White cell apoptosis in platelet concentrates

BACKGROUND: The aim of the present study was the evaluation of the apoptosis in residual white cells (WBCs) contained in platelet concentrates (PCs) and of the relationship of this apoptosis with the concentration of inflammatory cytokines in the medium and with platelet activation. STUDY DESIGN AND METHODS: Three independent methods were used to evaluated apoptosis in WBCs present in 9 PCs, either from single donors by apheresis (SD-PCs) or from pooled buffy coats (BC-PCs). All PCs were divided in two parts, one of which was irradiated. PCs were stored up to 4 days at room temperature, and samples were withdrawn daily for analysis of apoptosis, of platelet activation (surface and soluble CD62P), and of cytokine concentration (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, and tumor necrosis factor alpha). RESULTS: Apoptosis was found to occur with storage in both irradiated and nonirradiated units. Platelet activation increased with storage time and was higher in BC-PCs. The amount of released cytokines was rather variable among PC units. Only IL-8 was consistently found to increase with storage time. CONCLUSIONS: Apoptosis of residual WBCs occurred in PC units as a function of storage time. The amount and the time course of apoptosis seem to correlate with IL-8 release rather than with platelet activation or with the occurrence of febrile nonhemolytic transfusion reactions.     

Effects of filtration and gamma radiation on the accumulation of RANTES and transforming growth factor-β1 in apheresis platelet concentrates during storage

BACKGROUND: Platelet-derived biologic response modifiers (BRMs) including RANTES and transforming growth factor (TGF)-beta1 accumulate in platelet components during storage because of platelet activation, and they may play a causative role in nonhemolytic febrile transfusion reactions. The majority of PCs with high unit values are provided by single donor apheresis in Japan. STUDY DESIGN AND METHODS: RANTES and TGF-beta1 levels in platelet units prepared from single-donor apheresis platelet concentrates (apheresis PCs) and units from whole blood (buffy coat PCs) were investigated. The effects of prestorage and poststorage filtration and gamma radiation on the levels of RANTES and TGF-beta1 in the supernatant of apheresis PCs during storage were also examined. RESULTS: The levels of RANTES and TGF-beta1 increased during storage from Day 0 to Day 5. The levels of RANTES and of TGF-beta1 correlated with the platelet concentration (p<0.01), but not with the residual white cell concentration in apheresis PCs that were not white cell reduced by filtration (p>0.05). In addition, there was a correlation between RANTES and TGF-beta1 levels (p<0.01). In white cell-reduced apheresis PCs using negatively charged filters as well as in gamma-radiated apheresis PCs, the levels of these two BRMs-did not differ at any storage time from those of untreated apheresis PCs. Filtration of apheresis PCs with negatively charged filters after 3 days of storage significantly (p<0.05) reduced the levels of RANTES, but not of TGF-beta1. There was no reduction in the levels of RANTES and TGF-beta1 levels by positively charged filters. The RANTES levels in buffy coat PCs were slightly higher than but not significantly different from those of apheresis PCs during storage, except for the level on Day 1. There were no differences in the TGF-beta1 levels in apheresis and buffy coat PCs during storage. CONCLUSION: Prestorage filtration and gamma radiation had neither preventive effects on the accumulation of RANTES and TGF-beta1 nor adverse effects on platelet activation. Negatively charged filters might be useful for the reducing the levels of RANTES in stored apheresis PCs.     

Evaluation of pooled platelet concentrates using prestorage versus poststorage WBC reduction: impact of filtration timing

BACKGROUND: Concern for the undesirable consequences of transfusing passenger WBCs is leading to the general use of WBC-reduced platelet concentrates (PCs). However, the impact of prestorage versus poststorage WBC reduction on the quality of platelet products has not been clearly defined. STUDY DESIGN AND METHODS: Pooled PCs were WBC reduced before or after 5-day storage, by use of a WBC filter (PXL-8, Pall Corp.). Samples from pools were taken on days 1 and 5, before and after filtration, and on Day 9 of storage and assessed for cell counts, biochemical values, expression of platelet glycoproteins, thrombin generation, and content of IL-6, IL-8, TNFalpha, transforming growth factor beta1 (TGFbeta1), and anaphylatoxins C3a and C4a. RESULTS: Filtration of fresh and 5-day-stored pooled PCs via a PXL-8 filter was similarly efficient, rendering pools with low WBC counts (<1 x 10(6) cells) and high platelet recovery (>95%). No major changes were found in the metabolic behavior or the expression of platelet GPIb, GPIIb/IIIa, CD62, and CD63 in PCs filtered before or after storage. Filtration, either before or after storage, increased by less than 5 percent the proportion of CD62+ platelets. Moreover, no changes were found in the concentration of prothrombin fragments 1 and 2 and thrombin-antithrombin complexes in the pooled PCs derived from the time of filtration. Finally, prestorage WBC reduction abrogated the accumulation of IL-6 and IL-8, but it did not prevent that of anaphylatoxins C3a and C4a nor of TGFbeta1. However, filtration through a PXL-8 filter significantly reduced (40-90%) the amount of IL-8, C3a, and C4a in the filtrate. CONCLUSIONS: The timing of PXL-8 filtration of PCs has little impact on the efficiency of WBC reduction and on platelet recovery, and it does not seem to affect the quality of platelets or the generation of thrombin in the PCs. As regards the goal of reducing the amount of bioactive products in PCs, it remains uncertain as to whether prestorage WBC reduction fully eliminates the need for poststorage filtration. Prestorage filtration leads to low levels of IL-6 and IL-8 in PCs, but it does not impair the poststorage content of TGFbeta1 or anaphyla-toxins. By contrast, poststorage PXL-8 filtration removes significant amounts of C3a and C4a, and thus it might provide clinical benefits beyond those of prestorage WBC reduction.     

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

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: Platelet concentrations in 60 mL of HCPs were predictable according to Q_col (r² = 0.735). HCP yields varied from 0.9 to 3.2 × 10¹¹, depending on the desired final platelet concentrations in 60 mL, with an overall average of 1.92 × 10¹¹ (n = 32). Apheresis PCs had a mean platelet yield of 2.9 × 10¹¹ (1.3‐4.4 × 10¹¹, n = 20) and 3.9 × 10¹¹ (2.2‐5.8 × 10¹¹, n = 12) at concentrations of 1.3 × 10¹² per L for single‐needle and dual‐ needle procedures, respectively. Median WBC counts were 5.6 × 10³ for HCPs and 2.0 × 10⁴ for apheresis PCs, with >99 percent expected to be less than 1 × 10⁶. 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^oC was maintained at a level of >6.2 for more than 3 days for yields >1.6 × 10¹¹, less than 2 days for yields 1.6 to 2.2 × 10¹¹, and less than 24 hours for yields >2.2 × 10¹¹. HCPs showed good in vitro characteristics and could be stored for 1 to 3 days, depending on the total number of platelets collected. CONCLUSION: 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.     

Quality and clinical response to transfusion of prestorage white cell-reduced apheresis platelets prepared by use of an in-line white cell-reduction system

BACKGROUND: This study evaluated the quality and clinical effectiveness of white cell (WBC)-reduced apheresis platelets collected by the use of a new technology, fluidized particle-bed separation. STUDY DESIGN AND METHODS: In phase 1, six suitable donors underwent two separate plateletpheresis procedures on one occasion, each separated by less than 10 minutes. In random order, a control unit was collected with the COBE Spectra and a test unit with the Spectra Leukocyte-Reduction System (LRS). The quality of apheresis platelet components was assessed by an in vitro test panel, and residual WBCs were counted by Nageotte chamber and flow cytometric methods. For the in vivo studies, the test and control units were randomly labeled with either 51Cr or 111In at the end of storage and transfused simultaneously to the donor. Samples were taken for calculation of platelet survival and recovery. In phase II, 109 thrombocytopenic patients were given platelets collected by use of the Spectra LRS. RESULTS: Test platelets had significantly fewer residual WBCs (median 7.6 x 10(4)) than control platelets (median 3.9 x 10(5)), with equivalent in vitro function values. Test and control platelets had similar recovery and survival. Transfused platelets collected by use of the LRS achieved a mean 1-hour corrected-count increment of 19.3. CONCLUSION: The LRS collects platelet components with significantly lower WBC contamination without adverse effects on the function or in vivo survival of the platelets.     

WBC subset analysis of WBC-reduced platelet components

BACKGROUND: WBC-reduced platelet components may be prepared by filtration or apheresis processing. Both methods have previously been shown to result in a residual total WBC content <5 x 10(6) per component. However, there may be differences in the efficacy of these techniques for removing certain WBC subsets. STUDY DESIGN AND METHODS: Two multiparameter flow cytometric assays were developed and validated to perform WBC analysis on WBC-reduced platelets collected with two apheresis instruments (Amicus and COBE Spectra) and on 6 units of filtered pooled random-donor platelet concentrates. RESULTS: All components contained <1 x 10(5) WBCs. The COBE Spectra and Amicus apheresis platelet components contained more WBCs than did filtered pooled platelets (p<0.05). Lymphocytes (T and B), monocytes, and granulocytes were identified in all components. Granulocyte content was lowest in the Amicus components and filtered pools. Monocytes were lowest in filtered pools. Amicus platelet components had fewer granulocytes and monocytes than the COBE Spectra platelets. Amicus and COBE Spectra components contained more lymphocytes than the filtered pools. CONCLUSION: Multiparameter flow cytometry can be used to quantify and characterize WBCs in WBC-reduced platelet components. WBC reduction by filtration or apheresis was highly effective. WBCs from each subset were identified in all components. Although filtered pools had the lowest numbers of WBCs, the very low numbers observed in all components suggests that the absolute quantitative differences in WBC subset content are of questionable clinical significance.     

Comparison of plateletpheresis concentrates produced with Spectra LRS version 5.1 and LRS Turbo version 7.0 cell separators

BACKGROUND: The importance of transfusing WBC-reduced blood components is widely recognized, as it reduces the risk of alloimmunization and transfusion-transmitted CMV infections. The latest generation of cell separators allows the collection of WBC-reduced apheresis platelet concentrates (APCs). MATERIALS AND METHODS: Consecutive APCs (n = 232) were retrospectively evaluated: 163 collected with the Spectra LRS [leukocyte-reduction system] Version 5.1 (Group A) and 69 with the LRS Turbo Version 7.0 (Group B) (both: COBE BCT). Donor peripheral blood count, procedure data, platelet yield, collection efficiency (CE), and residual WBC count in APCs were recorded. RESULTS: The platelet yield was higher in Group B than in Group A: 5.5 +/- 1.4 versus 4.4 +/- 1.1, p<0.0001; residual WBCs were <5 x 10(6) in 99.4 percent of Group A APCs and in 97.1 percent of Group B APCs. CE was higher in Group B than in Group A: 51.4 +/- 8.7 versus 43.6 +/- 6.3, p<0.0001. Moreover, a correlation between predonation platelet count and platelet yield was observed in both groups. A double product (platelet yield >6.0 x 10(11)) was obtained in 28.9 percent of Group B APCs and in 9.2 percent of Group A APCs. CONCLUSIONS: The Spectra LRS Turbo version 7.0 release showed a better CE and resulted in a higher platelet harvest than did the LRS version 5.1. High predonation platelet counts allow a higher platelet yield.     

WBC content of platelet concentrates prepared by the buffy coat method using different processing procedures and storage solutions

BACKGROUND: The number of WBCs in platelet concentrates (PCs) prepared by the buffy coat (BC) method with different storage solutions can result in low (5 x 10(6)/unit) WBC levels by the use of careful centrifugation techniques without filtration. At present, most blood banks use filtration steps to meet these requirements. The difference in processing methods and suspension solutions prompted the investigation of the influence of the various procedures on the WBC and platelet content of PCs. STUDY DESIGN AND METHODS: PCs from 5 BCs were harvested without or with inline filtration (AutoStop BC, Pall Corp.) in either plasma (PCs-plasma) or platelet additive solution (PCs-PAS-2). After preparation, samples were taken for counting WBCs and platelets and for analyzing WBC subsets by flow cytometry using specific MoAbs. The WBCs were concentrated before analysis of the WBC subsets. Results less than 2.5 cells per microL were considered below the limit of accuracy of the subset analysis. RESULTS: All filtered PCs met the AABB standard of 5 x 10(6) per unit and the European guidelines of 1 x 10(6) per unit. None of the nonfiltered PCs met the European guidelines, but all met the AABB guidelines. All filtered units gave residual WBC counts below the detection limit for subset analysis. Filtered PCs-plasma gave significantly higher platelet counts than filtered PCs-PAS-2 or nonfiltered PCs (p<0.01, ANOVA). CONCLUSION: Careful centrifugation of pooled BCs, with plasma or PAS-2, can result in PCs with low WBC contamination levels. However, filtered PCs are superior, because of better WBC removal and higher platelet counts.     

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.     

WBC-reduced platelet concentrates from pooled buffy coats in additive solution: an evaluation of in vitro and in vivo measures

BACKGROUND: The use of a platelet additive solution (PAS-II, Baxter) may have benefits over plasma for storage of platelets. It was the aim of this study to develop a method to produce WBC-reduced platelet concentrates (PCs) in PAS-II with >240 x 10(9) platelets and <1 x 10(6) WBCs per unit, which can be stored for 5 days at pH >6.8 and that will give sufficient platelet increments after transfusion: a 1-hour CCI of >7.5 and a 20-hour CCI of >2.5. STUDY DESIGN AND METHODS: PCs were made from five pooled buffy coats and 250 g of PAS-II. After centrifugation the PCs were WBC-reduced with a filter (Autostop BC, Pall Biomedical) and stored in a 1000-mL polyolefin container. CCIs were assessed in stable hemato-oncologic patients after 5-day old PCs were transfused. RESULTS: Routinely produced PCs contained a median of 310 x 10(9) platelets (n = 5,363) with 3.5 percent containing <240 x 10(9) platelets, in a median volume of 320 mL (n = 11,834). The median number of WBCs was <0.03 x 10(6) (n = 694). The WBC count exceeded 1 x 10(6) in three PCs, but it was always <5 x 10(6), giving 99-percent confidence that more than 99.5 percent of the units will contain <1 x 10(6) WBCs. The pH remained >6.8 on Day 8, provided the concentration was below 1.1 x 10(9) platelets per mL (n = 32). After 28 transfusions in 28 patients, the 1-hour CCI was 12.6 +/- 4.3 (mean +/- SD, with 2/28 CCIs <7.5) and the 20-hour CCI was 8.9 +/- 5.6 (with 4/28 CCIs <2.5). Limitations of this study include the absence of a control group of patients receiving platelets stored in plasma and of in vivo radiolabeled survival studies, but a comparison of these data with previously published data suggested that the in vivo survival of platelets stored in PAS-II is less than that of platelets stored in plasma. CONCLUSION: The WBC-reduced PCs conformed to specifications. These WBC-reduced PCs could be stored at least 5 days with maintenance of pH, and they gave sufficient increments after transfusion to patients.     

Comparison of a new WBC-reduction system and the standard plateletpheresis protocol in the same donors

BACKGROUND: A cell separator (Spectra, Gambro BCT) with an integrated leukoreduction system (LRS) for producing WBC-reduced single-donor platelet concentrates has been shown to result in a slightly reduced collection efficiency as compared to the former Spectra system without LRS. A novel modified system for improved collection efficiencies (LRS Turbo, Gambro BCT) was evaluated. STUDY DESIGN AND METHODS: Each of 37 donors underwent plateletpheresis using the LRS Turbo (LRS-T) and the standard LRS (LRS) of the Spectra cell separator. The collection efficiency and WBC contamination of the different techniques were compared. Platelets were counted automatically and WBCs were counted by using one or two full grids of a Nageotte chamber. RESULTS: The preseparation and postseparation numbers of RBCs, WBCs, and platelets, as well as the number of collected platelets, did not differ for the two techniques. In the LRS-T separations, the collection efficiency was 112 percent of that in the LRS procedures. Median residual WBCs in the platelet components were 0.0256 x 10(6) per LRS-T procedure and 0.0253 x 10(6) per LRS procedure. The purity of the LRS-T components was not less than that of the standard LRS components, whereas the collection efficiency of the LRS-T was significantly greater, 44.9 percent versus 40.7 percent. CONCLUSIONS: The LRS-T procedures produced platelet concentrates with WBC-reduction capacity that is comparable to that obtained with the standard LRS procedures, which have previously been described as satisfying the most stringent criteria for WBC-reduced platelets. The new technique significantly improved the collection efficiency of the plateletpheresis procedure.     

Interleukin 1 beta and tumor necrosis factor levels in stored platelet concentrates and the association with gene polymorphisms

BACKGROUND: Cytokines (IL-1beta and TNF) generated by WBCs during storage of PLT concentrates have been associated with febrile nonhemolytic transfusion reactions. STUDY DESIGN AND METHODS: This study was undertaken to investigate whether there is an association between the polymorphisms of IL1B -511C/T and +3953C/T, IL1RN intron 2 VNTR and TNFA-308G/A genes and the increase of cytokines during the storage of PLT concentrates produced by plasma-rich PLTS (PRP-PC) or apheresis PLTs. RESULTS: Thirty PRP-PCs were studied and a progressive increase of IL-1beta and TNF during storage was revealed. IL1-beta and TNF levels were inversely correlated with the content of PLTs in PRP-PCs detected on Day 3 (p = 0.004) and Day 5 (p = 0.019), but not on Day 7. There was association of IL1B-511T polymorphism and IL-1beta levels (Day 5, p = 0.063, only tendency and on Day 7, p = 0.038, significant). There was no association of the other polymorphisms (IL1B+3953C/T, IL1RN intron 2 and TNFA-308G/A) with their respective cytokines. CONCLUSION: The great variation of cytokine levels in the plasma of PLT concentrates (PCs) during storage may also be caused by cytokine gene polymorphisms, as well as WBC contamination, material that the bags are made of, and storage time, as previously described.     

The influence of automated plateletpheresis on systemic levels of hematopoietic growth factors

BACKGROUND: Megakaryocytopoiesis and platelet production are regulated by several hematopoietic growth factors. The present study focuses on the effects of automated plateletpheresis on systemic levels of different hematopoietic growth factors. STUDY DESIGN AND METHODS: Platelet count, mean platelet volume, and serum levels of thrombopoietin, erythropoietin, interleukin-1beta, interleukin-6, and stem cell factor in 21 healthy donors were measured before platelet collection, after the first half of the apheresis procedure, at the end of apheresis, and on Days 1, 2, and 7 thereafter. RESULTS: Thrombopoietin levels (initial level: 49.5 +/- 25.5 pg/mL) showed a significant increase between measurements taken at the end of apheresis and Day 1 (56.9 +/- 26.7 pg/mL; p = 0.01). There was a highly significant decrease in stem cell factor levels during apheresis (p<0.0005), reaching preapheresis values (1679 +/- 210 pg/mL) on Day 1. A highly significant increase in erythropoietin levels (initial level: 7.5 +/- 4.0 U/L) was seen after apheresis (p<0.0005 on Days 1 and 2). The level remained significantly elevated until Day 7 (p = 0.004). Interleukin-1beta and interleukin-6 levels (before donation: 1.4 +/- 1.8 pg/mL and 1.1 +/- 0.7 pg/mL, respectively) did not change during the observation period. Thrombopoietin levels correlated consistently and inversely with stem cell factor levels after apheresis (Day 1, r = -0.46, p = 0.035; Day 2, r = -0.50, p = 0.02; Day 7, r = -0.50, p = 0.02). CONCLUSION: The data show a coordinated response of the hematopoietic system to platelet loss. It is suggested that the decrease in serum stem cell factor levels during apheresis reflects the consumption of stem cell factor by early hematopoietic progenitors that expand to initiate early megakaryocytopoiesis. The temporary increase in thrombopoietin is the result of platelet loss and serves as a stimulus for subsequent thrombopoiesis. The pronounced elevation of erythropoietin after apheresis suggests a role for this primarily erythropoietic cytokine in thrombopoiesis, too.     

Platelet storage lesion of WBC-reduced, pooled, buffy coat-derived platelet concentrates prepared in three in-process filter/storage bag combinations

BACKGROUND: With the implementation of universal WBC reduction in the United Kingdom, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) are used in routine production. The effects of three filter/storage bag combinations on platelet activation and microvesiculation and on the activation of coagulation were investigated. STUDY DESIGN AND METHODS: Using pooled BCs from the same donors, three filter/storage bag combinations (Autostop BC/CLX, Pall Biomedical; Sepacell PLX5/PL2410, Asahi Medical; and Imugard III-PL 4P/Teruflex, Terumo) were compared with unfiltered controls for their effects on microvesiculation and other storage-induced changes in platelets. Process efficiency was measured by platelet yield and residual WBC count. The storage changes were assessed: pH, activation of platelets measured by CD62P on the platelet surface and in supernatant plasma, quantitation of platelet-derived and RBC-derived microvesicles, cellular injury measured by annexin V in the supernatant plasma, and activation of the coagulation system measured by kallikrein-like and thrombin-like activities, prothrombin fragment 1+2, and thrombin-antithrombin complex. RESULTS: All three filters were comparable in terms of platelet recovery and WBC removal, and none induced immediate platelet activation or microvesiculation. With storage, platelet activation or microvesiculation increased in platelets prepared by all three filters and in unfiltered controls, but these effects were significantly less in the Imugard PCs than in controls. These findings were consistent with those for annexin V in the supernatant plasma, which were lower in Imugard PCs than in other products. Sepacell and Imugard filters reduced RBC-derived microvesicles to 50 percent of control levels, but the Autostop filter had no effect. On storage, levels of RBC-derived microvesicles in filtered products remained static, but levels in the unfiltered control doubled. Kallikrein- and thrombin-like activities were generated only by the Autostop filter without any further increment on storage. CONCLUSION: WBC-reduced pooled BC-PCs prepared by various filter/bag combinations were equivalent on Day 1 but differed during storage in terms of platelet activation or microvesiculation.     

Evaluation of a whole-blood WBC-reduction filter that saves platelets: in vitro studies

BACKGROUND: In this study, a new WBC-reduction in-line filter that removes WBCs but not platelets was evaluated. Three WBC-reduced blood components were prepared: RBCs, plasma, and platelet concentrates (PCs). STUDY DESIGN AND METHODS: Whole-blood components (n = 30) were filtered within 2 to 4 hours after collection and then were centrifuged and separated into RBCs, plasma, and WBC-reduced buffy coat. Saline-adenine-glucose-mannitol solution was added to the RBCS: The WBC-reduced buffy coats were stored overnight; on the following day, PCs were prepared from pooled WBC-reduced buffy coats and stored in a medium composed of approximately 35 percent CPD plasma and 65 percent platelet additive solution (T-Sol, Baxter). The WBC-reduction capacity of the filter, the recovery of cells after filtration, and the in vitro storage of RBCs (n = 10) and platelets (n = 6) were evaluated. RESULTS: Mean and maximum WBC counts after filtration were 0.08 x 10(6) and 0.3 x 10(6), respectively, per filtered whole-blood unit. Recovery of RBCs (mean values) after filtration was 90 percent in whole-blood components and 73 percent in RBCS: Recovery of platelets (mean values) was 81 percent after filtration and 66 percent in PCS: The in vitro storage study of RBCs showed results comparable with previously published data, except for a lower degree of hemolysis. In the in vitro platelet storage study, results were compared with those of standard preparations. In all essentials, similar results were found. CONCLUSION: The results of the present study suggest that effective WBC reduction meets current standards and satisfactory recovery after filtration. The storage characteristics for RBCs and PCs are similar to those of standard preparations. Use of a whole-blood in-line filter to save platelets is a new option for whole-blood processing, which may simplify WBC reduction and blood component preparation, as well as reduce costs in the future.     

Febrile and allergic transfusion reactions after the transfusion of white cell-poor platelet preparations

BACKGROUND: Nonhemolytic transfusion reactions (NHTRs) frequently occur after platelet transfusions. White cell (WBC)-derived inflammatory cytokines can cause these reactions, but they are rarely found in WBC-poor platelet preparations. Transfusion reactions were investigated with regard to the residual WBC content in the stored platelet concentrate in two consecutive study periods.
STUDY DESIGN AND METHODS: In the first study period, platelet concentrates were WBC-reduced by bedside filtration. In the second period, all platelet concentrates were filtered before storage. Recipients who experienced transfusion reactions were examined with regard to their main clinical symptoms during and after transfusion. In the supernatant of the involved platelet concentrates, concentrations of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)α, macrophage inflammatory protein 1α, and RANTES were analyzed.
RESULTS: The incidence of transfusion reactions remained steady when the transfusion regimen was changed from bedside filtration to prestorage WBC filtration (1.63% and 1.56%; p = 0.84). In both periods, NHTRs were predominantly of allergic origin. Inflammatory mediators IL-1β, IL-6, IL-8, and TNFα were detectable in only a minority of platelet components involved in NHTRs. Platelet concentrates involved in allergic reactions contained high concentrations of RANTES (668 ± 223 ng/mL).
CONCLUSIONS: Prestorage WBC filtration did not reduce the incidence of these reactions, and inflammatory cytokines were of minor relevance. The proinflammatory platelet-derived chemokine RANTES, which accumulates even in WBC-reduced platelet concentrates, was associated with allergic transfusion reactions. Platelet-derived mediators may be a key to understanding NHTRs.     

Peripheral blood progenitor cell grafts contain high levels of platelet-secreted mediators

BACKGROUND: The cytokine network in peripheral blood progenitor cell (PBPC) grafts may affect hematopoietic reconstitution or the risk of postransplant relapse of malignant disorders through effects on normal progenitor cells or contaminating malignant cells. Whether thrombopoietin (TPO), SCF, and platelet-secreted mediators are parts of this network was investigated. STUDY DESIGN AND METHODS: Peripheral blood and PBPC plasma samples were collected consecutively from patients with malignant disorders who underwent PBPC harvest. Blood samples were collected immediately before and after apheresis. Patients underwent mobilization by chemotherapy plus G-CSF, except for one patient who received only G-CSF. Plasma levels were also determined for healthy controls. RESULTS: PBPC grafts had greater levels of platelet-secreted platelet factor 4 (PF4), beta-thromboglobulin, and platelet-derived growth factor isoform AB, as compared with venous levels in patients and controls. Although platelet and PF4 levels in autografts were significantly correlated, the graft:blood ratio was higher for PF4 than for platelets. In both the patients' blood and the autografts, TPO levels were increased from the levels in normal controls. Blood and graft levels of SCF were within the normal range. CONCLUSION: The cytokine network of PBPC autografts includes increased levels of TPO and several platelet-derived mediators.     

Apheresis-induced platelet activation:comparison of three types of cell separators

BACKGROUND: Platelet-harvesting technology differs in various cell separators. Alteration in shear stress and biocompatibility of surfaces may give variable platelet activation and thereby affect the quality of the component. STUDY DESIGN AND METHODS: Four groups (n = 10) of single-needle apheresis procedures using three cell separators, were compared: 1) Spectra LRS, 90-minute harvesting time; 2) MCS+, 90-minute harvest; 3) Amicus, 90-minute; and 4) Amicus, 45-minute. Whole-blood samples were collected from the donors as were samples from the final components at intervals during the first 4 hours after cessation of the apheresis. Platelet activation status and platelet activation capacity after agonist stimulation were assessed by flow cytometry. RESULTS: No activated platelets were found in preapheresis and postapheresis samples from the donors. The platelets in the components from the Amicus (90-min) were significantly more activated than those in the other groups of components: that is, there was increased size of platelet aggregates, increased fraction of microparticles, increased degranulation, increased fibrinogen receptor activation, and decreased von Willebrand factor receptor expression. Moreover, the response of these platelets to agonist stimulation was reduced for all activation variables. CONCLUSIONS: After 90 minutes' processing time, platelets obtained with the Amicus cell separator were significantly more activated than platelets harvested with the Spectra and the MCS+.     

Periodic alternating interface positioning to lower WBC contamination of apheresis platelet concentrates: a multicenter evaluation

BACKGROUND: A new software version of a cell separator (AS TEC 204, Fresenius) providing WBC-reduced single-donor plateletpheresis concentrates was tested. STUDY DESIGN AND METHODS: Dual-needle apheresis procedures (n = 621) were performed in three centers, using either fixed interface positioning (FIP) or periodic alternating interface positioning (PAIP). The other separation parameters (e.g., anticoagulant:whole-blood ratio, and blood flow) were set individually. All platelet concentrates were evaluated for platelet yields and contaminating WBCs. RESULTS: The introduction of the PAIP resulted in a significant (p<0.001) reduction in contaminating WBCs (median, 30,000) from the numbers seen with FIP (median, 2,300,000) while maintaining the separation efficacy (47%) and separation time. Ninety-eight percent of all concentrates contained less than 5 x 10(6) WBCs per concentrate and 92 percent contained less than 1 x 10(6). CONCLUSION: Plateletpheresis using the AS TEC 204 cell separator with PAIP is a valid alternative to WBC reduction by filtration. It may provide WBC-reduced platelet concentrates without the additional cost of filters. However, the reliability of the WBC reduction is not yet advanced enough that PAIP can be employed without any monitoring of the end product.