reinfused to the subject following standard methods to determine double-label recovery and life span. Hypotheses tests were conducted using paired, repeated-measures analysis of variance.
RESULTS: Recovery for the anaerobically stored test RBC was significantly better than control at 6 weeks (p = 0.023). Test units at 9 weeks were not different than the 6-week control units (p = 0.73). Other in vitro measures of RBC characteristics followed the same trend. Two test units at 9 weeks had hemolysis of greater than 1 percent.
CONCLUSION: Anaerobically stored RBCs in OFAS3 have superior recovery at 6 weeks compared to the controls and equivalent recovery at 9 weeks with no change in life span. Anaerobic storage of RBCs may provide improved RBCs for transfusion at 6 weeks of storage and may enable extending storage beyond the current 42-day limit.     

A prospective, double-blind, randomized clinical feasibility trial of controlling the storage age of red blood cells for transfusion in cardiac surgical patients

BACKGROUND: Recent evidence demonstrates an association between duration of storage of red blood cells (RBC) and morbidity and mortality after cardiac surgery. We studied the feasibility of two different schemes for categorizing and randomizing age of RBC units transfused in cardiac surgical patients.
STUDY DESIGN AND METHODS: In Phase 1, 20 subjects were randomly assigned to standard of care (SOC) versus no RBCs with more than 21 days' storage duration. In Phase 2, 23 subjects were randomized to RBCs of 7 ± 4 versus 21 ± 4 days' storage duration. The age of study RBC units was masked.
RESULTS: In Phase 1, no patients received RBCs 31 days or older in SOC, and there was overlap in storage age shared in both arms so the predefined feasibility criteria were not met. In Phase 2, it was feasible to deliver specified age RBCs to the 7-day arm (achieved in 100% of subjects), but feasibility was not demonstrated for the 21-day arm (only 50% of subjects transfused with target age RBCs). Significant differences, however, were observed between the 7 ± 4- and 21 ± 4-day arms with respect to age of all RBC units (6 ± 2 vs. 18 ± 7, p = 0.0002) and maximum age (7 ± 2 vs. 20 ± 7, p < 0.0001).
CONCLUSION: Given the current storage age distribution of available RBC inventory, use of a SOC arm in future studies is unlikely to result in a large exposure to "old" blood. It is feasible to randomize patients to "younger" RBCs (3-11 days) but design strategies are needed to provide "intermediate-aged" or "old" blood as a comparator.     

In vitro and in vivo evaluation of LEUKOSEP HRC-600-C leukoreduction filtration system for red cells

BACKGROUND: Documentation of the benefits of leukoreduction has led to the increased use of this technique and the need for development of efficient and effective techniques for its accomplishment. This study investigated the in vitro properties and in vivo autologous radiolabeled recovery of leukoreduced red cells (RBCs) produced through a leukoreduction filtration system for RBCs (LEUKOSEP HRC-600-C, Hemerus Medical). STUDY DESIGN AND METHODS: Normal subjects donated 36 units of RBCs that were leukoreduced on Days 0, 3, or 5 through a "hands-off" technique. Biochemical studies were performed before and after filtration and at the end of 42 days of storage. Units leukoreduced on Days 0 or 5 were held until Day 42 and used for autologous radiolabeled return to determine recovery with 51Cr single-label radiolabeling techniques. RESULTS: Leukoreduction filtration was accomplished in 16.3 +/- 2 minutes on Day 0 at room temperature or 27 to 30 minutes on Days 3 or 5 after refrigeration. Leukoreduction efficiency was 4.6 +/- 0.6 log with a median residual white blood cell (WBC) content of fewer than 3.3 x 10(4) WBCs per unit. RBC recovery was 90 +/- 2 percent. Hemolysis was 0.34 +/- 0.16 percent at the end of 42 days of storage. The in vivo recovery of radiolabeled RBCs 24 hours after autologous return was 80.6 +/- 4.5 percent for RBC units leukoreduced on Days 0 and 5 combined. CONCLUSION: The LEUKOSEP HRC-600-C WBC reduction filtration system produced leukoreduced RBCs efficiently and effectively with acceptable poststorage biochemical measures and posttransfusion recovery after 42 days of storage.     

Cytokine generation in stored, white cell-reduced, and bacterially contaminated units of red cells

BACKGROUND: Proinflammatory cytokines were measured in the supernatant portion of stored, bacterially contaminated, and/or white cell (WBC)- reduced units of red cells (RBCs). Previous studies from this laboratory and others have shown that cytokines are generated in platelet concentrates during storage. This earlier work has been expanded to the study of stored RBCs. STUDY DESIGN AND METHODS: Units of AS-1 RBCs (n ? 10 non-WBC-reduced; n ? 10 WBC-reduced) were obtained from a regional blood center, and each was split on Day 3 of storage into three equal portions by sterile techniques. One portion was kept sterile (control), and the other two were inoculated with Yersinia enterocolitica and Staphylococcus aureus, respectively, at 1 to 3 colony-forming units per mL. The RBCs were stored at 1 to 6 degrees C for 42 days. Sequential samples were taken during storage and assayed for interleukin 8 (IL-8), interleukin 1 beta (IL-1 beta), interleukin 6, WBC count, and bacteria count. For the WBC-reduced group (n ? 10), WBC removal was done by filtration on Day 3 of storage, before bacterial inoculation. RESULTS: IL-8 was detected in the supernatant portion of all 42-day-old, non-WBC-reduced (mean WBCs ? 4760 ± 3870/μL) units of AS-1 RBCs at levels ranging from 63 to 1610 pg per mL. By contrast, at 2 to 3 days of storage, lower levels of IL-8 (range, 0-280 pg/mL) were detected in the same units. IL-8 levels increased progressively during storage in most (7/10) units. The highest mean levels of IL-8 were reached by outdate at Day 42. Y. enterocolitica-contaminated units had statistically higher levels of IL- 8, with a range of 170 to 2100 pg per mL, by 42 days of storage. S. aureus grew poorly in stored units of RBCs and failed to further stimulate cytokine production. No WBC-reduced unit (mean WBCs ? 0.5 ± 0.6/μL), even when contaminated with bacteria, had more than 260 pg per mL of IL-8. Although IL-1 beta was not detected in any unit of RBCs at 3 days of storage, it increased to low levels (5-13 pg/mL) in all units tested at 42 days. Interleukin 6 was not detected in any unit at any storage time. CONCLUSION: IL-8 and IL-1 beta accumulated in the supernatants of stored RBCs despite cold storage conditions. IL-8 reached levels > 1000 pg per mL in the supernatants of some RBC units. IL-1 beta increased to significant but low levels (< 13 pg/mL). WBC filtration early in storage prevented the accumulation of IL-8. The physiologic significance to transfusion recipients of IL-8 in RBC supernatants is currently unknown and deserves further investigation.     

Core temperature changes in resuspended red blood cells (RBCs) and pediatric RBCs removed from refrigerated storage

BACKGROUND: The 30-minute rule, whereby intact red blood cell (RBC) products may be returned to stock if returned to 4°C storage within 30 minutes of issue, was established many years ago. It was based on observations that the core temperature of units of whole blood removed from storage temperatures of 1 to 6°C, and left at room temperature, would reach 10°C at between 45 minutes and 1 hour.
STUDY DESIGN AND METHODS: Forty-one units of RBCs resuspended leukoreduced and 8 units of pediatric RBCs resuspended leukoreduced were exposed to ambient temperature for periods of time between 0 and 60 minutes. Core temperatures of all units were measured at 1-minute or 5-minute intervals.
RESULTS: Resuspended RBCs units reached a mean core temperature of 10°C at 15 minutes, 12.7°C at 30 minutes, and 15°C at 60 minutes. Pediatric RBCs reached a mean core temperature of 12.8°C at 15 minutes, 15.5°C at 30 minutes, and 17.8°C at 60 minutes.
CONCLUSION: In view of our results, and the range of RBC products now available, it may be timely for blood services to review and reduce the 30-minute rule.     

Stored red blood cell supernatant facilitates thrombin generation

BACKGROUND: Observational studies have reported that patients transfused with red blood cells (RBCs) have a worse clinical outcome than untransfused patients and that storage age of RBCs at the time of transfusion may be an independent predictor of this adverse clinical outcome.
STUDY DESIGN AND METHODS: Eight RBC concentrates in additive solution were studied over an 8-week storage period. The RBC supernatant was ultracentrifuged to concentrate microvesicles (RCMVs). RCMVs were studied by flow cytometry to identify phosphatidylserine (PS)-expressing RCMVs and in a thromboelastograph (TEG) using a modified assay to detect a thrombin facilitation effect.
RESULTS: For all products, the percentage of RCMVs that exhibited PS expression on Day 1 was 50 ± 13%, which increased with storage, and on Day 31 was 90 ± 4%. After 31 days, four of the eight products showed a thrombin facilitation effect as evidenced by a shortening of the TEG reaction (R) time of 1.3 ± 1.1 minutes, which persisted to Day 41. Data are the mean ± 1 SD. This TEG R shortening effect was neutralized by annexin V. No such effect was observed on, or before, Day 21.
CONCLUSIONS: Some stored RBCs release RCMVs, which express PS and are capable of facilitating thrombin generation in vitro. This provides a possible mechanism by which stored RBCs could promote adverse thrombotic or inflammatory effects.     

Level of platelet-derived cytokines in leukoreduced red blood cells is influenced by the processing method and type of leukoreduction filter

BACKGROUND: In contrast to the well-documented effect of white blood cells on the quality of red blood cells (RBCs), the effect of platelets (PLTs) has received little consideration. In this study, the PLT content and level of PLT-derived cytokines in RBCs prepared using different types of leukoreduction methods were investigated.
STUDY DESIGN AND METHODS: Buffy coat–poor RBCs and five types of leukofiltered (LF) RBCs, including RBCs prepared with a whole blood (WB) PLT-saving filter, were prepared and stored according to standard blood bank conditions. PLT content was measured on Day 1, and levels of PLT-derived cytokines were measured by enzyme-linked immunosorbent assay at nominated timepoints during 42 days of storage.
RESULTS: The PLT content of leukoreduced RBCs varied widely depending on the processing method and/or leukoreduction filter used, with some types of RBCs containing very low PLT counts while other units contained PLT counts comparable to those of unprocessed WB. The PLT content of RBCs directly influenced the concentration and accumulation of PLT-derived cytokines. Several PLT-derived factors exhibited significant accumulation throughout 42 days of storage. RBCs with high PLT content exhibited concentrations of RANTES (CCL5) and soluble CD40 ligand equivalent to those previously reported to show significant biologic and clinical effects.
CONCLUSION: The PLT content and levels of PLT-derived cytokines in leukoreduced RBCs are influenced by the processing method and types of leukoreduction filters used. It may be inappropriate to consider LF-RBCs prepared with different types of leukoreduction filters as equivalent products based on their differing levels of PLT factors.     

Supernatant from stored red cells activates neutrophils

Bioreactive substances including cytokines and lipids accumulate during storage of red blood cells (RBCs) but their clinical importance is uncertain. The goal of this study was to evaluate the effect of stored RBC supernatant on neutrophil activity in vitro . Packed RBCs (PRBCs) were collected and divided into two aliquots, one leukodepleted and the other nonleukodepleted. Plasma supernatant from PRBCs were collected on days 1, 8, 15, 29 and 35 and its effect on neutrophil expression of CD11b, CD16 and oxidative burst was measured by flow cytometry. Levels of tumour necrosis factor alpha (TNFα) and interleukin-8 (IL8) were also measured. The supernatant from PRBC units stored for greater than 15 days activated and primed neutrophils as evidenced by an increase CD11b and CD16 expression and oxidative burst. The greatest effect was seen in the oldest concentrates (35-day-old) ( P  < 0.008). Leukodepletion abrogated the effects of stored supernatant on CD11b and CD16 expression ( P  < 0.02) but did not reduce priming of the neutrophil oxidative burst ( P  > 0.1). Very low levels of IL8 and TNFα were detected in stored supernatants. Stored PRBC supernatant contains substances which directly enhance neutrophil expression of adhesion protein CD11b, CD16 and prime neutrophil oxidative burst. The exceedingly low level of IL8 and TNFα found in this study suggests that other factors may play a more important role in neutrophil priming and activation.     

Expression levels of CD47, CD35, CD55, and CD59 on red blood cells and signal-regulatory protein-alpha,beta on monocytes from patients with warm autoimmune hemolytic anemia

BACKGROUND: Animal models have shown that CD47-deficient mice develop severe autoimmune hemolytic anemia (AIHA) because the binding of red blood cell (RBC) CD47 to signal-regulatory protein (SIRP-α) on macrophages contributes to the inhibition of phagocytosis. In contrast, complement-inhibitory proteins such as CD35, CD55, and CD59 may protect RBCs against the lysis by complement.
STUDY DESIGN AND METHODS: With the use of flow cytometric analyses, the expression of CD47, CD35, CD55, and CD59 on RBCs and of SIRP-α,β on peripheral monocytes of 36 patients with warm AIHA (wAIHA; 23 with active wAIHA, 13 with wAIHA in remission) and 20 healthy subjects was evaluated.
RESULTS: The mean fluorescence intensities (MFIs) of the expression of CD47, CD35, CD55, and SIRP-α,β of active wAIHA patients, wAIHA in remission, and healthy subjects were not statistically different. Patients with active wAIHA showed significantly lower CD59 expression on RBCs than healthy individuals (MFI, 512.5 ± 59.6 vs. 553.7 ± 36.6; p = 0.009), while the CD59 expression in patients with wAIHA in remission was not significantly different from that of healthy controls (MFI, 538.4 ± 48.3 vs. 553.7 ± 36.6; p > 0.05). The expression of CD59 on RBCs of 3 patients who died from the wAIHA was lower than that seen on RBCs of healthy controls (MFI, 433.6 ± 69.6 vs. 553.74 ± 36.6; p = 0.0001).
CONCLUSIONS: Our data show that the expression of CD47 on RBCs and SIRP-α,β on monocytes of patients with wAIHA is not different from that seen in healthy individuals. In addition, we detected that patients with active wAIHA present low expression of CD59 and normal expression of CD35 and CD55 on their RBCs. Complement-regulatory proteins may play an important role in protecting RBC destruction through the activation of complement.     

Infusion of P-Capt prion-filtered red blood cell products demonstrate acceptable in vivo viability and no evidence of neoantigen formation

BACKGROUND: Transmission of variant Creutzfeldt‐Jacob disease (vCJD) is a major concern in blood transfusion. The P‐Capt filter has been shown to remove around 4 log ID₅₀ prion infectivity from prion‐spiked human red blood cells (RBCs). STUDY DESIGN AND METHODS: Two independent, single‐center, randomized, open‐label studies were designed to analyze the safety of P‐Capt–filtered RBCs. RBCs prepared from leukoreduced whole blood from 43 eligible subjects were randomly assigned to P‐Capt filtration and/or storage in plasma or SAGM and stored for 28 or 42 days. Stored RBCs were analyzed for in vivo 24‐hour recovery, hemolysis, metabolic variables, blood group antigen expression, neoantigen formation, and safety after autologous infusion. RESULTS: Mean P‐Capt filtration times for leukoreduced RBCs were 41 (SAGM) to 51 (plasma) minutes. Thirteen of 14 subjects receiving P‐Capt–filtered RBCs had 24‐hour RBC recoveries of 75% or more after 42‐day storage, with a mean hemolysis of less than 0.6%. No loss of RBC antigen expression or formation of neoantigens was observed. In both studies, RBCs had white blood cell counts of less than 1 × 10⁶/unit after leukofiltration. P‐Capt prion filtration provided an additional greater than 0.8 log leukoreduction. No serious or unexpected adverse events were observed after infusion of P‐Capt–filtered full‐volume RBC units. CONCLUSIONS: P‐Capt–filtered, stored RBCs demonstrated acceptable viability and no detectable neoantigen expression, immunogenic responses. or safety issues after infusion of a complete unit. The additional filtration time and modest reduction in RBC content are within acceptable levels for implementation in countries with transfusion transmission of vCJD.     

Rejuvenation of stored human red blood cells reverses the renal microvascular oxygenation deficit in an isovolemic transfusion model in rats

BACKGROUND: Storage of red blood cells (RBCs) results in various biochemical changes, including a decrease in cellular adenosine triphosphate and 2,3-diphosphoglycerate acid. Previously it was shown that stored human RBCs show a deficit in the oxygenation of the microcirculation in the gut of anesthetized rats. In this study, the effect of RBCs on rat kidney oxygenation and the effect of rejuvenation of stored RBCs on their ability to deliver oxygen were investigated.
STUDY DESIGN AND METHODS: Washed RBCs, derived from leukoreduced RBCs stored in saline-adenine-glucose-mannitol, were tested in an isovolemic transfusion model in rats after hemodilution until 30 percent hematocrit (Hct). The cells were derived from RBCs stored for up 3 days or from RBCs stored for 5 to 6 weeks with or without incubation in Rejuvesol to rejuvenate the cells. Renal microvascular oxygen concentrations (µPO₂) were determined by Pd-porphyrin phosphorescence lifetime measurements.
RESULTS: Isovolemic transfusion exchange of 5- to 6-week-stored RBCs resulted in a significantly larger decrease in renal µPO₂ than RBCs stored for up to 3 days: 16.1 ± 2.3 mmHg versus 7.1 ± 1.5 mmHg, respectively (n = 5). Rejuvenation of stored RBCs completely prevented this deficit in kidney oxygenation. The differences in oxygen delivery were not due to different recoveries of the human RBCs in the rat circulation.
CONCLUSION: This study shows that the storage-induced deficit of human RBCs to oxygenate the rat kidney microcirculation at reduced Hct is completely reversible. Prevention of metabolic changes during storage is therefore a valid approach to prevent this deficit.     

Automated collection of double red blood cell units with a variable-volume separation chamber

BACKGROUND: Automated collection of blood components offers multiple advantages and has prompted development of portable devices. This study sought to document the biochemical and hematologic properties and in vivo recovery of red cells (RBCs) collected via a new device that employed a variable-volume centrifugal separation chamber. STUDY DESIGN AND METHODS: Normal subjects (n = 153) donated 2 units of RBCs via an automated blood collection system (Cymbal, Haemonetics). Procedures were conducted with wall outlet power (n = 49) or the device's battery source (n = 104). Units were collected with or without leukoreduction filtration and were stored in AS-3 for 42 days. The units were assessed via standard biochemical and hematologic tests before and after storage, and 24 leukoreduced (LR) and 24 non-LR RBCs were radiolabeled on Day 42 with Na(2)(51)CrO(4) for autologous return to determine recovery at 24 hours with concomitant determination of RBC volume via infusion of (99m)Tc-labeled fresh RBCs. RESULTS: Two standard RBC units (targeted to contain 180 mL of RBCs plus 100 mL of AS-3) could be collected in 35.7 +/- 2.0 minutes (n = 30) or 40.3 +/- 2.7 minutes for LR RBCs (n = 92). An additional 31 collections were conducted successfully with intentional filter bypassing. RBC units contained 104 +/- 4.1 percent of their targeted volumes (170-204 mL of RBCs), and LR RBCs contained 92 percent of non-LR RBCs' hemoglobin. All LR RBCs contained less than 1 x 10(6) white blood cells. Mean hemolysis was below 0.8 percent (Day 42) for all configurations. Adenosine triphosphate was well preserved. Mean recovery was 82 +/- 4.9 percent for RBCs and 84 +/- 7.0 percent for LR RBCs. CONCLUSIONS: The Cymbal device provided quick and efficient collection of 2 RBC units with properties meeting regulatory requirements and consistent with good clinical utility.     

Release of WBC-derived IL-1 receptor antagonist into supernatants of RBCs: influence of storage time and filtration

BACKGROUND: Transfusion-associated immunodepression may be related to the transfer of immunoinhibitory cytokines with blood components. STUDY DESIGN AND METHODS: After evidence of increasing concentrations of IL-1 receptor antagonist (IL-1RA) but not of IL-10 was obtained in supernatants of stored RBC units that were WBC-reduced by centrifugation (C-RBCs) in a pilot study, IL-1RA concentrations were determined weekly in supernatants of C-RBCs and in units that underwent prestorage WBC reduction by in-line filtration (F-RBCs) over a 49-day storage. For assessing total IL-1RA content, complete cell lysis by repeated freezing and thawing was done. The results were related to the changes in WBC count during storage. The dependency of IL-1RA content on preparation procedures was assessed. RESULTS: The prestorage IL-1RA concentration in C-RBCs (859 +/- 218 pg/mL) was significantly higher than in F-RBC (75 +/- 13 pg/mL). Whereas no changes were seen in F-RBCs during storage, IL-1RA levels in C-RBC supernatants drastically increased to levels about 50 times those in normal plasma (16,327 +/- 2,686 pg/mL on Day 49). Follow-up analysis revealed stringent correlation between IL-1RA release into supernatants and the current loss of WBCs (r = 0.79, n = 42; p<0.001). The total IL-1RA content did not change during storage and was directly dependent on prestorage WBC count. Preparation procedures altered the IL-1RA content only by WBC reduction. CONCLUSION: The immunosuppressive cytokine IL-1RA is transmitted by RBCs in relation to WBC content and storage time.     

Packed blood cells stored in AS-5 become proinflammatory during storage

BACKGROUND: Studies have shown that packed blood cells (PBCs) stored in AS-1 (Adsol, Baxter) and AS-3 (Nutricel, Medsep Corp.) accumulate proinflammatory substances, which may contribute to increased complications from allogeneic blood transfusion. This study assessed whether supernates from PBCs stored in AS-5 (Optisol, Terumo Corp.) prime neutrophils (PMNs), activate platelets (PLTs), and accumulate proinflammatory cytokines and PMN granule constituents.
STUDY DESIGN AND METHODS: PBC units were prepared in AS-5 from nonleukoreduced (NLR) and leukoreduced (LR) whole-blood units and stored at 4°C. Supernates from samples of PBCs collected at various storage times were analyzed by multiplex enzyme-linked immunosorbent assay for proinflammatory cytokines and myeloperoxidase (MPO) and were incubated with type-matched blood, which was assessed by flow cytometry for expression of CD11b on PMNs, CD62P on PLTs, and formation of PMN-PLT aggregates.
RESULTS: Supernates from NLR PBCs stored for at least 14 days elevated CD11b expression on PMNs and the number of PMN-PLT aggregates compared to supernates from collection day PBCs. The magnitude of these effects correlated with storage age. Supernates from LR PBCs did not elicit these responses. Expression of CD62P on PLTs was not affected by supernates from either NLR or LR PBCs. Levels of interleukin (IL)-1β, IL-6, IL-8, IL-18, NAP-2, MCP-1, RANTES, and MPO were elevated in supernates from 28- and 42-day NLR units. Tumor necrosis factor α and MIP-1α did not increase, and cytokine levels in LR PBC units did not increase.
CONCLUSION: Units of NLR PBCs stored in AS-5 become increasingly proinflammatory as a function of storage time. Leukoreduction prevents this change.     

Selective effects of CPAP on sleep apnoea-associated manifestations

Background   Visceral adiposity and obstructive sleep apnoea (OSA) may be independently associated with daytime sleepiness/low performance, insulin resistance, hypercytokinaemia, and/or hypertension. The objectives of this study are to simultaneously test these associations at baseline and after 3 months of continuous positive airway pressure (CPAP) therapy.
Materials and methods   Sixteen obese men with OSA; 13 non-apnoeic, obese controls, and 15 non-obese controls were monitored in the sleep laboratory for four consecutive nights. Objective measures of daytime sleepiness and performance, serial 24 h plasma measures of interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), TNF receptor 1 (TNF-r1) and adiponectin, fasting blood glucose and insulin, visceral adiposity and blood pressure were obtained. Sleep apnoeics were re-assessed using the same protocol after 3 months of CPAP.
Results   At baseline, IL-6, TNF-r1, and insulin resistance were highest in OSA patients, intermediate in obese controls, and lowest in non-obese controls ( P  < 0·05). Visceral fat was significantly greater in sleep apnoeics than obese controls and predicted insulin resistance and IL-6 levels, whereas OSA predicted TNF-r1 levels ( P  < 0·05). CPAP decreased daytime sleepiness and blood pressure ( P  < 0·05), but did not affect fasting glucose or insulin or around the clock adiponectin, IL-6, TNF-α, or TNF-r1 levels.
Conclusions   In obese sleep apnoeics, visceral fat is strongly associated with insulin resistance and inflammation. CPAP decreases sleepiness and moderates hypertension but does not affect visceral adiposity, insulin resistance, hypoadiponectinaemia or hypercytokinaemia, all of which are independent risk factors for cardiovascular disease and diabetes.     

Effect of polymerized-type I collagen in knee osteoarthritis. I. In vitro study

Background  Polymerized-type I collagen (polymerized-collagen) is a down-regulator of inflammation and a tissue regenerator biodrug. The aim of this study was to evaluate its effect in co-cultures of cartilage and synovial tissue from patients with knee osteoarthritis (OA).
Materials and methods  Cartilage and synovial tissue from five patients with OA were co-cultured for 7 days in the presence or absence of 1% polymerized-collagen. To determine proteoglycans content, tissues were stained with alcian blue technique. Pro- and anti-inflammatory cytokines [interleukin (IL)-1β, IL-8, IL-10, IL-12, tumour necrosis factor (TNF)-α, and interferon (IFN)-γ] and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants by ELISA and results were normalized by total protein concentration. Cartilage oligomeric matrix protein (COMP), type II collagen, TNF-α, IL-10 and Ki-67 expression were determined by immunohistochemistry.
Results  Polymerized-type I collagen induced an increase of 3- to 6fold cell proliferation (Ki-67), proteoglycans content, and COMP and type II collagen expression, whereas it inhibited IL-1β and TNF-α production. IL-10 levels were up-regulated in treated vs. untreated cultures. No differences were found on IL-8 or TIMP-1 levels in supernatants from polymerized-collagen-treated co-cultures when compared with untreated cultures. IL-12 and IFN-γ were undetectable.
Conclusion  The addition of polymerized-type I collagen to cartilage and synovial tissue co-cultures induced up-regulation of chondrocytes proliferation and cartilage extracellular matrix proteins production (COMP, type II collagen and proteoglycans) as well as an anti-inflammatory cytokine (IL-10) and the down-modulation of pro-inflammatory cytokines (IL-1β and TNF-α). It is possible that this mechanism might contribute to induce tissue regeneration and down-regulation of inflammation in OA.     

IMMUNOHEMATOLOGY: Storage of murine red blood cells enhances alloantibody responses to an erythroid‐specific model antigen

BACKGROUND: Red blood cell (RBC) alloimmunization can be a serious complication of blood transfusion, but factors influencing the development of alloantibodies are only partially understood. Within FDA‐approved time limits, RBCs are generally transfused without regard to length of storage. However, recent studies have raised concerns that RBCs stored for more than 14 days have altered biologic properties that may affect medical outcomes. To test the hypothesis that storage time alters RBC immunogenicity, we utilized a murine model of RBC storage and alloimmunization. STUDY DESIGN AND METHODS: Blood from transgenic HOD donor mice, which express a model antigen (hen egg lysozyme [HEL]) specifically on RBCs, was filter leukoreduced and stored for 14 days under conditions similar to those used for human RBCs. Fresh or 14‐day‐stored RBCs were transfused into wild‐type recipients. The stability of the HOD antigen and posttransfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti‐HEL immunoglobulin levels. RESULTS: Transfusion of 14‐day‐stored, leukoreduced HOD RBCs resulted in 10‐ to 100‐fold higher levels of anti‐HEL alloantibodies as detected by enzyme‐linked immunosorbent assay than transfusion of freshly collected, leukoreduced RBCs. RBC expression of the HOD antigen was stable during storage. CONCLUSIONS: These findings demonstrate that HOD murine RBCs become more immunogenic with storage and generate the rationale for clinical trials to test if the same phenomenon is observed in humans. Length of storage of RBCs may represent a previously unappreciated variable in whether or not a transfusion recipient becomes alloimmunized.     

Is red blood cell rheology preserved during routine blood bank storage?

BACKGROUND: Red blood cell (RBC) units stored for more than 2 weeks at 4°C are currently considered of impaired quality. This opinion has primarily been based on altered RBC rheologic properties (i.e., enhanced aggregability, reduced deformability, and elevated endothelial cell interaction), during prolonged storage of nonleukoreduced RBC units. In this study, the rheologic properties and cell variables of leukoreduced RBC units, during routine blood bank storage in saline‐adenine‐glucose‐mannitol, were investigated. STUDY DESIGN AND METHODS: Ten leukoreduced RBC units were stored at the blood bank for 7 weeks at 4°C. RBCs were tested weekly for aggregability, deformability, and other relevant variables. RESULTS: RBC aggregability was significantly reduced after the first week of storage but recovered during the following weeks. After 7 weeks aggregability was slightly, but significantly, reduced (46.9 ± 2.4‐44.3 ± 2.2 aggregation index). During storage the osmotic fragility was not significantly enhanced (0.47 ± 0.01% phosphate‐buffered saline) and the deformability at shear stress of 3.9 Pa was not significantly reduced (0.36 ± 0.01 elongation index [EI]). The deformability at 50 Pa was reduced (0.58 ± 0.01‐0.54 ± 0.01 EI) but remained within reference values (0.53 ± 0.04). During 5 weeks of storage, adenosine triphosphate was reduced by 54% whereas mean cell volume, pH, and mean cell hemoglobin concentration were minimally affected. CONCLUSIONS: RBC biochemical and physical alterations during storage minimally affected the RBC ability to aggregate and deform, even after prolonged storage. The rheologic properties of leukoreduced RBC units were well preserved during 7 weeks of routine blood bank storage.     

Exploratory in vitro study of red blood cell storage containers formulated with an alternative plasticizer

BACKGROUND: The plasticizer di‐2‐ethylhexyl phthalate (DEHP) is a common component in medical plastics. There is motivation to replace this component; however, DEHP is necessary to prevent excessive hemolysis in stored red blood cells (RBCs). Our objective is to evaluate a candidate replacement plasticizer (Hexamoll, di‐isononyl cyclohexane‐1,2‐dicarboxylic acid [DINCH], BASF Corp.) compared to DEHP in an in vitro feasibility study. We hypothesize that the candidate will provide at least equivalent protection against hemolysis for RBCs stored for 42 days and periodic mixing of RBCs will add additional protection against hemolysis. STUDY DESIGN AND METHODS: Whole blood was collected into citrate‐phosphate‐dextrose; combined into pools of 2 ABO identical whole blood units; and divided, leukoreduced, centrifuged, and separated into plasma and RBCs. Additive solution was added, and the RBCs were stored for 42 days at 1 to 6°C. In three parts of this study, split pools were paired as DINCH‐polyvinyl chloride (PVC) with weekly mixing versus DINCH‐PVC with no mixing, DINCH‐PVC mixed versus DEHP‐PVC no mix, and DINCH‐PVC versus DEHP‐PVC with neither mixed. A standard panel of in vitro RBC characteristics was determined on Days 0 and 42. RESULTS: Mixing DINCH‐PVC weekly improved Day 42 hemolysis (0.36 ± 0.07% vs.0.56 ± 0.15%, p = 0.002), and mixed DINCH‐PVC bags were noninferior to unmixed DEHP‐PVC bags (p ≤ 0.05). DINCH‐PVC bags stored without weekly mixing were inferior to unmixed DEHP‐PVC bags for hemolysis on Day 42, although no individual bag exceeded 0.8% hemolysis. CONCLUSION: Periodic mixing of RBCs stored in DINCH‐PVC provides additional protection against hemolysis. Unmixed DINCH‐PVC bags were inferior to DEHP‐PVC bags for prevention of hemolysis, but remain a candidate for replacement DEHP in RBC storage bags.