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BACKGROUND: Universal leukodepletion (LD) has been implemented in the United Kingdom to reduce the risk of transfusion-transmitted variant Creutzfeldt-Jakob disease. If LD causes microvesiculation of blood cells, however, potentially infectious membrane-associated prion could reach the final products. STUDY DESIGN AND METHODS: We have measured microvesicles (MV) derived from red cells (RBC-MV), platelets (PLT-MV), and white blood cells (WBC-MV) and cellular prion protein (PrPc) in blood components produced by four whole-blood, five RBC, three PLT, and two plasma LD filters and three plateletpheresis techniques. RESULTS: RBC-MV and PLT-MV were either unaltered or reduced by all processes, with PLT-MV reduced 10-fold by RBC LD and greater than 300-fold by plasma LD. WBC-MV were reduced or unchanged by RBC and PLT LD and reduced by plasma LD. Whole-blood filtration appeared to increase MVs derived from granulocytes, but the load in the final components was comparable to that in processed RBCs in additive solution. PrPc was reduced by whole-blood, RBC, and plasma LD and unchanged by PLT techniques. There were differences between various filters and techniques, which were generally minor compared to the overall effects. CONCLUSION: These findings suggest no detrimental effects of LD processes in terms of generation of MVs or PrPc release.

Background Sickle cell trait donations can block leukodepletion (LD) filters or fail to LD, but the variables affecting blockage are unclear. Study design and methods To identify critical variables for further study, the relationship was investigated between filter blockage and donor characteristics, processing conditions, PLT and coagulation system activation, and microvesicle formation in donations with (n = 63) and without (n = 40) sickle trait. With eight filter types whole blood was LD either at ambient temperature on Day 0 or after overnight 4 degrees C hold. Markers of PLT activation (CD62P and CD63 expression and soluble CD62P) and coagulation activation (activated FXII and prothrombin fragment 1 + 2 [F1 + 2]), RBC microvesicles, blood gases, and residual WBCs were measured. Results All Day 0 filtrations blocked (n = 7). On Day 1, no filter tested was 100 percent successful, with most achieving an approximate 50 percent success rate. Two filters blocked consistently and an additional filter did not block, but resulted in 50 percent of units with high residual WBC counts (30 x 10(6)-394 x 10(6)/unit). Day 1 filtration was not improved if performed at 4 degrees C. Donor RBC variables and prefiltration measures varied little between blocked and successful filtrations except pO2, where 9 of 17 blockages had a pO2 of less than 5.0 kPa, compared with 0 of 13 completed filtrations. F1 + 2 levels increased after filtration in sickle trait units, a consequence of slow flow rate. Conclusion Filter blockage in sickle trait donors cannot be predicted by donor characteristics or filter type and is not related to PLT or coagulation activation, but can be reduced by storing units at 4 degrees C before filtration.

Background and Objectives With the implementation of universal white blood cell (WBC) reduction in the UK, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) and apheresis methods are used routinely for the production of WBC-reduced PCs. While these strategies meet the specification for WBC reduction (< 5 × 106 WBCs/unit), the products from these processes may differ depending on the process employed and its performance. The aim of this study was therefore to investigate whether PCs prepared using various WBC-reduction processes are sufficiently depleted of WBCs to limit cytokine accumulation during storage and to assess if cytokine levels detected in platelet products can serve as indicators of acceptable platelet activation as a result of the WBC-reduction process. Materials and Methods We measured the levels of cytokines predominantly derived from WBCs [e.g. interleukin-8 (IL-8)] and platelets [e.g. regulated on activation, normal, T-cell expressed, and secreted (RANTES) and transforming growth factor-β1 (TGF-β1)] under the present experimental conditions in different WBC-reduced PCs, i.e. PCs prepared from three different WBC-reduction filters and control non-filtered PCs using pooled BCs from the same donors and three apheresis types. Supernatant plasma was collected at the beginning (day 1) and end (day 5) of the shelf life of each PC, and the cytokine content was determined using appropriate enzyme-linked immunosorbent assays (ELISAs). Process efficiency was assessed by platelet yield and residual WBC count. Results We found that products from the apheresis process involving a filtration step (Haemonetics MCS+) showed a lower cytokine content on both day 1 and day 5 in comparison with the fluidized bed (COBE Spectra) or elutriation (Amicus) processes. WBC reduction of BC-PCs of the same origin using three different filters showed comparable levels of cytokines on day 1 in all units. After storage for 5 days, the levels of IL-8 remained essentially unchanged in filtered BC-PCs but increased by more than threefold in control non-filtered BC-PCs, suggesting IL-8 release by residual WBCs present in the control PCs. The concentration of platelet-derived cytokines such as RANTES and TGF-β1, however, increased significantly in all filtered and control non-filtered PCs during the storage period. Conclusion These results show that markers of cytokine release from both WBCs and platelets are useful indicators of the performance and efficacy of the WBC-reduction process and of platelet quality.

The conventional and a new marker of global cellular lesion (Annexin V) are used to assess the processing/storage-induced changes in four types of RBC and filtered blood at 4 degrees and 22 degrees C, stored for a period of 35 days, in multi-satellite packs. It appears that mechanical trauma and presence of leucocytes and residual platelets have potential to increase levels of all markers of storage lesion, but to a variable extend. We have also provided new evidence that multi-satellite packs can be safely used for up to 35 days for small volume transfusion to sick premature infants, in a well-managed system by administering several transfusions from the same donation, hence reducing donor exposure.

The causes of leucodepletion failure are multifactoral and can be related to haematological variability in blood donors or donation, defective filters, poor specimen handling or ageing, and/or the presence of non-adhering leucocyte/platelets. Since refiltering removes all types of leucocytes, including the populations appearing as extra gated events, we have developed a practical method for refiltering the failed leucodepleted components on standard filters and back-flushing the second filter to assess the nature of the WBC sub-population. In practice, recovered leucocytes from red cell filters and whole blood mainly consist of neutrophils. Those from platelet and plasma filters were mainly lymphocyte with considerable differences depending on the type of leucodepletion process. Atypical leucocytes are often seen in some pre-/post-cellular leucofiltered components. These appear characteristically as small WBC with a lower affinity for filter matrix, or as cell fragment, pinched leucocyte or apoptotic cells. Different reagents in use show variable sensitivity in identifying these extra gatal events. Storage of leucodepleted samples also induces different types of abnormality in leucocyte dot plot. A useful practical approach for characterisation of the nature of leucocyte sub-populations causing failure in leucodepleted components is provided.

Different types of filters are currently used for leucodepletion of red cell concentrates. These filters meet the specification for leucoreduction ( 6 leucocytes/ATD) but the quality of the final product may differ depending on the performance of the filters for effective removal of both leucocytes, platelets and possibly cytokines which are associated with transfusion reactions. We measured the levels of three representative cytokines: IL-8, RANTES and TGF- β 1, in red cell concentrates prior to and subsequent to the filtration procedure on day 1 and after a storage period of 35 days. Low levels of IL-8 (10–24 pg/ml) in the control unfiltered concentrates on day 1 which increased by approximately twofold on storage. Filtration reduced the levels of IL-8 on day 1 and day 35, in filtered concentrates in comparison with their control unfiltered counterparts. Leucoreduced concentrates produced by three different filters showed similar IL-8 levels on day 1 and day 35. However, concentrates prepared using another type of process showed a twofold increase in IL-8 levels on storage in comparison with day 1. None of the concentrates tested contained any detectable RANTES and TGF- β 1 suggesting a minimal platelet content. These results indicate that a combination of IL-8, RANTES and TGF- β 1 are useful quality indicators for validation of leucoreduced red cell preparations.

182 full double donations were collected, using 7-9 cycles with an in-house guideline based on donor HCT, and platelet count. 95.6% of donations were within specifications for platelet yield and 100% were leucodepleted. To overcome the potential for red cell spill-over when the platelet peak is too small, automated continuous monitoring of machine performance has been introduced reducing the potential for spill-over. To understand the impact of the continuous exposure of platelets to the filter, the quality of the first and second part of the collection was monitored using a new set of markers for the platelet storage lesion (activation, microvesiculation, cellular injury, complement and response to ADP). The results were equivalent and compared well with routine practice. Our new protocol allows a wide range of donors to be selected and the highest percentage of products to meet the UK specification with consistency. It is recommended that it can be used for double dose platelets with routine SPM.

This exercise focused on performance of NBS quality monitoring establishments with respect to enumeration of low leucocyte and other quality indexes of platelet concentration. Paired identical leucodepleted platelet samples, spiked with WBC (20 cells/microl) in 'vacuette' or 'pouch' were assessed by participants (n = 20) on days 1, 2 and 5. For low WBC counting, all laboratories gave estimates within acceptable range (+/-25%) and good agreement between storage and assay methods was observed on days 1 and 2. Day 5 results showed greater variability. Under improved performance criteria (+/-15%), only one laboratory under-estimated at days 1 and 2. Similarly, other parameters demonstrated good agreement between storage methods on days 1 and 2. At day 5, mean results were often significantly different to previous days. Improved performance target (+/-15%) will allow identification of non-conformers.

It is currently unclear to what degree methylene blue in combination with removal, of cells from plasma, by filter, can directly influence the loss of active components of plasma and whether the co-precipitation of FVIII/vWf with fibrinogen/fibronectin is affected by combined methylene blue and light treatment (MBLT). These questions are investigated using the Fenwal system. Our results indicate that up to 15% of the FVIII and IX are lost due to exposure of plasma to filters and methylene blue (MB). The illumination leads to a further 10–15% loss of all other major clotting factors. Factor XI appears to be highly sensitive to the MBLT-process, while inhibitors of the coagulation system are less affected. MBLT did not grossly influence the distribution of fVIII/vWf:Ag between cryoprecipitate and cryosupernatant using a paired control/test protocol, although the fVIII/vWf recovery is reduced in MBLT samples. The three commercially available MBLT processes differ in terms of operational aspects. These may have some impact on overall quality/safety and bioequivalency.

The aim of this study was to evaluate the quality of leucodepleted (LD) fresh-frozen plasma (FFP) produced using one of five whole blood filters (Baxter RS2000 & RZ2000, NPBI T2926, Macopharma LST1 and Terumo WBSP) or two plasma filters (Pall LPS1 and Baxter FGR7014). Whole blood or plasma was filtered within 8 h of collection at an ambient temperature. Samples were taken pre- and post filtration for analysis of coagulation factors and complement activation (n = 7--12 for each type of filter). All filtered units (209--286 ml) contained < 5 x 10(6) residual leucocytes and < 30 x 10(9)/l platelets. Statistically significant losses of factors V, VIII, IX, XI and XII and increases in markers of coagulation activation were observed (0--21%), which were dependent on filter type. None of the filters had a significant effect on von Willebrand factor (VWF) multimeric distribution or the activity of VWF and factors II, VII or X. The effect on levels of C3a appeared to be related to the filter surface charge: positively charged filters resulted in C3a generation, whereas negatively charged resulted in C3a removal. None of the observed changes are likely to be clinically significant unless subsequent processing of plasma (such as pathogen inactivation) results in further losses of coagulation factors.

BACKGROUND: Implementation of universal WBC reduction of blood components means that automated analytical methods may be the only satisfactory way for production laboratories to meet increased testing requirements. STUDY DESIGN AND METHODS: A multicenter study on the performance of a microvolume fluorimeter (IMAGN 2000, Becton Dickinson) was undertaken on 519 RBC, 353 platelet, and 27 fresh plasma units. RESULTS: WBC counts for the RBC samples ranged from 0.02 to 6.94 × 106 per unit (mean, 0.57) as determined by FC and from 0.02 to 5.53 × 106 per unit (mean, 0.40) as determined by MVF with a mean FC bias of +0.15 × 106 WBCs per unit, and discrepancies outside the 95% limits of agreement were mainly associated with higher FC counts. The series of platelet samples showed means of 0.90 (range, 0.06-19.45) and 0.66 (range, 0.01-18.95) × 106 WBCs per unit for FC and MVF methods, respectively. FC and MVF results were in good agreement at low counts, although significant discrepancies were noted at higher counts. Overall, for the platelet units, there was a mean FC bias of +0.34 × 106 WBCs per unit. The intermethod agreement exceeded 99 percent for both types of blood component when the single (both UK and United States) decision point of 5.0 × 106 WBCs per unit was applied. The mean WBC counts for the 27 analyzed fresh plasma units were 61.8, 56.0, and 46.0 per μL by Nageotte hemocytometry, FC, and MVF, respectively. CONCLUSIONS: This evaluation found that the level of intersite consistency for FC was relatively poor compared to that for MVF. The results nevertheless validated the broad equivalence of FC and MVF results for the current Council of Europe and UK/US decision points of

With the implementation of universal leucodepletion, an in-line, negatively charged LRF6H leucodepleting filter became an essential part of the Haemonetics MCS+ plateletpheresis system. A larger-scale (968) study using the standard protocol revealed a 2.79% leucodepletion failure rate (standard5 x 10(6) leucocytes per adult therapeutic dose). Factors influencing the efficacy of the filter were investigated. The pH of the filtrate was 7.0, the temperature 28 degrees C and filtration rate 80 ml/min. Reduction of the filtration rate to 30 ml/min (784 doses) reduced leucodepletion failure to 0.38%. Measurement of the leucocyte count, pre- and post-filtration of the platelet products, revealed that donations from 1% of donors contained substantially larger numbers of leucocytes in pre-filter samples (300-1500/microl) than in control samples (35-70/microl). This number tends to increase progressively with subsequent donations in these individuals, leading to leucodepletion failure, whilst peripheral leucocyte counts remain normal. The new continuous filtration protocol (version C) using a less impact filter LRF-XL and a lower (7 ml/min) head pressure was also effective but failure still occurred twice on one of the donors who persistently showed high pre-filter count. We conclude that leucodepletion failures in the Haemonetics system are related to both donor leucocyte (i.e., being light and non-adherent) and operational/filter performance.

BACKGROUND: Statistical process control is required for monitoring of the WBC-reduction process. This study focused on some factors that may influence the outcomes of statistical process monitoring, such as WBC-reduction technologies, the anticoagulant used, and WBC-counting technologies, by using two types of software. STUDY DESIGN AND METHODS: Data were collected from January to September 1999, before the implementation of universal WBC reduction. The effects of three major factors were investigated: methods of preparation, the addition of EDTA to the sample, and the WBC-counting technologies used (microvolume fluorimetry, flow cytometry, and Nageotte chamber). The WBC-reduction process capability was assessed by two types of software, EZQC (Gambro BCT) and NWA (Northwest Analytical). In addition, the differences between various sets of results were compared by the t test or ANOVA. RESULTS: There was no statistical difference (at the 0.05 level of significance) in WBC content when the three types of platelets in citrate samples were compared with EDTA samples. In general, the Nageotte chamber appeared to count the lowest, and microvolume fluorimetry appeared to count lower than flow cytometry. There were minor but significant methodologic differences between the software packages. However, these differences had negligible effects on the percentage of conforming components at both

Levels of factor VIII (FVIII) and fibrinogen were assessed in control cryoprecipitate and cryoprecipitate prepared in two centres from plasma subjected to methylene blue (MB) photochemical virus inactivation. The level of coagulation FVIII activity was reduced in plasma by approximately 30% after MB photoinactivation, with only 44% (centre A) and 31% (centre B) of units meeting the current UK specification of 0·7 iu/ml. A revised specification of 0·5 iu/ml is suggested. Losses of less than 11% were seen for von Willebrand factor (VWF)-related activities. Cryoprecipitate prepared from group O or group A MB-treated plasma contained 27–40% less FVIII than control units. This reflected the lower levels in MB-treated plasma. The concentrating power of the cryoprecipitation process was not reduced for FVIII or fibrinogen in MB-treated units. MB cryoprecipitate from centre A still met the UK guideline specification for FVIII and fibrinogen content, whereas at centre B only 62·5% of the group O cryoprecipitates contained > 70 iu FVIII/unit. This may reflect the lower product volume and lower FVIII content of group O plasma used at centre B and suggests that maintenance of total coagulation factor recovery in MB-treated cryoprecipitate will require the higher product volume.

With the implementation of universal leucodepletion in UK all leucodepletion processes have gone through a standard process qualification and quality improvement. The Haemonetics MCS system is a well established automated platelet collection system for the production of double dose leucoreduced platelet concentrate (WBC approximately 70x10(6)/dose). Recently an automated post collection filtration harness system has been introduced (MCS plus LDP) in which platelets are filtered, using an in-line PALL polyester filter (LRFH6 PALL) to reduce the WBC level to below 5x10(6) WBC/dose. This system passed our Phase I evaluation process based on 20-40 runs. However, some changes in the final volume of the products were needed to conform to national guidelines. Large scale trials using the new volume adjusted protocol revealed occasional failure in the leucocyte content. Therefore, 100% testing had to be implemented on all products. A national evaluation was carried out to determine whether changing the filter to a more efficacious one, the LRFXL (PALL) or slowing the filtration flow rate can influence the overall outcome. To reduce donor variability, known donor population were used with identical apheresis conditions. A more consistent and systematic drop in leucocyte content was observed by reducing the flow rate whereas a similar failure (i.e. 1-3%) rate was found both in controls and LRFXL when using the standard head pressure, which is recommended by the manufacturer. A similar failure rate was found using three different low leucocyte counting technologies (Nageotte, flow cytometry and Imagn 2000). It is recommended that a process qualification/validation program should be implemented when even a small modification in the collection system is introduced.

Double dose leucodepleted PC without filtration is considered to be the most cost-effective way of preparing leucodepleted PC in a reasonable time. The procedure lends itself to a multicomponent system and production of hyperconcentrate and dry platelets, with < 10-15 ml plasma in final product and viral inactivation without considerable loss of in vitro platelet functions. Platelet concentrates obtained by various procedures are highly heterogeneous, even if a standard protocol is used for the preparation. Therefore, standard/standardisation in both production and testing procedures remain a challenging area in order to obtain comparative results. Attention needs to be focused on growing and complex technical features of preparation and on the use of new filter material in terms of biocompatibility and the related effects of activated factors on function of platelets and leucocytes. Both the production process and storage containers appear to contribute to various cellular lesion and generation of some biological response modifiers such as complements, cytokines and microparticles. In this respect it is relevant to adopt a multiparameter analysis for the validation of platelet quality as some markers of platelet storage lesion have different affinity to various surfaces, leading to false under estimation. Further development work is still needed in preparation and usage of dry platelet, platelet alternative and bacterially safe products. The underlying conditions of the transfused patients is also an important issue in this respect, it is interesting to note that patients with high IL8 levels have a substantially lower platelet recovery.

An observational study was carried out to assess the effect of bacterial growth on changes in platelet cellular indices and leucocyte content. Platelet derived from pooled buffy coats, with and without an additional leucocyte removal step by filtration and platelet derived from Cobe LRS system were used. These were spiked with two doses of several types of bacteria (1 and 50 CFU) in a paired control and test experiment. The changes in cellular parameters were monitored by an automated cell counter (Sysmex SE-9000) and the concentration of the residual leucocyte were evaluated by two automated techniques, based on DNA staining principles (flow cytometry-EPICS-XL Coutter and Imagn 2000). Our results indicate that initially bacterial growth is associated with a decrease in leucocyte count, followed by a concomitant increase in platelet large cell ratio, possibly due to aggregate formation. During the prolonged storage a dramatic increase in pseudoleucocytes was observed by both Imagn 2000 and flow cytometry techniques with an abnormal dot plot around FL3 regions in the latter counting method, making the true identification of native leucocytes rather difficult. It is concluded that bacterial growth is associated with both changes in platelet cellular indices and development of cellular aggregates and/or partially fragmented cells with DNA binding properties appearing as pseudoleucocytes. Further work on the true nature of so called `pseudoleucocyte' is in progress.

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) (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. 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.

There is ample evidence for the presence of microvesicles (MV) of different sizes and functions in various blood components. A variety of mechanisms have been proposed for the formation of MV. These include mechanical injury, shear stress, cell activation, activation of complements, hypoxia, and the cell aging process. While MV share many biological properties and surface receptors of their parental cells, they demonstrate significant differences in membrane asymmetry of the inner membrane phospholipid, in particular phosphatidylserine (PS). This provides high-affinity binding sites for the components of the prothrombinase complex. To what extent these MV contribute to hemostatic effectiveness, immudomodulation, and some untoward effects of the transfused blood components remains to be fully elucidated. Several methods for qualitative and semiquantitative characterization of MV are now available. Although in most cases it is necessary to separate MV from the intact cells for improved characterization, recent advances in flow cytometry make it possible to accurately differentiate MV in the presence of their parental cells on the basis of light scattering and fluorescent intensity. This review focuses on four main areas of MV in blood components: (1) the proposed mechanisms of platelet vesiculation, (2) factors influencing the formation of MV, (3) laboratory analysis of MV, and (4) the clinical impact of the presence of MV in blood components. Key Words: Microvesicte—Vesicutation—Biood component—Ptatelets—Transfusion.

With the increasing demand for leukoreduction of blood components and the implementation of universal leukoreduction in several countries, the problems associated with leukocyte filtration of sickle cell trait blood have been revisited. Currently, there is no unified standard practice for sickle cell trait donors. Different blood centers adopt different policies. While some defer these donors from red cell component donation, some do not. Some screen all ethnic African donors for hemoglobin S (Hb S), others do not. Furthermore, there are differences in views of whether sickle cell trait red cells should be considered as equivalent to non-sickle cell trait red cells. Some blood centers do not give red cells from a sickle cell trait donor to the newborn or patients undergoing general anesthesia as a preventative measure. In this presentation, we discuss the epidemiology of the sickle gene, the sickling process, problems associated with leukoreduction of sickle cell trait whole blood and red cells, and some unresolved issues concerning donor referral and the usage of sickle cell trait blood.

Sickle cell trait donations can block leukodepletion (LD) filters or fail to LD, but the variables affecting blockage are unclear.To identify critical variables for further study, the relationship was investigated between filter blockage and donor characteristics, processing conditions, PLT and coagulation system activation, and microvesicle formation in donations with (n = 63) and without (n = 40) sickle trait. With eight filter types whole blood was LD either at ambient temperature on Day 0 or after overnight 4 degrees C hold. Markers of PLT activation (CD62P and CD63 expression and soluble CD62P) and coagulation activation (activated FXII and prothrombin fragment 1 + 2 [F1 + 2]), RBC microvesicles, blood gases, and residual WBCs were measured.All Day 0 filtrations blocked (n = 7). On Day 1, no filter tested was 100 percent successful, with most achieving an approximate 50 percent success rate. Two filters blocked consistently and an additional filter did not block, but resulted in 50 percent of units with high residual WBC counts (30 x 10(6)-394 x 10(6)/unit). Day 1 filtration was not improved if performed at 4 degrees C. Donor RBC variables and prefiltration measures varied little between blocked and successful filtrations except pO2, where 9 of 17 blockages had a pO2 of less than 5.0 kPa, compared with 0 of 13 completed filtrations. F1 + 2 levels increased after filtration in sickle trait units, a consequence of slow flow rate.Filter blockage in sickle trait donors cannot be predicted by donor characteristics or filter type and is not related to PLT or coagulation activation, but can be reduced by storing units at 4 degrees C before filtration.

The BSE outbreak has led to public safety concerns and current methods for the stunning and slaughter of cattle have been questioned. It has been suggested that the use of current procedures on an animal with BSE could contaminate the carcass with prions. One of the ways in which potential contamination could occur is by haematogenous dissemination of brain tissue during the stunning. Most cattle are stunned with a penetrating captive bolt gun (CBG) prior to slaughter. The use of CBGs may damage intracranial blood vessels and dislodge brain tissue. In order to assess the potential risk, the following investigations were carried out. Firstly, we have identified fragments of brain tissue in the jugular venous blood of cattle slaughtered after the use of penetrating CBGs [1]. The results support the view that there is a risk of haematogenous dissemination of CNS tissue with the use of penetrating CBGs. We have also carried out an investigation into the indirect effects of emboli on some haematological parameters including the release of Annexin V, a global marker of cellular injury [2]. Secondly, we have attempted to enrich the membrane bound CNS from the soluble form by centrifugation and whole blood filtration. A comparison is also made between the distribution of CNS marker and Annexin V. The inhibitory effect of Annexin V on prothrombinase and activation of coagulation factors by tissue factor were also investigated and briefly reported in this paper.

Background . WBC counting, an essential part of quality monitoring of WBC-reduced blood components, is carried out logistically within 48–72 h of collection. The between-laboratory variability and effects of 24–48 h storage were investigated using three major counting technologies. Study design and methods . Samples of RBC and platelets with WBC in the range 0–50/μl were transported by courier. WBC counting was performed on days 1 and 2, by IMAGN 2000, flow cytometry and Nageotte, initially using local protocols and then using a national flow protocol. Up to 15 laboratories participated in each exercise. Results . For "real failed leucodepleted" red cell products, higher levels of variability were observed for flow and Nageotte, as compared to IMAGN. For spiked RBC samples at critical decision making point (3–20 WBC/μl), between-laboratory the coefficients of variation (CVs) were low for IMAGN and were the highest for Nageotte. Flow cytometry CVs were generally high but improved subsequent to standardisation of sampling and the gating strategy. A similar pattern in the variability of results was observed for platelet concentrates. Sign tests using all samples (carried out for each method in each exercise; 25 in total) demonstrated no overall tendency for larger WBC counts to be recorded on day 1 when compared to day 2, although this difference was significant ( p Conclusions . We conclude that while a good performance is achieved using validated automated technologies for low residual leucocyte counting, the unification of reagents and standardisation of sampling and gating strategies are essential in obtaining interchangeable results. Unfixed RBC and platelet samples can generally be stored for 48 h before WBC counting.

Background : The levels of residual red blood cells (RBC) and platelets (PLT) in WBC-reduced plasma are often below the lower detection limit of automated blood cell counters. This study established a novel flow cytometric method for the enumeration of residual RBC and PLT in plasma. Furthermore, their levels in WBC-reduced plasma prepared by using various filters were investigated. Materials and methods : WBC-reduced plasma was prepared from two sources: (i) filtration of buffy-coat reduced plasma using dock-on Baxter, Pall and Maco Pharma plasma filters; (ii) filtered whole blood using integral Asahi RZ2000, Maco Pharma LST1, NPBI, and Pall WBF2 whole blood filters. Residual RBC and PLT counts were assessed by using a TruCount tube (Becton Dickinson) containing a known number of lyophilized fluorescent beads. RBC and PLT were labelled with dual monoclonal antibodies, anti-CD41-R-phycoerythrin and anti-glycophorin A-fluorescein isothiocyanide, and analyzed by flow cytometer. Results : The flow cytometric method used in this method can detect residual RBC, PLT as well as RBC-MV simultaneously. The sensitivity of the assay was 50×106cells/l with the coefficient of variations ⩽10%. Baxter and Maco Pharma plasma filters consistently reduced both RBC, RBC-MV and PLT to below 50×10 6 /l. Plasma derived from day 1 RZ2000 filtered whole blood contained PLT below 50×106cells/l, whereas day 0 NPBI filtered whole blood showed the highest level of residual PLT. Conclusion : A sensitive and accurate method for the detection of low levels RBC, RBC-MV, and PLT was established to measure their levels in WBC-reduced plasma. The procedure is simple and practical for routine quality monitoring of plasma, as well as for setting a new specification for WBC-reduced plasma.

The relationship between the platelet storage lesion (PSL) and programmed cell death (apoptosis) is poorly understood. Nevertheless, there is some experimental evidence that platelets contain most of the components of the apoptosis machinery and both the apoptotic process and the PSL lead to platelet activation and microvesiculation with expression of phosphatidyl serine (PS) on the outer layer of cell membrane, a hallmark of all nucleated cells. The PS exposure is believed to contribute to the development of inflammatory or immunomodulation process, to the regulation of haemostatic balance and the ultimate clearance of dead or fragmented cells from the circulation. While there is no doubt that apoptosis, as a form of genetically encoded programmed cell death in nucleated cells, is triggered by several signalling stimuli at the nuclear level, there is some doubt as to whether platelets, as enucleated cells have retained the memory of the "parental" megakaryocytes for apoptosis or whether platelet mitochondrial DNA has a major role in both the apoptotic process and the PSL. The storage lesion occurs during processing and storage subsequent to mechanical trauma, hypoxic conditions or exposure to cold. In this brief report some observational evidence is provided in support of the notion that the PSL and apoptosis may be related to each other, despite the fact that, in contrast to the 'parental' megakarocyte, the platelets appear to survive upon stimulation with a high concentration of protein kinase inhibitors such as staurosporine (STS), in the presence of cycloheximide (CHX) which inhibit protein synthesis. This is a model which is often used to regulate the level of survival signals. The possible relevance of platelet microvesiculation to transfusion practice is briefly discussed.

A 54-year-old double dose donor failed leucodepletion by Cobe LRS for five sequential donations, without triggering the "machine failure alarm". All collections were uneventful but final products appeared lipaemic. The last two donations were studied: (i) by collecting the first half of each donation into one bag and the other half into a second bag. In both cases, the product in bag one was leucodepleted and that in bag two "failed" leucodepletion. (ii) By examining the filterability of the final products using an LRF6 filter. Leucodepletion was satisfactory but showed a slightly higher level of leucocytes as compared to control or lipaemic donations, possibly due to the presence of an interfering substance in the platelet product. (iii) By examining the cellular content of the LRS cone and comparing it with those obtained from lipaemic donations from other donors. Only the test cone contained a large mass of waxy white material. It is postulated that this material was gradually filling the cone and increasingly interfering with its leucodepleting function as the donation progressed. (iv) To monitor pre/post whole blood as well as the products for the presence of abnormal leucocyte subsets. The only difference was the presence of some extra abnormal dot plot. The intensity of these extra events which was reduced both upon donation and filtration. We conclude that, in this case, failure of leucodepletion was clearly donor rather than machine or process related. The implementation of this investigative study may help in the characterisation of the potential cause of leucodepletion failures.

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 mi-crovesiculation 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.

This study evaluated Cobe Trima for donor and operational acceptability, the quality and storage stability of the blood components collected, and the clinical responses to transfusion. The study was carried out in 2 phases; phase 1 assessed the efficiency of red cells and platelet collection, and the characteristics of the components collected before and after storage. Phase 2 was an evaluation of operational issues and the in vitro characteristics of the red cells and platelet concentrates at the time of transfusion in respect to their cellular content, and leucocyte (interleukin IL-6 and IL-8) and platelet-derived (Rantes) cytokine levels. Cytokine levels were also measured in the donors before and after the collection procedure and in patients both before and after transfusion. The clinical responses to a small number of transfusions were assessed. The Cobe Trima was found to be straightforward to use by the operators, although additional operator training was required to manage occasional uncertainty with alarm messages. It was acceptable to the donors except for the occurrence of citrate reactions in 3/6 donors in phase 1; this problem persisted in phase 2 (6/15 donors), and needs to be addressed in the future. All blood components met UK product specifications apart from 2 platelet concentrates, 2 red cell concentrates, and one unit of FFP; the red cell and platelet concentrates had good storage characteristics. The 2 procedures, which resulted in low platelet yields, were due to occlusion of the plasma line; the method for installation of the harness has been subsequently modified to prevent this. 2 red cell concentrates showed haemolysis; the reason for this was not established. The Factor VIII level was satisfactory in plasma and the cellular content was low. The responses to 12 platelet transfusions were expected as in a group of haematology patients, and no immediate adverse effects were reported with any of the transfusions. Leucocyte-associated (IL-8 and IL-6) and platelet-associated (Rantes) cytokine levels were not elevated in donor samples taken before or after the collection procedure, or in the red cell and platelet concentrates at the time of issue. Pre- and post-transfusion IL-8 levels were raised in one patient with non-immune platelet refractoriness, and normal in 2 patients with excellent or almost satisfactory responses to platelet transfusions raising the question as whether IL-8 could be used as a laboratory marker for non-immune platelet refractoriness due to infection.

Released annexin V, an intracellular platelets glycoprotein, was used to determine the cellular injury which occurred during storage of platelet concentrates. Twenty-eight units of leuco-reduced apheresis platelet concentrates, obtained without leucocyte filtration, were analysed. Released annexin V showed a significant correlation with EDTA-induced shape changes of platelet (r = 0.62, P < 0.01) while poor correlation was found between released annexin V and pH or MPV. The combination of released annexin V with dMPV provides excellent markers of the platelet storage lesion for quality monitoring, based on morphological/functional integrities and cellular injury, which are of direct relevance to clinical efficacy of platelet concentrates.

D URING THE PAST 2 decades, the demand for platelet concentrates (PC) has been steadily increasing because of more intense treatment schedules as well as widening indications for the use of chemotherapy. This has. placed considerable pressure on the logistics of supply. The emphasis on reducing donor exposure, caused by the possibility of transfusion associated alloimmunisation and leucocyte borne infection, t has led to an increase in the use of apheresis and leucoreduced PC. In re@onse to these requirements, measures have been taken that are aimed at maximum resource use, for example: by splitting a high platelet yield unit, collected by a cell separator, into two therapeutic doses (-->240 • 109/dose); development of new containers and storage media to permit prolonging the length of storage period; and the development of new generation of leukocyte filters as welLas modem apheresis procedures to achieve high purity leukoreducedproducts (~1 • 106/unit). In parallel to the above development, the bottom and top (BAT) bag system, 2 combined with semiautomated blood component separation procedures, have been used to optimize platelet recovery from whole blood and to standardize the yield. This has enabled the consistent production of PC with an average dose of 300 X 109 platelets/unit and leucocyte content 0.05 X 109/unit from a pool of four ABO compatible buffy coats. Storage in synthetic media has been proposed 3 with the practical advantages of reducing the rapid accumulation of lactate and the removal of some plasma components, cell-released enzymes, and soluble antigens including HLA; therefore improving the viability of platelets as well as reducing potential transfusion reactionsJ -6 Despite these continual process improvements and major advances in establishing optimized storage conditions, the platelet storage lesion (PSL) as manifested by the loss. of function and changes in morphology associated with vacuolisation and plate

Modern blood transfusion services are striving to attain the highest standards through continual quality improvement and ensure that a formalised quality system is in place in the organisation for time management and waste reduction. With respect to the production of platelet concentrates (PCs), semi-automated and automated procedures are now available which are convenient and allow the processing of multiple packs simultaneously or the production of multiple doses of PCs with low leucocyte content. Nevertheless, PCs are highly heterogeneous on the basis of cellular content and storage stability. Statistical process control (SPC) is essential to ensure that optimal quality is maintained. In the light of new developments in platelet production, their clinical effect should be reappraised. This article highlights some of the key features in the production of PCs, focusing on basic concepts and quality indicators which reflect in vivo functions. The potential for bacteriological contamination and future production strategies are briefly mentioned.

We describe a new approach for assessing filtration-induced changes in cellular indices of platelet concentrates at the beginning and the end of storage, using pairs of identical packs. The results revealed that post-filtered products did not store as well as their counterparts. Filtration did not induce any significant changes on aggregation as determined by spontaneous aggregation nor was there a disparity between leucocyte peroxidase/basophil count. We recommend filtration on day 2 which causes minimal loss of platelets and less change in the mean platelet volume.