Background and Objectives: Our requirements for leukocyte–depleted platelet concentrates (LD–PC) for an adult patient are: platelets >240×109, leukocytes <5×106, volume of 150–400 ml; and at the end of storage a pH between 6.8 and 7.4 and presence of the swirling effect. Our aim was to develop a standardized, semiautomated method for the production of LD–PC, by pooling of buffy coats (BC), and prestorage leukoreduction by filtration. Materials and Methods: Whole blood was collected in Top and Bottom systems, and separated automatically with the Compomat™ G3 equipment into a red cell concentrate, a plasma and a BC. Subsequently, a pool of 5 BC was made, and 200 g plasma from one of the donors was added. Then, after soft spin centrifugation, the platelet rich plasma was leukocyte depleted by filtration using the Autostop™BC filter, and stored in a 1,000 ml polyolefin platelet storage bag. Results: BC (n = 60) had a volume of 51±2 ml (mean ± SD) with a hematocrit of 0.44±0.03 l/l and contained 80±5% of the platelets and 74±12% of the leukocytes of the whole blood. Routinely prepared LD–PC (n = 15,037) contained a median of 341×109 platelets (range 49–599×109), with only 104/15,037 (0.7%) containing fewer than 240×109 platelets; the median volume was 263 ml (range 134–373 ml). In 118/917 (13%) LD–PC leukocytes were observed in the Nageotte hemocytometer, but only twice exceeding 1×106 leukocytes per unit, and none exceeding 5×106 (median <0.6×106; range <0.6–1.41×106). Storage experiments of the LD–PC (n = 12) revealed adequate oxygenation and maintenance of pH and swirling effect up to 9 days. Conclusions: This method warrants with 99% confidence that LD–PC contain more than 240×109 platelets; with 97.5% confidence that 100% of the LD–PC contain <5×106 leukocytes, and with 95% confidence that more than 99% of the LD–PC contain fewer than 1×106 leukocytes; these LD–PC can be stored satisfactorily for up to 9 days.

The purpose of the present overview was to determine the factors influencing the removal of infectious agents from red cell concentrates by filtration. In general, the efficacy of the filtration method depends on the physical as well as the functional properties of blood cells. These properties are highly influenced by the changes exerted on the blood cells during blood collection, processing and storage and the filtration method itself. In particular, the removal of infectious agents of red cell concentrates by filtration will be determined by the type of virus and therewith the binding towards leukocytes, the type of bacteria and holding period before filtration, the deformability of infected cells and the disintegration of cells in the filter.

Background: Posttransfusion complications can be prevented by pretransfusion removal of donor white cells from platelet concentrate. The filtration used for this removal seems to have little effect on platelet function and activation, but more information is needed on its effect on function during subsequent long-term storage of concentrate.Study Design and Methods:The effect of prestorage filtration of buffy coat-prepared platelet concentrates (PCs) on platelet function, metabolism, and activation was investigated. A pool of three PCs, each made of four buffy coats, was split into three equal volumes; two were filtered over two different filters and the third served as a control. Variables monitored immediately after filtration and during the subsequent 8-day storage period at 22 degrees C included aggregation upon stimulation with collagen and/or ADP, platelet adhesion capacity to collagen and fibrinogen in flowing blood, nucleotide content of and nucleobase release by the platelets, expression of activation-dependent antigens, and beta-thromboglobulin release by the platelets.Results: No differences were observed between the PCs filtered over two different filters and the nonfiltered control PCs immediately after filtration and during storage, except for a selective removal (20%) of beta-thromboglobulin by one filter.Conclusion: PCs prepared from a pool of four buffy coats can be filtered and subsequently stored for 8 days (starting +/- 24 hours after whole blood collection) without detriment to platelet function, metabolism, or activation.

Vox SanguinisVolume 67, Issue s3 p. 159-160 LEUKOCYTE-POOR BLOOD PRODUCTS: FILTRATION MECHANISMS I. STENEKER, I. STENEKER Red Cross Blood Bank Amsterdam P.O. Box 9137 1006 AC Amsterdam The NetherlandsSearch for more papers by this author I. STENEKER, I. STENEKER Red Cross Blood Bank Amsterdam P.O. Box 9137 1006 AC Amsterdam The NetherlandsSearch for more papers by this author First published: July 1994 https://doi.org/10.1111/j.1423-0410.1994.tb04567.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume67, Issues3July 1994Pages 159-160 RelatedInformation

Die Entnahme von Spenderblut in Mehrfachbeutelsystemen ermöglicht die Blutkomponentenauftrennung in einem geschlossenen System [1]. Die hergestellten Blutkomponentenkonserven können im jeweils geeigneten Temperaturbereich gelagert werden, behalten damit ihre optimale Qualität und stehen für eine ‚Hämotherapie nach Maß‘ in der Patiententherapie zur Verfügung.

The effect of filtration on the quality of platelet concentrates (PC) during storage was investigated. Two leukocyte depletion filters (Pall PL50HF and Sepacell PL-10A) were applied to filter PC made from a pool of 4 buffy coats. For each experiment 3 PC were pooled and divided into 3 identical PC to eliminate differences between the PC. Two PC were filtered, and the third PC served as an unfiltered control. A total of 12 experiments was performed. Before filtration, volumes of the PC were 263 +/- 11.7 ml (mean +/- SD). Platelet and leukocyte counts per PC were 241 +/- 25.9 x 10(9) and 7.2 +/- 1.8 x 10(6), respectively. After filtration leukocyte counts did not exceed 5 x 10(4) in any of the PC. In the PC filtered with the Pall PL50HF the mean platelet loss was approximately 14% and with the Sepacell PL-10A, 17%. During a 9-day storage period the pH, PO2, PCO2, bicarbonate, lactate and glucose concentration and LDH release as well as the morphology, examined by the swirling effect and microscopically, were not significantly different in filtered and unfiltered units. Filtration through the 2 investigated leukocyte depletion filters for PC did not adversely affect in vitro viability of the platelets during storage.

Quality assurance (QA) should result in meeting the specifications and regulations of regulatory, accrediting and organizational policies. For Europe the guidelines for good manufacturing practice (GMP), good laboratory practice and good clinical practice of the European Community (EC) are mandatory whereas in the United States the requirements are put down in federal regulations of the Food and Drug Administration (FDA). All guidelines refer to standard operating procedures (SOPs) which should contribute to compliance to regulations, consistency of procedures and processing, training of staff, control over manufacturing processes, effcient procedures, and communication about errors and complaints. Most transfusion centers and blood banks are facing a tremendous task to convert their organization according to these regulations founded on pharmaceutical production facilities. The major difference between processing in blood banks and in the pharmaceutical industry is that the source material for blood components is blood from individual donors, which may vary substantially in biological composition. Thus, every unit of whole blood is a batch. Moreover, quality checks are not easy to apply because invasion of blood products for sampling means sacrificing the, usually scarce, products and few blood banks can allow discarding of blood products because of quality failures. On the other hand if products are not tested, the reproducibility of processing cannot be warranted. Because of these problems quality checking of blood products has for a long time been a hidden issue. In many guidelines quality checks on a minimum of 4 units per month are required, sometimes out of processing 1,000 or more processed products. From a statistical point of view this number will never reflect the quality of the products. To draw up an inventory blood bank experts were invited by answering a number of questions to express their opinions on the implementation of QA programs according to the above requirements and especially give their methods of quality control (QC) of the blood products and the number of products tested.

The effect of platelets on the removal of white cells (WBCs) from 16 to 24‐hour‐old red cell (RBC) concentrates by filtration was studied. RBC concentrates with various concentrations of platelets and WBCs were filtered on a cellulose acetate column filter and on three polyester flatbed filters. The microscopic study revealed that lymphocytes and most monocytes were captured in the smaller pores of the fiber network, irrespective of the brand of filter, the type of filter material, or the prefiltration platelet amount in the RBC concentrates. In contrast, efficient granulocyte depletion depended on granulocyte‐platelet interaction and on the filter material. In the presence of platelets, granulocytes were captured in the top part of the column filter or in the coarse layers of two of the flatbed filters, where platelets covered the fibers. Platelet depletion of the RBC concentrates prior to filtration diminished the contribution of these parts of the filters to granulocyte capture. A larger part of the column filter or the fine layers of the flatbed filters were now required for granulocyte capture. In one of the flatbed filters, granulocyte‐platelet interaction occurred mainly in the fine layers, which ended in blockage of this filter after the filtration of variable volumes (250‐600 mL) of standard RBC concentrates. A quantitative estimation of the effect of platelets on the WBC‐reduction capacity found that all three flatbed filters had a highly significant decrease (p = 0.001) in WBC‐reduction capacity for platelet‐depleted or buffy coat‐depleted RBC concentrates, as compared with standard RBC concentrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukocytes and platelets are known to adhere to a variety of materials [1]. Already in 1928, Fleming used cotton wool to remove leukocytes from small amounts of blood [2]. This was the basis for the preparation of leukocyte-poor red cell concentrates (RBC) for transfusion many years later [3,4]. In 1972 Diepenhorst et al. [3] described the first leukocyte depletion filter which was composed of a column filled with tightly packed cotton wool fibers, providing a network with equally distributed pores. The retention of both granulocytes and lymphocytes in this cotton-wool filter was independent of Ca2+ and Mg2+ and hematocrit and slightly temperature dependent.

Polyurethane membrane filters and filters coated with poly(ethyleneimine) were used to investigate the influence of leukocyte adhesion during filtration. Treatment of the filters with an aqueous solution of 1% (w/v) poly(ethyleneimine) (PEI) led to the introduction of amine groups at the filter surfaces, as was confirmed by X-ray photoelectron spectroscopy. The modification procedure did not significantly change the porous structure in the filters, as was demonstrated by SEM and porometry. Using 14C-labeled poly(ethyleneimine) it was shown that nearly a complete coverage (approximately 0.1 mg/m2) was achieved that did not desorb from the filter surface during contact with blood plasma. When the filtration was carried out with purified leukocytes in the absence of red cells, platelets, and blood plasma, the number of cells removed by modified filters (> 95%) was significantly higher as compared to the removal with unmodified filters (approximately 80%). However, no significant differences between the filters were found when the filtration was performed with whole blood. This finding was unexpected, because it was shown before that immobilization of poly(ethyleneimine) on solid polyurethane film, surfaces promoted the adhesion of leukocytes from whole blood. Apparently, the adhesive properties of the PEI diminish during filtration. Filter coating of commercial leukocyte filters composed of polyester fibers also had no effect on the removal of leukocytes from whole blood. It was postulated that morphological factors, such as filter shape, roughness, tortuosity, and porosity rather than the physicochemical properties of the filter surface influence cell adhesion to the filter surface, and through that the filtration process.

The leukocyte depletion capacity and performance of 5 filters designed for filtration of red cell concentrates (RCC) were compared by counting leukocytes, measuring red cell volumes and by histological examination of the filters after use. To eliminate interdonor differences, 5 buffy-coat-poor RCC were pooled (in each of 10 experiments) and subsequently split up into the original bags. The RCC were passed over the Cellselect filter, a column filled with cellulose acetate, and over flat-bed polyester filters: the Cellselect Optima, the Pall RC 50, the Leukostop and the Sepacell R-500. The filtration was shortest with the Pall RC 50 (p less than 0.001 compared to the other 4 filters). Leukocyte removal was most effective with the cellulose acetate filter (p less than 0.01 compared to the other 4 filters) followed by the Cellselect Optima polyester filter (p less than 0.02 compared to the remaining 3 filters). Residual leukocytes did not exceed 50 x 10(6) for any brand of filter. Red cell recovery was similar for all 5 filters with mean values from 86.1 to 89.2%. The leukocyte numbers, counted in Türk's solution or in propidium iodide, gave comparable values in hemocytometers applying light microscopy or fluorescent microscopy, respectively. Histological examination showed that lymphocytes were mainly removed by trapping, whereas granulocytes showed a variable pattern: adhesion in presence of platelets or trapping.

The mechanisms of white cell (WBC) reduction in 16-hour-old CPDA-1 red cell (RBC) concentrates by filtration on a column filter and on three different flatbed filters were studied by electron microscopy, with special emphasis on cell-to-cell interaction, cell damage, and interaction of blood cells with the material. Generally, lymphocytes were removed by mechanical sieving and monocytes by adherence and mechanical sieving. Granulocyte depletion occurred by mechanical sieving, direct adhesion to the fibers, and indirect adhesion to activated and spread platelets. In the column filter, most granulocytes were captured by adhesion. In the coarse layers of two of the flatbed filters, indirect adhesion was most prominent, whereas direct adhesion was most prominent in the other flatbed filter. For the most part, granulocytes were captured by direct adhesion in the fine layers, but in one flatbed filter, capture apparently occurred by mechanical sieving. The results of this study suggest that the efficiency and the mechanism of WBC reduction depend on the physicochemical characteristics of the non-woven materials in the filters as well as the cellular composition of the RBC concentrates.

Three third‐generation white cell (WBC)‐depletion filters based on polyester layers with decreasing pore size were investigated. In the coarse layers, unaggregated granulocytes, monocytes, and platelets and aggregates of these cells were captured in close contact with the fibers. This indicates that the depletion of granulocytes, monocytes, and platelets in the coarse layers of the filters is due in part to activation and adhesion with the formation of cell clusters on the fibers. In Filter I and Filter II platelets were not found in the fine layers, whereas in Filter III, 70 percent of the platelets were detected as unaggregated platelets on the fibers of the fine layers. More than 95 percent of the lymphocytes captured in the three filters occurred as single cells in the fine layers, and over 60 percent of these could be recovered. This suggests that the depletion of lymphocytes depended on trapping of the cells in the fiber network. All three filters captured HLA‐DR‐positive lymphocytes in the top layers, a finding that supports earlier reports that the transfusion of filtered red cell concentrates reduces HLA alloimmunization. More lymphocytes and granulocytes were found in the last layer of Filters II and III than of Filter I. Therefore, the risk of white cell leakage is probably lowest for Filter I. Red cells were found as red cell aggregates in the fine layers of Filters I and II, whereas almost no red cells were detected in Filter III. It was shown that the three filters studied were similar in their removal of WBCs, but differed in their mechanisms of removal.

As part of a study on the mechanisms of leukocyte filtration, the influence of pore size distribution on filter efficiency was investigated. Conventional leukocyte filters are not suitable for model studies, as these filters are composed of tightly packed synthetic fibers, with a poorly defined porous structure. Therefore, open cellular polyurethane membranes with pore size distributions varying from approximately 15 to 65 microns were prepared. Filtration experiments with stacked packages of these membranes showed that leukocytes are best removed (greater than 99%) by filters with a pore size distribution of 11-19 microns. These pore sizes approach the size of leukocytes (6-12 microns). However, due to fast clogging, blood flow through these filters is rapidly reduced, which results in a low filter capacity. With an asymmetric membrane filter, in which the pore size decreases from about 65 to 15 microns in the direction of blood flow, both moderate removal of leukocytes (greater than 80%) and maintenance of flow (approximately 0.2 mL/s) are obtained. This results in efficient leukocyte removal. From cell analysis of both filtrate and filter, it is concluded that adhesion rather than sieving is the major filtration mechanism. Thus, further optimization of the filter may be achieved by surface modification.

A histological and immunohistochemical investigation of slices from the Cellselect® leukocyte filter showed that the capture of lymphocytes within these filters depended on trapping of the cells in the cellulose acetate fiber network more than on adherence. Probably, additional mechanisms played a role in granulocyte removal. Microaggregates of granulocytes and platelets were found at the top of the Cellselect leukocyte filter. Therefore, it is likely that granulocyte and platelet removal, at least in part, is due to the formation of cell clusters on the surface of the fibers. Although a part of the original, nonfiltered, leukocytes already showed morphologic characteristics of cell necrosis, more necrotic leukocytes were detected within the filter.