Fresh Platelet Concentrates Research Articles

Evaluation of platelets componentized with the Reveos Automated Blood Processing System and stored for 7 days at room temperature in a non-Di(2-ethylhexyl) phthalate (DEHP) platelet pooling set.

Platelet concentrates (PCs) used for transfusion can be produced by apheresis or derived from whole blood (WB). The Reveos device is the first US Food and Drug Administration-approved automated blood processing system that can produce PCs. In this work, we evaluated the quality and function of Reveos-collected PCs stored for 7 days at room temperature. WB was collected from healthy donors and componentized on the day of collection (Fresh) or after an overnight hold (Overnight). PCs were produced (n = 7 Fresh; n = 6 Overnight), stored at room temperature in plasma, and evaluated on days 1 and 7 for quality metrics, platelet activation, clot formation, and aggregation response. Platelet count was comparable between Fresh and Overnight PCs. A drop in pH was reported in Fresh day 7 PCs (p < .001, vs. day 1) but not in Overnight. Overnight units displayed the lowest levels of P-selectin expression (p = .0008, vs. day 7 Fresh). Reduced clot strength and increased lysis were observed in both Fresh and Overnight units on day 7 (vs. day 1). Overnight-hold PCs resulted in the highest clot strength on day 7 (p = .0084, vs. Fresh). No differences in aggregation were reported between groups. Reveos-processed PCs produced from overnight-hold WB performed better in hemostatic function assays and displayed reduced activation compared to fresh WB-derived PCs, although both PC groups maintained platelet quality throughout storage. Utilization of overnight WB for PC preparation with Reveos holds promise as an alternative method of producing platelets for transfusion purposes.

Platelet Refractory Cases after Transfusion of Fresh and Stored Platelet in all Children

Introduction: Acute leukemia is the most common form of cancer in children, platelet transfusion is needed for the chemotherapy of acute lymphoblastic leukaemia children. Platelets are transfused for therapeutic and prophylactic purposes. Transfusion of stored platelet concentrates (up to 5 days) has been demonstrated to be as effective as transfusion of fresh platelet concentrates (upto 24 hours). Platelet refractory cases were also observed in this study that was detected by Corrected Count Increment (CCI) and Percent Platelet Recovery (PPI) at 1hour and 24 hours. Aim of the study: The aim of this study was to observe the platelet refractory cases after transfusion of fresh and stored platelet in acute lymphoblastic leukemia (ALL) in a tertiary care hospital, Dhaka. Methods: A cross-sectional study was conducted in the Department of Clinical Pathology, Paediatric Haematolo- Oncology and transfusion Medicine BSMMU, Department of Haematology and Paediatric Haemato-Oncology in Dhaka Medical College Hospital from March 2010 to February 2011 where 81 children diagnosed with acute lymphoblastic leukaemia were taken as the study population. Non probability purposive sampling was used by fulfilling the inclusion criteria. Ethical consideration was taken by the BSMMU ethical review committee. A data sheet with two parts (Part A and Part B) was designed with a view to collect data from the patients to be enrolled in the study. Data were analyzed using the SPSS version 25.0. Results: Out of 81 in 47 children with acute lymphoblastic leukemia (ALL), fresh platelet concentrates (FPC) (upto 24 hours) or day-0 platelets were transfused. In 34 children, stored platelet concentrates (SPC) (days 1–5) were transfused. In 27 cases, platelet concentrates were transfused both fresh and stored. In FPC and SPC, the corrected count increment at one hour (CCI 1h) was 20.5×109/L and 18.9×109/L respectively. FPC and SPC values for the mean corrected count increment at 24 hours (CCI 24h) were 15.5×109/L and 13.8×109/L respectively. Platelet refractory cases were 15(18.5%) out of 81 cases transfused. Platelet refractory cases were 7(15%) out of 47 cases transfused with FPC and 8(23%) out of 34 cases transfused with stored platelet concentrate (SPC). There were no significant differences (P&lt;0.05) between FPC and SPC according to platelet refractoriness. Conclusion: In vivo quality of freshly generated platelet (upto 24 hours) versus stored platelet concentrates (up to 5 days) were determined after 1 hour and 24 hours. 1-5 day’s stored PRP-PCs are as effective as fresh PRP-PCs for sufficient platelet increment.

A Comparative Study between Fresh and Stored Platelet Concentrate after Transfusion in same ALL patients in a Tertiary Hospital

Background: Platelet transfusion is needed for the treatment of ALL children. Researchers have shown that transfusion of stored platelet concentrates (up to 5days) is as effective as fresh platelet concentrates (24 hours). Objectives: The present study was conducted to access the comparison between fresh and stored platelet concentrate after transfusion in same all patients in a tertiary hospital. Materials and methods: The study was a cross-sectional descriptive study which was conducted in department of Clinical Pathology, Paediatric Haemato-Oncology and transfusion Medicine BSMMU, and Department of Haematology and Paediatric Haemato-Oncology in Dhaka Medical College Hospital. Over a period of March 2010 to February 2011.The sample size was 81. A data sheet with two parts (Part A and Part B) was designed with a view to collect data from the patient to be enrolled in the study .The data were analyzed using the SPSS version 25.0. Results: Out of 81 patients fresh platelet concentrates (FPC) or day-0 platelets were transfused in 47 acute Lymphoblastic leukaemia (ALL) children. Stored platelet concentrates (day 1-5) were transfused in 34 children. In 27cases both fresh and stored platelet concentrates were transfused. Corrected count increment at 1 hour (CCI 1h) in FPC and SPC were 20.5 and 18.9 respectively. Mean corrected count increment at 24 hour (CCI 24h) FPC and SPC were 15.5 and 13.8 respectively. There were no significant differences between FPC and SPC P&gt;0.05. Conclusions: It was observed that 1-5 day's stored platelet-rich-plasma platelet concentrate (PRP-PC) was as effective as fresh PC to obtain acceptable platelet increment in childhood acute lymphoblastic leukemia. Furthermore, corrected count increment and percentage platelet recovery are important tools to evaluate platelet refractoriness.

Underlying Inflammation Impacts Immune Modulation Following Exposure to Platelet Concentrates and Packed Red Blood Cells

IntroductionTransfusion of platelet concentrates (PC) and packed red blood cells (PRBC) are lifesaving treatments for patients suffering from thrombocytopenia, haematological disorders, severe trauma and cancer. Platelets play a central role in mediating coagulation and wound healing, inflammation and innate immune responses. Red blood cells are crucial for delivery of oxygen to surrounding tissues and translocation of carbon dioxide in the lungs. In Australia, PC and PRBC are routinely stored for up to 5- or 42- days respectively prior to transfusion. Storage related changes of PC and PRBC may contribute to adverse patient outcomes and include increased release of biological mediators (sCD40L, IL-8), partial platelet activation, loss of 2,3-diphosphoglycerate and ATP, change in oxygen dissociation and altered morphology. Despite the therapeutic benefits of PC and PRBC transfusion, clinical studies suggest transfusion-associated modulation of the recipient immune response contributes to adverse outcomes including infection and mortality. Underlying inflammation is an important factor contributing to the development of transfusion-related acute lung injury (TRALI), a pro-inflammatory response, whereas in vitro transfusion models largely report leucocyte suppression following exposure to PC and PRBC. The relationship between the level of underlying inflammation and the impact on cell function post-transfusion remains unclear. We investigated the relationship between inflammation and modulation of dendritic cell (DC) and monocyte phenotype following exposure to PC and PRBC.MethodsAn in-vitro transfusion model was used to investigate changes in DC and monocyte phenotype following exposure to PC-supernatants (PC-SN) or PRBC-supernatants (PRBC-SN) in co-culture with a range of lipopolysaccharide concentrations (LPS; 0 µg/mL (no LPS), 0.25 µg/mL, 0.5 µg/mL, 0.75 µg/mL, 1 µg/mL LPS) to model different levels of underlying inflammation. Based on results of recent randomised controlled trials, we used fresh PC- and PRBC- SN. Freshly collected whole blood was cultured with RPMI media and day 2 (D2) PC-SN or D2 PRBC-SN at doses representing a 1 (10%), or 2-3 (25%) unit transfusion. Golgi-plug (containing Brefeldin-a) was used to inhibit protein release to facilitate intracellular staining of cytokines (IL-6, IL-8, IL-10, IL-12, IL-1α, TNF-α, MIP-1α, MIP-1β, MCP-1, IP-10) from DC and monocytes using via flow cytometry. Changes in monocyte and DC cytokine and chemokine production at different concentrations of LPS were assessed by a repeated measures one-way ANOVA with Tukey's post-test (P<0.05).ResultsCulture with LPS alone resulted in a concentration dependent decrease in the production of most monocyte and DC cytokines. Modelling a low dose (1 unit) transfusion with D2 PC-or PRBC- SN resulted in a reduction in monocyte and DC cytokine production in the presence of 0.25 µg/mL and 0.5 µg/mL LPS. This effect was less evident when higher concentrations of LPS (0.75 µg/mL, 1 µg/mL) were used. When modelling a high dose (2-3 unit) transfusion, exposure to D2 PC-or PRBC- SN resulted in a predominant suppression of monocyte and DC responses at all LPS concentrations tested.ConclusionOur data provides evidence that the level of underlying inflammation differentially affects DC and monocyte responses following exposure to PC-SN or PRBC-SN. Dose in combination with level of underlying inflammation significantly impacts monocyte and DC responses which may contribute and increase the risk of transfusion-associated poor patient outcomes including infection. DisclosuresNo relevant conflicts of interest to declare.

Platelet Count Increment after Transfusion of Fresh and Stored Platelet Concentrates in Children with Acute Lymphoblastic Leukaemia

Background: Therapeutic platelet transfusion is necessary to prevent bleeding due to thrombocytopenia in childhood acute lymphoblastic leukaemia (ALL). Platelet concentrates may be transfused as fresh platelet concentrate immediately or with five days of storage after collection Objectives: To compare the platelet count increment between fresh and stored platelet concentrate transfusion in children with acute lymphoblastic leukaemia. Methodology: This observational study was carried out in the Department of Clinical Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, in collaboration with the Department of Transfusion Medicine and Paediatric Haemato-Oncology, BSMMU, Dhaka. Seventy-two children diagnosed as acute lymphoblastic leukaemia were included in this study. ABO identical whole-blood derived platelet-rich-plasma platelet concentrates (PRP-PCs) were transfused within 24 hours of preparation or 1-5 days of storage. Effectiveness of platelet transfusion was carried out by measuring platelet count prior to transfusion, subsequently 1 hour and 24 hours of transfusion. Result: Post transfusion platelet count at 1 hour and 24 hours with fresh platelet concentrates (FPC) and stored platelet concentrates (SPC) were not statistically significant. Post transfusion platelet increment at 1 hour and 24 hours were also similar in both FPC and SPC. Corrected count increment (CCI) at 1 hour in FPC and SPC were 20.5±10.1 and 18.6 ±6.5 respectively and was statistically not significant (P: 0.168). CCI at 24 hours were 15.5±9.1 and 13±5.8 which were also statistically not significant. Percentage platelet recovery at 1 hour and 24 hours in both FPC and SPC were statistically not significant. But these values in SPC were consistently lower than that of FPC. All the values of FPC and SPC were well apart from the lower limit of CCI and percentage platelet recovery (PPR). It was also observed that 85.1% satisfactory platelet transfusion was achieved with FPC and rest of 14.9% was platelet refractory cases. In 79.5% cases satisfactory platelet transfusion were achieved with SPC and refractory cases were 20.5 %. Conclusion: One to five day’s stored PRP-PCs are as effective as fresh PRP-PCs to achieve satisfactory platelet increment.

Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat–derived platelet concentrates

BackgroundPlatelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat–derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. MethodsPLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat–derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). ResultsOf the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. ConclusionPLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use.

Platelet concentrate transport in pneumatic tube systems – does it work?

Blood sample transport via pneumatic tube systems (PTS) reduces the turnaround time of laboratories, but it might influence analysis results. Its effect on platelet concentrates (PCs) is not known. Platelet function was investigated after single and multiple PTS transport in comparison with storage and irradiation. Optical and impedance aggregation, CD-62, and microparticles changed as a result of storage, but not due to transport. Irradiation lowered platelet function independently. Multiple transport impaired thrombin receptor-activating peptide-induced aggregation. This investigation demonstrates the feasibility of PTS transport. As platelet function depends on storage, it may be more important to transfuse fresh PCs.

Supernatant of stored platelets causes lung inflammation and coagulopathy in a novel in vivo transfusion model

AbstractTransfusion-related acute lung injury is suggested to be a “2-hit” event resulting from priming and activation of pulmonary neutrophils. Activation may result from infusion of lysophosphatidylcholines (LysoPCs), which accumulate during storage of blood products. In the present study, we developed a syngeneic in vivo transfusion model to test whether storage of platelet concentrates (PLTs) results in lung injury in healthy rats as well as in a “2-hit” model using lipopolysaccharide-pretreated rats. In addition, the effect of washing of platelets was studied. In healthy rats, transfusion of aged PLTs caused mild lung inflammation. In LPS-pretreated rats, transfusion of aged PLTs, but not fresh PLTs, augmented pulmonary systemic coagulopathy. When PLTs components were transfused separately, supernatant of aged PLTs, but not washed aged platelets, induced pulmonary injury in the “2-hit” model. Supernatants of aged PLTs contained increased concentrations of LysoPCs compared with fresh PLTs, which enhanced neutrophil priming activity in vitro. We conclude that transfusion of aged PLTs induces lung inflammation in healthy rats. In a “2-hit” model, aged PLTs contribute to pulmonary and systemic coagulopathy, which may be mediated by LysoPCs, which accumulate in the supernatant of PLTs during storage.

Transfused stored platelets have the same haemostatic function as circulating native platelets

As thrombelastography (TEG) measures haemostasis in whole blood, we used this instrument to study whether transfused platelets (PLTs) have the same haemostatic function compared to native circulating PLTs. Further, we studied the effect of storage time on the haemostatic potential of platelet concentrates (PCs). During the decrease in PLT count after chemotherapy, TEG parameters were measured serially until the transfusion trigger was reached in 92 patients. TEG parameters for different ranges of native circulating PLTs could be assessed, which were compared to ranges obtained in the thrombocytopenic period in which the patient received PLT transfusions. Finally, we compared the haemostatic potential of fresh PCs (1-3 days) with PCs with longer storage time (4-5 days). No differences could be found in haemostatic potential between native PLTs and transfused stored PLTs (all P-values > or = 0.1). The transfusion of fresh PLTs demonstrated better haemostatic effects than longer stored PLTs, measured 1 h after transfusion. Both the time until a fixed level of clot firmness was reached (K-time) and the rate of clot growth (alpha angle) were superior for fresh PCs. TEG is able to monitor the haemostatic effects of PLT transfusion, with comparable haemostatic properties of native circulating and transfused stored-PLTs. Further, our data suggest that limited storage time is associated with a better haemostatic capacity. However, before TEG can be applied as a qualitative test in PLT transfusion, further research is needed with focus on clinical outcomes like bleeding episodes.

Canine platelet transfusions

To review potential platelet storage options, guidelines for administration of platelets, and adverse events associated with platelet transfusions. Data sources included original research publications and scientific reviews. Transfusion of platelet concentrates (PCs) plays a key role in the management of patients with severe thrombocytopenia. Currently PCs are stored at 22 degrees C under continuous gentle agitation for up to 5 days. Chilling of platelets is associated with rapid clearance of transfused platelets, and galactosylation of platelets has proven unsuccessful in prolonging platelet survival. Although approved by the American Association of Blood Banks, cryopreservation of human platelets in 6% DMSO largely remains a research technique. Pre-storage leukoreduction of PCs has reduced but not eliminated acute inflammatory transfusion reactions, with platelet inflammatory mediators contributing to such reactions. Canine plateletpheresis allows collection of a concentrate with a high platelet yield, typically 3-4.5 x 10(11) versus <1 x 10(11) for whole blood-derived platelets, improving the ability to provide sufficient platelets to meet the recipient's transfusion needs. Cryopreservation of canine platelets in 6% DMSO offers immediate availability of platelets, with an acceptable posttransfusion in vivo platelet recovery and half-life of 50% and 2 days, respectively. While data on administration of rehydrated lyophilized platelets in bleeding animal models are encouraging, due to a short lifespan (min) posttransfusion, their use will be limited to control of active bleeding, without a sustained increase in platelet count. Fresh PC remains the product of choice for control of bleeding due to severe thrombocytopenia or thrombopathia. While cryopreservation and lyophilization of canine platelets offer the benefits of immediate availability and long-term storage, the compromise is decreased in vivo recovery and survival of platelets and some degree of impaired function, though such products could still be life saving.

Cryopreservation of Canine Platelets

Platelet cryopreservation allows long-term storage and immediate availability of transfusion products. The addition of a preparation inhibiting platelet activation (Thrombosol, in 2% dimethyl sulfoxide [DMSO]) will enhance in vitro function and prolong in vivo survival of cryopreserved platelets compared with those preserved in 6% DMSO. Thirty-three research dogs. Prospective study. Eleven fresh canine apheresis platelet concentrates (PCs) were each split into 3 units: fresh and cryopreserved in 6% DMSO or Thrombosol. Platelet analysis, performed 1-10 weeks postfreezing, included in vitro functional testing and in vivo survival assessed by administration of biotinylated platelets. Platelet aggregation was diminished in cryopreserved PC. Cryopreserved platelets could be activated, as based on mean thrombin-stimulated P-selectin expression (6% DMSO, 23.0%; Thrombosol, 18.4%), although to a lesser extent than fresh PC (49.1%) (P < .0001). The mean maximum in vivo platelet recovery for fresh PC was 80.3%, significantly greater than recovery for 6% DMSO (49.2%) and Thrombosol PC (43.7%) (P< or = .001). The half-life (days) of fresh PC (3.8 +/- 0.4) was significantly (P < .002) greater than that of 6% DMSO (1.9 +/- 1.0) and Thrombosol (2.4 +/- 1.1) PC, with no difference (P= .3) between cryopreserved PC. Cryopreservation of canine platelets using Thrombosol did not provide any advantage over preservation using 6% DMSO. Cryopreserved platelets can be activated in vitro and provide therapeutic benefit when fresh platelets are unavailable. Further studies are needed to assess their in vivo hemostatic function.

Current methods of assessing platelet function: relevance to transfusion medicine

Current methods of assessing platelet function: relevance to transfusion medicine

Additive effects between platelet concentrates and desmopressin in antagonizing the platelet glycoprotein IIb/IIIa inhibitor eptifibatide

Platelet (PLT) glycoprotein (GP) IIb/IIIa receptor antagonists have demonstrated efficacy in decreasing ischemic complications of percutaneous coronary intervention and/or unstable angina. In case of bleeding, the drug can be stopped and PLT transfusions can be given. This crossover study tested the additive effects of PLT concentrates (PCs) after desmopressin (DDAVP) infusion in antagonizing the anti-PLT effects of GPIIb/IIIa inhibitors and aspirin. After eptifibatide and aspirin infusion (at standard dosages), 10 healthy volunteers received DDAVP or placebo. Thereafter, increasing amounts of PLTs from fresh single-donor apheresis concentrates were added in vitro to blood samples of all volunteers to increase PLT counts by 30 x 10(9), 60 x 10(9), or 120 x 10(9) per L. Adding platelets in vitro further improved PLT function after DDAVP: it shortened collagen-adenosine diphosphate closure times (p < 0.01), to normal ranges as measured by the PLT function analyzer (PFA-100). In contrast, normal PLT function could not be restored even when PLT counts were increased by 50 percent (120 x 10(9)/L) in the placebo group. Combined use of PLTs from fresh apheresis PC and DDAVP additively enhances recovery of normal PLT function after eptifibatide infusion. Such a strategy may help to avoid excessive transfusion of PC.

Room temperature storage and cryopreservation of canine platelet concentrates

SUMMARY Objective To determine whether in vitro viability and function, and microbiological sterility, of canine platelet concentrates (PC) could be retained during storage at 20 to 24 C (room temperature [RT]) for up to 7 days and cryopreservation for 6 months. Animals 14 mature dogs. Procedure PC prepared by centrifugation of fresh blood were stored for 7 days at RT with continuous agitation. An aliquot of each was cryopreserved with 6% dimethyl sulfoxide at −74 C for 6 months. Fresh PC (day 0) were tested by microbial culture and measurement of platelet count, mean platelet volume, pH, glucose and lactate concentrations, lactate dehydrogenase activity, response to hypotonic stress, and aggregation. Tests were also performed on PC stored at RT on days 3, 5, and 7, and on the cryopreserved aliquots after thawing. Results After 7 days at RT, microbial growth was not evident, and decrease in platelet number was not significant. On the basis of pH and glucose and lactate concentrations, metabolic activity was maintained throughout RT storage. On the basis of mean platelet volume and lactate dehydrogenase activity, platelet swelling and membrane damage had occurred. Aggregatory responses decreased during RT storage, and platelets recovered poorly from hypotonic stress. After cryopreservation for 6 months, microbial growth was not evident, but platelet numbers were significantly decreased. Mean platelet volume and lactate dehydrogenase activity were significantly greater, compared with values for day-0 PC. Crypreserved platelets aggregated poorly and did not respond to hypotonic stress. Conclusions Platelet viability and microbiological sterility are retained in canine PC stored for 7 days at RT, but platelet function progressively decreases and day-7 platelets are substantially damaged. Crypreservation of PC results in considerable damage, compared with that of PC stored at RT. Clinical Relevance Similar to human PC, canine PC stored at RT for up to 5 days can be recommended for treatment. (Am J Vet Res 1997;58:1338–1347)

Clinical and laboratory experience with erythrocyte and platelet preparations from a 0.5CPD Erythro-Sol opti system.

Red blood cells stored as concentrates or suspensions in additive solutions change rapidly their oxygen affinity mainly due to the loss of 2,3-diphosphoglycerate (2,3-DPG). When collected in CPD with half of the normal concentration of citrate and citric acid (0.5CPD) and stored in a new additive solution (Erythro-Sol), 2,3-DPG is better maintained. No studies of the oxygen affinity of red cells stored under these conditions have been published. In Erythro-Sol, red cells have a satisfactory in vivo recovery for 49 days but the conditions after 28 days, within which time most red cell units are transfused, have not been investigated. Of importance is also to be able to make platelet concentrates (PCs) from 0.5CPD blood. Little data are available concerning the clinical usefulness of platelets prepared from 0.5CPD buffy coats (BCs). Blood was collected in 0.5CPD, held at 20 degrees C for 3-4 h, then separated with the bottom-and-top technique into red cells, plasma and BC. In a storage experiment with 6 U the 2,3-DPG and P50 values were determined weekly and a number of in vitro parameters were tested on day 28. In 6 donors the in vivo recovery and survival of red cells were determined using a single-chromium technique. Transfusions of 212 0.5CPD-Erythro-Sol red cell units were given to hematological patients under supervision. PCs derived from pools of 0.5CPD BCs suspended in PAS2 (T-Sol) were transfused to 20 thrombocytopenic patients and compared with CPD-BC-PCs suspended in PASI. Corrected count increments (CCI) were determined. The erythrocyte 2,3-DPG and P50 values were normal or slightly subnormal initially but increased to supernormal levels during the 1 week, and remained at these levels for a further 1-3 weeks; the 2,3-DPG was two thirds of normal after 28 days, the P50 was 3.72 +/- 0.28 kPa after 14 days and 2.84 +/- 0.41 after 28 days (mean +/- SD). The P50 values corresponded closely (r2 = 0.903) to 2,3-DPG. The in vivo recovery of 4-week-stored red cells was 89.6 +/- 5.5% and the T50 was 32.2 +/- 2.0 days. No adverse effects were observed in the transfusions. The CCI values did not differ between test and control groups; in both, 3- to 5-day-stored PCs gave lower CCI than fresh (0-2 days) PCs. Patients with acute myeloid leukemia AML (n = 11) had significantly lower CCI values than patients with myelodysplastic syndrome, myeloma and lymphoma (n = 9; CCI 1 h: p = 0.001; CCI 24 h: p = 0.006). Red cells stored in Erythro-Sol sustain a normal or slightly lowered oxygen affinity for 2-4 weeks, their viability is excellent, and they are well tolerated in clinical transfusions. Platelets prepared from 0.5CPD-BCs cause CCI, of the same magnitude as CPD-BCs.

The expression of p-selectin during collection, processing, and storage of platelet concentrates: relationship to loss of in vivo viability.

Recent studies suggested that platelet activation with surface expression of p-selectin on stored platelets may be related to a loss of viability. At present, there has been no thorough investigation of the extent or significance of p-selectin expression during the collection, processing, and storage of platelet concentrates (PCs) under various conditions. Platelet surface expression of p-selectin (CD62) was determined on fixed platelet samples using fluorescein-conjugated monoclonal antibodies. Platelet viability was assessed by autologous transfusion of platelets stored for 5 days and labeled with either 51Cr or 111in. Little (2-10%) platelet expression of p-selectin was found in whole blood and platelet-rich-plasma preparations, whereas PCs showed a substantial increase in p-selectin expression to levels of 20 to 30 percent. Both fresh PCs and those stored for 5 days, obtained with one cell separator (MCS, Haemonetics) showed substantially lower levels of p-selectin expression than PCs from two other cell separators (Spectra, COBE, and CS-3000 with TNX-6, Baxter Healthcare). Exposure of platelets to EDTA, cold, or a pH below 6.2, conditions that are known to result in the loss of viability upon transfusion, produced substantial and irreversible p-selectin expression. PCs with a pH of 6.2 to 6.8 (conditions in which no loss of viability has been demonstrated) also showed pronounced p-selectin expression, which returned to control values after incubation at 37 degrees C in plasma at pH 7.0 to 7.2. With storage under current conditions the in vivo studies (n = 61) demonstrated a rather poor correlation between p-selectin expression and the percentage of recovery (r = -0.25) but a somewhat better correlation with survival (r = -0.42). Better correlations were observed with the extent of shape change, lactate, and hypotonic shock response. These studies show that p-selectin expression on the platelet surface is a predictor of platelet viability, although the extent of shape change and the hypotonic shock response may be more sensitive.

A multicenter evaluation of reproducibility of swirling in platelet concentrates

Eleven laboratories participated in a study investigating whether swirling can be used in monitoring platelet discoid morphology in platelet concentrates (PCs). In each laboratory, two readers observed the swirling pattern in 1- to 5-day-old PCs, in PCs containing spherical platelets obtained by 4 degrees C storage (spherical PCs), and in PCs containing known proportions of spherical and discoid platelets. Swirling was reported as positive, intermediate, or negative. Moreover, platelet morphology was evaluated by oil-phase microscopy. Swirling of spherical PCs was found to be positive and negative in 5 and 68 percent of cases, respectively. Conversely, swirling of fresh PCs was positive and negative in 83 and 2 percent of cases, respectively. Results in PCs containing known amounts of added spherical platelets were intermediate between those in fresh and spherical PCs. The observed agreement between the two readers (number of concordant readings/total readings) was 67 percent. Most disagreements concerned positive versus intermediate or intermediate versus negative evaluations, whereas a positive versus negative disagreement was observed in only 1 percent of cases. The percentages of discs found by microscopy showed greater variation among the different laboratories, indicating that this measurement should be better standardized. Evaluation of swirling seems promising for large-scale quality control of PCs. Further studies are needed to confirm this hypothesis.

Effect of amphotericin B and fluconazole on platelet membrane glycoproteins.

Fever, chills, and reduced platelet recovery may result when platelets are transfused simultaneously with amphotericin B. Amphotericin B reportedly increases the pitting of membranes in stored platelets. The effects of amphotericin B and another antifungal agent, fluconazole, on platelet membrane glycoproteins (GP) were examined by the incubation of split aliquots of fresh and stored platelet concentrates (PCs) with these drugs for 3 days in storage bags. To determine the effect of storage, PCs were stored for 5 days, and aliquots removed on Days 1 through 5 were placed in platelet storage bags with 4 micrograms per mL of amphotericin B for 2 to 6 hours. Membrane glycoprotein expression was assessed by flow cytometry with fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed against the following antigens: GPIb (CD42b), CD63 (an activation protein), P-selectin (CD62), and GPIIb/IIIa (CD41a). Amphotericin B produced a concentration-dependent decrease in the surface binding of CD42b MoAb with no consistent changes in the binding of CD41a, CD63, or CD62 MoAbs after a 3-day exposure. Stored but not fresh PCs showed decreased binding of MoAb CD42b after a 6-hour exposure to amphotericin B (4 micrograms/mL). Fluconazole produced no changes. When the binding of MoAb CD42b to permeabilized platelets was used to measure total platelet content, amphotericin B (4 micrograms/mL) decreased MoAb CD42b binding to a similar degree in fresh and stored platelets. Inhibition of aggregation to ADP and collagen and ADP and epinephrine was seen in stored but not fresh PCs. Therapeutic levels of amphotericin B resulted in partial loss of total platelet GPIb in fresh and stored PCs, but decreased surface expression of platelet membrane GPIb only in stored platelets. This difference between fresh and stored platelets may be related to the limited reservoir of GPIb available for redistribution to the membrane in the previously stored PCs and may account for the decreased recovery of transfused platelets observed in some patients receiving amphotericin B.

EDTA Reveals the Aggregation State and Functional Integrity of Platelets

Dear Sir, The clinical use of platelet concentrates (PC) is influenced by custom and preference rather than by the functional quality of the preparation.' The Transfusion Service has long recognised the need for high quality components of consistent and definable composition. It is accepted however that the minimum testing requirements for PC are based exclusively on cell counting and pH, rather than on functional integrity. Our Centre, which is one of the largest PC producers in the UK, has been active in seeking a test that would reflect platelet functionality. We have developed a standardized and innovative quality index, reflecting the morphological and functional integrity of platelets. The test is based on changes that occur when platelets in citrated samples are suspended in a solution containing EDTA (0.5 ml in a 4 ml EDTA tube). Thus using paired & EDTA samples, the differences in the recorded cellular indices (e.g. dMPV, using a Technicon H*l Cell Analyser) can be calculated to provide accurate information on platelet quality. We have already shown that dMPV correlates with a number of other functional tests.' This procedure has now been in use in our Centre for four years resulting in the following new observations: 0 Changes in aggregation state: The use of paired sampling and the calculation of the index dPLT gives an indication of the relative state of aggregation. In addition, as is already known, the inclusion of EDTA tubes means that a true platelet count is obtainable as EDTA disperses reversible aggregates. 0 Improved cellular discrimination: In citrated samples, clumps and aggregates may appear as pseudo-leuco/erythrocytes. The difference in peroxidase/basophil counts and altered differential counts are indicators of this phenomenon and can be used in optimising processes in order to reduce clumping. 0 Changes in size distribution: Using this technique, we have been able to observe clear differences in the quality (based on MPV and delta indices) of recovered platelets, apheresis platelets, and recently those prepared by the pooled buffy coat method. In the latter, we were able to observe at an early stage in development that larger platelets were obtained, more akin to apheresis than recovered. 0 Platelet storage stability: At least two populations of platelets have been observed in fresh routine PC, with progressive merging to one population towards the end of their 5 day shelf life. Alongside this, the decline in dMPV is related to PC age in an inverse logarithmic manner. We believe that this test fulfils the need for sensitivity, specificity and simplicity in platelet function testing. The use of these new platelet indices heralds a new era of more uniform and better controlled PCs, all to the eventual benefit of the patient.

Functional capacity of transfused platelets estimated by the Thrombostat 4000/2

Post-transfusion platelet increment is a useful test for the evaluation of transfusion efficacy. It does not, however, give information about the in vivo platelet function. We have evaluated the in vitro bleeding time (IVBT) using the Thrombostat 4000/2 with ADP as activating agent in 60 platelet transfusions given to 17 chemotherapy-induced severely thrombocytopenic patients. Determinations were performed before and after transfusion of platelet concentrates (PC) prepared from buffy coat. Both fresh and stored platelets resulted in significant reductions of the IVBT already 10 minutes after completed platelet transfusion. In the majority of patients there was a correspondence between IVBT and platelet increment. However, in 30% of the cases there was no improvement of the IVBT despite an increase of the platelet count. Fresh PCs were more effective than stored. The IVBT seems to represent a clear-cut improvement in the possibilities for evaluating and monitoring the effect of platelet transfusion.