Standard Platelet Concentrates Research Articles

Two novel platelet biotinylation methods and their impact on stored platelet concentrates in a blood bank environment.

Storage of platelet concentrates (PCs) has an impact on platelet quality and possibly affects their functions after transfusion. The influence of processing and storage conditions of PCs on their in vivo function upon transfusion is unknown. One option for investigating this question is to implement an ex vivo labeling of human platelets, to analyze them after transfusion into heathy volunteers and/or patients. In this study, we developed two labeling methods employing biotin. Two methods of biotinylation were compared to a control (standard PC). The "Bio-Wash" process used washing steps to label all platelets within the PC; for the other method, "Bio-Direct," one fifth of the PC were directly labeled without washing steps. The control and the two biotinylated PCs were analyzed over 7 days of storage. Labeling efficiency, platelet counts, phenotypes, and functions, along with time and costs, were evaluated to select the best process. Both methods achieved a stable labeling through the storage, with similar platelet counts and metabolism in comparison to control PCs. Bio-Wash showed higher activation phenotype and lower aggregation response in comparison to the Bio-Direct method. The Bio-Direct was performed within 1.5 h versus 3 h for the Bio-Wash. However, the Bio-Direct required 12 mg of biotin instead of 8 mg for the other process. We set up two methods of biotinylation that can be easily implemented in a blood bank environment. The Bio-Direct process was preferred to the Bio-Wash because of its similarity, from a functional and phenotypic point of view, with standard PCs.

High‐density lipoprotein 3 and apolipoprotein A‐I alleviate platelet storage lesion and release of platelet extracellular vesicles

Stored platelet (PLT) concentrates (PLCs) for transfusion develop a PLT storage lesion (PSL), decreasing PLT viability and function with profound lipidomic changes and PLT extracellular vesicle (PL-EV) release. High-density lipoprotein 3 (HDL3 ) improves PLT homeostasis through silencing effects on PLT activation in vivo. This prompted us to investigate HDL3 and apolipoprotein A-I (apoA-I) as PSL-antagonizing agents. Healthy donor PLCs were split into low-volume standard PLC storage bags and incubated with native (n)HDL3 or apoA-I from plasma ethanol fractionation (precipitate IV) for 5 days under standard blood banking conditions. Flow cytometry, Born aggregometry, and lipid mass spectrometry were carried out to analyze PL-EV release, PLT aggregation, agonist-induced PLT surface marker expression, and PLT and plasma lipid compositions. Compared to control, added nHDL3 and apoA-I significantly reduced PL-EV release by up to -62% during 5 days, correlating with the added apoA-I concentration. At the lipid level, nHDL3 and apoA-I antagonized PLT lipid loss (+12%) and decreased cholesteryl ester (CE)/free cholesterol (FC) ratios (-69%), whereas in plasma polyunsaturated/saturated CE ratios increased (+3%) and CE 16:0/20:4 ratios decreased (-5%). Administration of nHDL3 increased PLT bis(monoacylglycero)phosphate/phosphatidylglycerol (+102%) and phosphatidic acid/lysophosphatidic acid (+255%) ratios and improved thrombin receptor-activating peptide 6-induced PLT aggregation (+5%). nHDL3 and apoA-I improve PLT membrane homeostasis and intracellular lipid processing and increase CE efflux, antagonizing PSL-related reduction in PLT viability and function and PL-EV release. We suggest uptake and catabolism of nHDL3 into the PLT open canalicular system. As supplement in PLCs, nHDL3 or apoA-I from Fraction IV of plasma ethanol fractionation have the potential to improve PLC quality to prolong storage.

Rapid detection of contaminant bacteria in platelet concentrate using differential impedance

Current FDA-approved culture-based methods for the bacterial testing of platelet concentrate (PC) can yield false-negative results attributed to Poisson-limited sampling errors incurred near the time of collection that result in undetectable bacterial concentrations. Testing PC at the point of issue (POI) extends the incubation period for any contaminant bacteria increasing the probability of detection. Data are presented from time-course experiments designed to simulate POI testing of bacterially contaminated PCs at different stages of growth using differential impedance sensing. Whole-blood-derived PCs were typically spiked with low numbers of bacteria (approximately 100CFU/ml) and incubated under standard PC storage conditions. Each infected unit was evaluated every two hours over a 12-h period. All samples were treated with a chemical compound that induces stress in the bacterial cells only. The development of any bacterial stress was monitored by detecting changes in the dielectric properties of the PC using differential impedance. Differential impedance measurements and corresponding cell counts at the different time-points are presented for six organisms implicated in post-transfusion-septic reactions. All infected PCs were detected once contaminant bacteria reached concentrations ranging between 0·6×10(3) and 6×10(3) CFU/ml irrespective of the phase of growth. Results were obtained within 30min after the start of the assay and without the need for cell lysis or centrifugation. Differential impedance sensing can detect bacterial contamination in PC rapidly at concentrations below clinical thresholds known to cause adverse effects.

Novel flow cytometric screening method for bacterial contamination of red blood cells: a proof‐of‐principle evaluation

Platelet concentrates (PCs) are the major source of transfusion-transmitted bacterial infection but the significantly higher number of transfused red blood cells (RBCs) has resulted in a similar occurrence of possible transfusion-associated bacterial infections as those of recipients transfused with platelets. The aim of this study was adaption of the BactiFlow (BF) flow cytometric rapid PC screening method for the detection of bacterial contamination in RBCs. The BF assay was used to detect and count bacteria based on esterase activity in viable cells in RBCs. The initial sample preparation for RBCs included the lysis of RBCs, followed by enrichment of bacteria by centrifugation and subsequent standard PC sample preparation. Two-hundred RBC units were analyzed by BF and the BacT/Alert automated culture system. Furthermore, RBCs were spiked with eight different bacteria to monitor bacterial growth kinetics. The BF showed an excellent correlation to conventional plate count results after RBC sample processing. The diagnostic sensitivity of the assay was determined to fewer than 500 counts/mL. Analysis of RBC units showed concordant negative results by BF and culture. Growth kinetics of bacteria were successfully monitored using flow cytometry, showing that Yersinia enterocolitica or Serratia liquefaciens had the capability to grow in RBCs to high counts. Our study demonstrates the successful modification and application of a rapid flow cytometric screening method for bacterial contamination in RBCs. The BF assay represents a powerful basic tool to study bacterial contamination, potential screening strategies of RBCs, and the clinical outcome of screened RBCs prepared for transfusion.

HLA Antigens Shed from the Surface of Synthetic or Naturally Occurred Platelet-Derived Microparticles During Storage of Platelet Concentrate

The demand for standard platelet concentrates (PCs) has continued to increase in the recent years. Infusible platelet membranes (IPM) prepared from new or outdated human platelets have been developed as an alternative to standard PCs, with the additional advantage of long shelf life and increased viral safety. Reduction of HLA antigens on the IPM has been assigned as one of the probable advantages of this product. In re-examining this issue, we studied the existence of HLA class I on the surface of IPM microparticles. In comparison we also surveyed HLA expression on the surface of the naturally occurred platelet-derived microparticles (nPMPs) during 7days storage. Intended for producing IPM, PCs obtained from Iranian blood transfusion organization were lysed; virally inactivated with wet heat in the presence of a heat stabilizer and then sonicated. IPMs were separated using centrifugation and liquid-stored in 4°C. The expression of HLA class I antigens was surveyed using flow cytometry technique. HLA molecules were present on the microparticles. Shedding of HLA antigens was demonstrated from the surface of the both liquid-stored IPM and nPMPs during storage. Storage of IPM in 4°C was accompanied with significant reduction of HLA molecules. It seemed that achievement of HLA-free IPM could be impossible unless chloroquine treated platelets were used to prepare these microvesicles.

Production and characterization of liquid-stored and lyophilized reconstituted human infusible platelet membranes

During recent years, the need for platelet concentrate (PC) has increased. Infusible platelet membranes (IPM) have been developed as an alternative to standard PCs, with the additional advantage of long shelf-life and increased viral safety. In this study, IPM construction and the morphological and biological features of these microvesicles were surveyed to determine their binding capacities in vitro. Thirty-five PC units prepared by the Iranian Blood Transfusion Organization were used to produce IPM. Platelets were lysed by repeated cycles of freezing and thawing, virally inactivated with wet heat in the presence of different concentrations of sodium octanoate as a heat stabilizer, and then sonicated. IPM were separated, kept at 4°C or lyophilized, and examined for binding to collagen and von Willebrand factor (VWF). IPM retained the binding capacity for collagen and VWF, and the extent of VWF binding was dependent on the concentration of the heat stabilizer. Additionally, a higher binding capacity was demonstrated for liquid-stored compared with lyophilized IPM. This study revealed the potential of IPM microvesicles to mimic the binding features of platelets in vitro.

Impact of Platelets and Fresh Frozen Plasma in Contrast to Red Cell Concentrate on Unstimulated and Stimulated Cytokine Release in an In Vitro Model of Transfusion

Transfusion of blood may contribute to immunomodulation. Leuco-depleted standard blood products are supposed to result in less immunomodulation compared with whole blood. To determine the influence of leuco-depleted blood products on the cytokine response, red blood cell concentrates (RBC), fresh frozen plasma (FFP) and platelet concentrates (PC) were investigated in an in vitro model of blood transfusion. Leuco-depleted standard blood bank RBC, FFP and PC were mixed in vitro with AB0 compatible venous blood from healthy volunteers in ratios of 3:1, 1:1 and 1:3. Specimens were incubated in presence or absence of lipopolysaccharide, 1 mug/ml. After 24 h of incubation cytokine release of tumour necrosis factor (TNF)-alpha and interleukin-10 (IL-10) was measured in cell culture supernatants by means of enzyme-linked immunsorbent assay. Addition of RBC, FFP and PC to venous blood from healthy volunteers led to a significant and dose-dependent increase in spontaneous TNF-alpha and IL-10 release. After endotoxin stimulation, RBC, FFP and PC significantly suppressed the TNF-alpha response, while the stimulated release of IL-10 tended to increase, reaching significance only after high doses of FFP. Addition of leuco-depleted blood products changed the spontaneous and stimulated cytokine response in an in vitro model of transfusion. These data may suggest a possible contribution of transfused FFP and PC to immunomodulation after transfusion similar to RBC.

Evaluation of Different Preparation Procedures of Pathogen Reduction Technology(Mirasol®)-Treated Platelets Collected by Plateletpheresis

Background: The Mirasol® pathogen reduction technology (PRT) for platelet concentrates (PC) uses riboflavin and UV light (270–360 nm). We evaluated the impact of PRT on platelets in comparison to standard single-donor PC. Material and Methods: Platelets were resuspended in autologous plasma. After 2 h rest without agitation, PC were split into an untreated control unit (C-PC) and an immediately treated unit (T-PC) (series I). In series IV, split PC were stored under agitation over night before PRT was carried out. Platelet quality was assessed by pH, glucose consumption, lactate production rate, LDH, soluble sCD62p and CD62p expression with and without TRAP (thrombin receptor-activating peptide) over 7 days. Results: Series I: On day 5, pH values were lower for T-PC (6.8 ± 0.2 vs. 7.4 ± 0.1, C-PC), accompanied by a higher glucose consumption rate of 0.069 ± 0.016 vs. 0.035 ± 0.006 mmol/10¹² platelets/h and lactate production rate of 0.126 ± 0.031 vs. 0.063 ± 0.011 mmol/10¹² platelets/h. CD62p using TRAP was lower for T-PC (50 ± 11 vs. 62 ± 14%). Baseline activation was higher in T-PC (35 ± 12 vs. 28 ± 15%). Longer initial rest time had no impact on these results (series II/III/IV). Conclusion: PRT leads to an increase of platelet metabolism and activation independent of the length of the initial rest times. PC resuspended in autologous plasma should be stored at maximum up to day 5.

Complexes leucoplaquettaires en tant que marqueur d’activation dans les concentrés plaquettaires standards

Objectives Several in vitro laboratory tests to assess the quality control of platelet concentrates (PC) are available. Some of them have a good correlation with the platelet recovery index. To assess the quality control of standard PC prepared in our blood bank, we measured the blood gas and the degree of platelet activation. Materials and methods SPC were prepared by the PRP method. Fifty-five SPC (45 SPC at day one of storage and 20 SPC at day five of storage) were analysed. Blood gas (pH, PO 2, PCO 2 and bicarbonate concentration) in the SPC were measured by blood gas automate. Platelet activation profile were determined by measuring the percentage of platelet expressing the CD62p (% CD62) and the percentage of platelet-leukocyte aggregate (% PLA). Results The pH values of all studied SPC were comprised between 7.0 and 7.6. SPC at day 1 of storage have a significantly higher pH than those at day 5 of storage (7.5 ± 0.05 versus 7.3 ± 0.14; p < 0.001). The % CD62p were higher in SPC at day five compared to the SCP at day one without reaching a statistical significance (28.4 ± 15% versus 24.3 ± 9.7%, p = 0.052). The percentage of PLA were higher in SPC at day one compared to SCP at day five although this difference is not statistically significant (22.2 ± 7.5% versus 17.9 ± 8.0%; p = 0.23). Conclusion Preparation and storage procedure adopted in our centre did not significantly affect the quality SPC. Our study is the first to assess the PLA in PC. Studies assessing the PLA are warranted to appreciate the clinical impact of this parameter.

The Functional Integrity of Platelets in Volume-Reduced Platelet Concentrates

Premature and low-birth-weight infants usually require small-volume platelet transfusions to treat thrombocytopenia. Also, infants undergoing open-heart surgery with extracorporeal circulation and with compromised cardiac function are at risk for excessive intravascular volume. The small-volume platelet substitution can be achieved by dispensing an aliquot from the unit of a standard single-donor platelet concentrate (PC). Alternatively, there is an indication for volume reduction of PCs to maximize the number of platelets transfused in the smallest possible volume. We determined the spontaneous and induced activation of platelets before and after volume reduction in 20 consecutive single-donor-apheresis PCs. After a mean storage time of 2 days, the PCs were plasma-depleted by centrifugation. Spontaneous, adenosine diphosphate (ADP)-induced, and collagen-induced activation were determined by flow cytometry. Furthermore, ADP- and collagen-induced aggregation were measured. A total of 33.8% of platelets in standard PCs were activated spontaneously. Volume reduction of PCs led to a mild but significant increase of spontaneous activation of platelets (43.2%). Additionally, volume reduction resulted in an impaired ADP-induced aggregability of platelets, whereas collagen induction was unaffected. Transfusion of volume-reduced PCs is an effective alternative to use of standard PCs in patients at frequent risk for excessive intravascular volume, because equal volumes increase the platelet count twice as effectively.

Effects of high platelet concentration in collecting and freezing dry platelets concentrates

Objective. To evaluate, in vitro, the effects of collecting and cryopreserving fresh dry platelet concentrates (PCs). Material and methods. Standard and dry PCs were collected in the same apheresis procedure. PCs were evaluated by mean platelet volume (MPV), pH, glucose and LDH levels. Activation was examined by flow cytometry using anti-CD41, anti-CD42 and anti-CD62p monoclonal antibodies and annexin binding assay. Platelet function was assessed by aggregation using ADP, collagen and arachidonic acid as agonists. Dry PCs were compared to standard PCs and to cryopreserved dry PCs. We also compared the use of ThromboSol (TS) to 5% DMSO as cryoprotectives. Results. Dry PCs presented a significantly reduced pH and glucose ( p<0.001), increased LDH levels and CD62p expression ( p<0.001) and diminished aggregation response to ADP ( p<0.001). Platelet cryopreservation was associated with platelet lysis, activation and loss of function. Dry PCs cryopreserved with TS were associated with statistically higher LDH levels ( p<0.001) and a higher percentage of annexin binding ( p=0.005), in addition to a lower number of CD42 positive platelets ( p=0.01). Conclusion. Dry PCs should be rapidly frozen after collection to avoid a fall in pH and platelet activation. 5% DMSO performed better than TS to cryopreserve dry PCs.

Réaction frissons–hyperthermie et transfusion de concentrés plaquettaires standard : étude prospective

Fever–shivers reaction (FSR) is the most frequent transfusion immediate incident related to platelet transfusions. The aim of our prospective study was to assess the frequency of the different immediate incidents, especially the frequency and the causes of the FSR, observed during the transfusion of standard platelet concentrates (SPC). For each FSR, analysis of causes included: a bacterial culture of the implicated SPC, a blood culture and HLA antibody screening (lymphocytotoxicity assay) among the patients. In the study period, 34 patients were followed during 74 transfusions. Ten immediate incidents were noted; FSR: N = 8, erythema–urticaria: N = 1 and nausea-vomit: N = 1. The FSR was observed in 6 patients who received 56 SPC. Analysis of causes of this reaction revealed that: HLA antibodies were present in one patient; bacterial contamination was not found neither among the patients nor in the implicated SPC, and the risk of the FSR occurrence rose with increased storage time of the SPC transfused. Indeed, a significant difference was noted between the mean age of the SPC implicated in the FSR and the mean age of those not implicated ( P = 0,0028). In conclusion, the FSR is a frequent incident observed during SPC transfusions. In the majority of cases, the cause of this reaction was not identified. Further studies will be necessary to better understand the physiological mechanisms of the FSR.

Evaluation of Leukocyte‐Depleted Platelet Concentrates Obtained by In‐Line Filtration

Abstract Background and Objectives: The use of leukocyte‐depleted platelet concentrates (PCs) is justified, yet questions remain regarding their properties. We have assessed the in vitro quality of WBC‐reduced PCs obtained by using a new in‐line filter. Materials and Methods: Twenty blood units were randomized for standard component preparation, or for processing including in‐line leukodepletion of platelet‐rich plasma using an ATS™ PL Pall filter. The resultant PCs were compared during storage for several in vitro platelet quality parameters, content of cytokines and anaphylatoxins, and coagulation markers. Results: In‐line filtration of platelet‐rich plasma through ATS PL was highly efficient rendering PCs with 99.93% less white blood cells (WBCs) than standard PCs. During storage for up to 9 days, leukocyte‐depleted units displayed platelet content, biochemical parameters, and platelet surface expression of glycoproteins lbα, llb/lla, CD62 and CD63, similar to those of standard units. However, in‐line leukocyte‐reduced PCs displayed no significant accumulation of IL‐6 and IL‐8 during storage in contrast to standard units. Moreover, while storage promoted complement activation with C3a and C4a liberation in both WBC‐reduced PCs and standard units, the concentration of these anaphylatoxins after the 9‐day storage period was higher in the former group. Finally, all units, standard and leukocytereduced, displayed a similar storage‐promoted rise in TGF‐β1, and a mild prolongation of activated partial thromboplastin and prothrombin times. Conclusion: Inline filtration during component preparation appears as an easy and effective procedure for obtaining prestorage leukocyte‐depleted PCs. During subsequent blood bank storage, these WBC‐reduced PCs display, in comparison with standard PCs, normal in vitro platelet properties, decreased accumulation of cytokines IL‐6 and IL‐8, and increased complement activation.

Evaluation of Leukocyte–Depleted Platelet Concentrates Obtained by In–Line Filtration

Background and Objectives: The use of leukocyte–depleted platelet concentrates (PCs) is justified, yet questions remain regarding their properties. We have assessed the in vitro quality of WBC–reduced PCs obtained by using a new in–line filter. Materials and Methods: Twenty blood units were randomized for standard component preparation, or for processing including in–line leukodepletion of platelet–rich plasma using an ATS^TM PL Pall filter. The resultant PCs were compared during storage for several in vitro platelet quality parameters, content of cytokines and anaphylatoxins, and coagulation markers. Results: In–line filtration of platelet–rich plasma through ATS PL was highly efficient rendering PCs with 99.93% less white blood cells (WBCs) than standard PCs. During storage for up to 9 days, leukocyte–depleted units displayed platelet content, biochemical parameters, and platelet surface expression of glycoproteins Ibα, IIb/IIa, CD62 and CD63, similar to those of standard units. However, in–line leukocyte–reduced PCs displayed no significant accumulation of IL–6 and IL–8 during storage in contrast to standard units. Moreover, while storage promoted complement activation with C3a and C4a liberation in both WBC–reduced PCs and standard units, the concentration of these anaphylatoxins after the 9–day storage period was higher in the former group. Finally, all units, standard and leukocyte–reduced, displayed a similar storage–promoted rise in TGF–β₁, and a mild prolongation of activated partial thromboplastin and prothrombin times. Conclusion: In–line filtration during component preparation appears as an easy and effective procedure for obtaining prestorage leukocyte–depleted PCs. During subsequent blood bank storage, these WBC–reduced PCs display, in comparison with standard PCs, normal in vitro platelet properties, decreased accumulation of cytokines IL–6 and IL–8, and increased complement activation.

Preparation and storage characteristics of white cell-reduced high-concentration platelet concentrates collected by anapheresis system for transfusions in utero.

Important concerns with regard to in utero platelet transfusions are avoidance of volume overload and the immunomodulatory effects of residual white cells (WBCs). This study evaluated a modification of a leukocyte-reduction system (LRS, Spectra, COBE BCT) for apheresis, which collects high-concentration WBC-reduced platelets (HCPs) for in utero transfusion. The LRS procedure was modified by running the platelet collection pump at specified low flow rates (Q(col)) for the first part of the procedure, collecting HCPs by gently purging them from the LRS chamber into a designated collection bag and then restoring the original LRS procedure settings to collect a second standard apheresis platelet concentrate (PC). Two centers carried out 32 procedures. Platelet yield, residual WBCs, and in vitro platelet function studies were evaluated. Platelet concentrations in 60 mL of HCPs were predictable according to Q(col) (r(2) = 0.735). HCP yields varied from 0.9 to 3.2 x 10(11), depending on the desired final platelet concentrations in 60 mL, with an overall average of 1. 92 x 10(11) (n = 32). Apheresis PCs had a mean platelet yield of 2.9 x 10(11) (1.3-4.4 x 10(11), n = 20) and 3.9 x 10(11) (2.2-5.8 x 10(11), n = 12) at concentrations of 1.3 x 10(12) per L for single-needle and dual- needle procedures, respectively. Median WBC counts were 5.6 x 10(3) for HCPs and 2.0 x 10(4) for apheresis PCs, with >99 percent expected to be less than 1 x 10(6). HCP in vitro characteristics were equivalent to those of apheresis PCs at 24 hours after collection. In vitro performance declined over storage as a function of HCP yield. HCP pH at 22(o)C was maintained at a level of >6.2 for more than 3 days for yields >1.6 x 10(11), less than 2 days for yields 1.6 to 2.2 x 10(11), and less than 24 hours for yields >2.2 x 10(11). HCPs showed good in vitro characteristics and could be stored for 1 to 3 days, depending on the total number of platelets collected. A standard apheresis PC and an HCP requiring no secondary processing can be collected with the Spectra LRS. The platelet concentration may be determined by clinical need. HCPs meet the requirements for components that are transfused in utero.

Impact of Storage at 22°C and Citrate Anticoagulation on the Cytokine Secretion of Mononuclear Leukocytes

Abstract Background and Objectives: Peripheral blood mononuclear cells (PBMC) and cytokines accumulating in platelet concentrates (PC) during storage have been implicated in causing non‐hemolytic transfusion reactions, in particular after prolonged PC storage. We investigated the impact of major storage parameters, such as storage time, anticoagulation and temperature, on the capability of PBMC to secrete cytokines. Materials and Methods: In a first study, PBMC from whole blood donations (n = 16) were exposed to standard PC storage conditions: 22°C, citrate phosphate dextrose (CPD) anticoagulation. Secretion of the cytokines interleukin‐1β (IL‐1β), IL‐2, IL‐6 and interferon‐γ (IFN‐γ) on mitogenic stimulation was measured directly after blood donation and after 1, 3 and 5 days of storage. In a second study, paired whole blood samples (n = 24) were investigated for the mitogenic induction of IL‐2, IL‐6, IFN‐γ and IFN‐α. Cytokine values were compared with respect to incubation temperature (22 vs. 37°C) and anticoagulant (CPD vs. lithium‐heparin). Results: PBMC stored at 22°C with CPD anticoagulation showed an altered cytokine secretion pattern in comparison to freshly donated cells. After storage for 3 days, IL‐2 release was decreased (p &lt; 0.05) and after 5 days secretion of all cytokines was significantly decreased, though still detectable (p &lt; 0.01). In the second study, CPD anticoagulation resulted in a significantly impaired cytokine secretion compared with lithium‐heparin. Cytokine secretion at 22°C were significantly lower (p &lt; 0.001) compared with 37°C for both anticoagulants. Conclusion: Storage of PBMC at 22°C with CPD leads to an impaired cytokine response, but PBMC are still capable of secreting cytokines after 5 days of storage. PBMC remain a potential source of cytokines even after 5 days of storage in CPD at 22°C.

Impact of Storage at 22 °C and Citrate Anticoagulation on the Cytokine Secretion of Mononuclear Leukocytes

Background and Objectives: Peripheral blood mononuclear cells (PBMC) and cytokines accumulating in platelet concentrates (PC) during storage have been implicated in causing non-hemolytic transfusion reactions, in particular after prolonged PC storage. We investigated the impact of major storage parameters, such as storage time, anticoagulation and temperature, on the capability of PBMC to secrete cytokines. Materials and Methods: In a first study, PBMC from whole blood donations (n = 16) were exposed to standard PC storage conditions: 22 °C, citrate phosphate dextrose (CPD) anticoagulation. Secretion of the cytokines interleukin-1β (IL-1β), IL-2, IL-6 and interferon-γ (IFN-γ) on mitogenic stimulation was measured directly after blood donation and after 1, 3 and 5 days of storage. In a second study, paired whole blood samples (n = 24) were investigated for the mitogenic induction of IL-2, IL-6, IFN-γ and IFN-α. Cytokine values were compared with respect to incubation temperature (22 vs. 37 °C) and anticoagulant (CPD vs. lithium-heparin). Results: PBMC stored at 22 °C with CPD anticoagulation showed an altered cytokine secretion pattern in comparison to freshly donated cells. After storage for 3 days, IL-2 release was decreased (p < 0.05) and after 5 days secretion of all cytokines was significantly decreased, though still detectable (p < 0.01). In the second study, CPD anticoagulation resulted in a significantly impaired cytokine secretion compared with lithium-heparin. Cytokine secretion at 22 °C were significantly lower (p < 0.001) compared with 37 °C for both anticoagulants. Conclusion: Storage of PBMC at 22 °C with CPD leads to an impaired cytokine response, but PBMC are still capable of secreting cytokines after 5 days of storage. PBMC remain a potential source of cytokines even after 5 days of storage in CPD at 22 °C.

Studies on platelets exposed to or stored at temperatures below 20 degrees C or above 24 degrees C.

Platelet concentrates (PCs) may be subjected to temperatures outside 20 to 22 degrees C during shipping or storage, which may have an adverse effect on platelet quality. These studies systematically evaluated the effect of short-term exposure (< or = 24 hours) of platelets to temperatures above 22 degrees or below 20 degrees C as part of standard 5-day PC storage at 22 degrees C, as well as the effect of long-term storage (5 days) at 24 and 26 degrees C. For the short-term exposure studies, up to 6 units of Day 1 standard PCs were mixed, split, and returned to the containers. Test units were then stored without agitation in an incubator at a specific temperature (4, 12, 16, or 18 degrees C) for various times up to 24 hours, after which they were stored with agitation at 22 degrees C. One unit acted as control and was stored at 20 to 22 degrees C throughout the 5-day storage period. Loss of platelet discoid shape was determined photometrically by the extent of shape change assay, by an increase in apparent platelet size by morphologic evaluation, and by swirling. A gradual loss of platelet discoid shape occurred at temperatures below 20 degrees C. For similar periods, a greater difference between test and control PCs was observed in units held at 4 degrees C than in those held at 16 degrees C. The data were fitted to an equation to relate platelet discoid shape (% of control) to exposure temperature and time. Assuming that a 20-percent decrease or more in the extent of shape change assay represents a significant loss in platelet viability, the equation predicts that such a loss occurs when the platelets are exposed to 16 degrees C for > or = 16 hours, to 12 degrees C for > or = 10 hours, or to 4 degrees C for > or = 6 hours, whereas exposure to 18 degrees C for < or = 24 hours has no significant effect. Storage for 5 days at temperatures < or = 26 degrees C was not associated with any significant reduction in platelet discoid shape or other measures of platelet quality. There was a gradual loss of platelet discoid shape at exposure temperatures < 20 degrees C, which worsened as temperatures decreased and exposure times increased to 24 hours. This relationship can be described in an equation that could be used as a guideline for allowable exposure conditions.

PSORALEN‐MEDIATED VIRUS PHOTOINACTIVATION IN PLATELET CONCENTRATES: ENHANCED SPECIFICITY OF VIRUS KILL IN THE ABSENCE OF SHORTER UVA WAVELENGTHS

Treatments with psoralens and long-wavelength ultraviolet radiation (UVA, 320-400 nm; PUVA) have shown efficacy for virus sterilization of platelet concentrates (PC). Our laboratory has employed the psoralen derivative 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), and we have found that platelet integrity is best preserved when rutin, a flavonoid that quenches multiple reactive oxygen species, is present during AMT/UVA treatment of PC. In this report, we examine the effects of different UVA spectra under our standard PC treatment conditions (i.e. 50 micrograms/mL AMT, 0.35 mM rutin and 38 J/cm2 UVA). Added vesicular stomatitis virus (VSV; > or = 5.5 log10) was completely inactivated with the simultaneous maintenance of the platelet aggregation response (> 90% of control) when a UVA light source with transmission mainly between 360 and 370 nm (narrow UVA1) was used. In contrast, with a broad-band UVA (320-400 nm; broad UVA) light source, the aggregation response was greatly compromised (< 50% of control) with only a minor increase in the rate of VSV kill. With this lamp, platelet function could be improved to about 75% of the control by adding a long-pass filter, which reduced the transmission of shorter (< or = 345 nm) UVA wavelengths (340-400 nm; UVA1). At equivalent levels of virus kill, aggregation function was always best preserved when narrow UVA1 was used for PUVA treatment. Even in the absence of AMT, and with or without rutin present, narrow UVA1 irradiation was better tolerated by platelets than was broad UVA.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro evaluation of a new filter for leucocyte depletion of platelet concentrate during component preparation.

A newly developed filter for prestorage leucocyte depletion of platelet concentrates (PC) was studied. The filter is designed for leucocyte depletion during the preparation of the pool PC from platelet rich buffy coats. In all the leucocyte depleted PC (LD-PC) leucocyte depletion was satisfactory. 19 of 20 units of LD-PC had a leucocyte content below 3 x 10(5) per PC and 1 contained 8 x 10(5) leucocytes. The standard PC contained 2.53 x 10(8) (0.87 x 10(8)-15.3 x 10(8); n = 20) leucocytes per PC (median and range). The quality of the LD-PC was evaluated by measuring platelet activation, platelet morphology, and pre- and poststorage pH. There were no differences in any of the parameters studied.