White cell-reduced platelet concentrates prepared by in-line filtration of platelet-rich plasma

BACKGROUND: The importance of white cell (WBC) reduction in platelet concentrates (PCs) for component quality is undetermined. STUDY DESIGN AND METHODS: Eleven paired components, each derived from one of two whole-blood units given by a single donor on the same day, were studied. One PC was WBC reduced by filtration with an in-line, integral, prototype filter, and the other was produced from unfiltered platelet-rich plasma (PRP) by a standard method. In vitro tests performed on Day 1 and Day 5 were blood gases, plasma lactate, glucose, platelet ATP, mean platelet volume, morphology score, hypotonic stress ratio, extent of shape change in response to ADP, and beta- thromboglobulin. After 5 days of storage, each component pair was labeled with 51Cr or 111In and transfused for the estimation of percent recovery and survival. RESULTS: PCs using the in-line, prototypic filter had a platelet loss of approximately 15 percent and a variable 1 to 3 log10 reduction (average, 95%) in WBC content. The variation in filter WBC removal was related to PRP WBC content and indicated that the filter did not have the capacity for a 3 to 4 log10 removal when PRP WBC content exceeded 1 × 10(8). The in vitro and in vivo measures of platelet quality showed no meaningful differences between filtered and unfiltered PCs by paired t test. The mean differences in posttransfusion percent recoveries and survivals were 0.9 +/? 2.9 percent and 4 +/? 13 hours, respectively. Additional studies were performed using a larger filter with improved capacity. Those studies (n = 18) showed a significant improvement in filtration time and platelet yield and a consistent 3 to 4 log10 reduction in WBCs. Filtration time was 6.6 +/? 2.7 minutes, total PC WBCs were 9.6 +/? 4.6 × 10(4), and total PC platelets were 7.8 +/? 1.8 × 10(10) (mean +/? SD). CONCLUSION: Prestorage filtration of PRP and the preparation of filtered platelets do not result in any significant beneficial or adverse effect on subsequent platelet quality. With the large-capacity filter, consistent WBC reduction and good platelet yields are achieved.     

White cell reduction in platelet concentrates and packed red cells by filtration: a multicenter clinical trial. The Trap Study Group

BACKGROUND: Most previous studies on white cell (WBC) reduction by filtration have been small-scale studies conducted under tightly controlled laboratory conditions. Their results would be the ideal, rather than what might be expected during routine operation. STUDY DESIGN AND METHODS: To obtain information on routine filtration of blood components, data have been collected from a large-scale, ongoing, multicenter clinical trial designed to determine the effectiveness of WBC reduction in or ultraviolet B radiation of platelet concentrates before transfusion in preventing platelet alloimmunization and platelet transfusion refractoriness. The WBC content of blood components both before and after filtration was determined by automated cell counters and a manual propidium iodide-staining method, respectively. Platelet and red cell losses during filtration were measured. RESULTS: The average platelet losses after filtration were 24 +/? 15 percent and 20 +/? 9 percent for apheresis platelets and pooled platelets, respectively. The frequencies at which filtered platelet concentrates contained high levels of residual WBCs (> 5 × 10(6)) were 7 percent and 5 percent for apheresis platelets and pooled platelets, respectively. Further analysis of the platelet filtration data showed that greater numbers of total initial WBCs in the pooled platelets were associated with increased percentages of filtration failure (> 5 × 10(6) residual WBCs). For packed red cells, the average losses during filtration were 23 +/? 4 percent and 15 +/? 3 percent for CPDA-1 units and Adsol units, respectively. The frequencies at which filtered red cells contained > 5 × 10(6) residual WBCs were 2.7 percent for one type of filter and 0.3 percent for another type of filter. CONCLUSION: There were significant losses of platelets during filtration, which could add to the costs of WBC reduction and lead to possible increases in donor exposures. Filtration failures still occurred, despite careful observation of the standard filtration procedures. The number of total WBCs in pooled platelets before filtration has been identified as an important factor in determining the success of WBC reduction.     

Changes in blood counts after the administration of granulocyte-colony- stimulating factor and the collection of peripheral blood stem cells from healthy donors

BACKGROUND: After the collection of granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells from healthy donors, the donor platelet counts fall. However, the magnitude and duration of this decrease are not known. STUDY DESIGN AND METHODS: Sixty healthy people were given G-CSF (5, 7.5, or 10 micrograms/kg/day) for 5 days (Days 1–5), and 1 peripheral blood stem cell component was collected on Day 6. The platelet count, white cell count, absolute neutrophil count, hematocrit, and red cell count were measured before administration of G-CSF (Day 0), before collection of peripheral blood stem cells on Day 6, and on Days 8, 10, 13, 16, and 20. RESULTS: The platelet count fell from 261 +/? 47 × 10(9) cells per L on Day 0 to 159 +/? 30 × 10(9) cells per L on Day 8 (p < 0.0001) and reached its lowest level on Day 10 (146 +/? 30 × 10(9)/L; p < 0.001). Compared to Day 0 levels, the platelet count was lower on Day 13 (185 +/? 49 × 10(9)/L, p < 0.001), was the same on Day 16 (270 +/? 53 × 10(9)/L), and was greater on Day 20 (333 +/? 60 × 10(9)/L, p < 0.0001). The white cell count returned to pretreatment values on Day 13, and the absolute neutrophil count returned to pretreatment values on Day 10 (Day 0 white cell count = 6.05 +/? 1.59 × 10(9)/L and Day 0 absolute neutrophil count = 3.97 +/? 1.52 × 10(9)/L). On Day 20, both were less than pretreatment values (white cell count = 5.14 +/? 1.24 × 10(9)/L, p = 0.0007 and absolute neutrophil count = 3.20 +/? 1.24 × 10(9)/L, p = 0.0036). The red cell counts on Day 16 (4.52 +/? 0.41 × 10(12)/L) and Day 20 (4.42 +/? 0.39 × 10(12)/L) were less than Day 0 values (4.73 +/? 0.43 × 10(12)/L, p = 0.008 and p < 0.0001, respectively). The hematocrit on Day 20 (39.2 +/? 3.2%) was also less than that on Day 0 (41.2 +/? 4.8%; p = 0.01). The changes in these blood counts were not affected by the dose of the G-CSF. CONCLUSION: After stimulation with granulocyte-colony-stimulating factor and the collection or peripheral blood stem cells, the platelet counts in normal donors were decreased for at least 7 days (Days 6–13). Two weeks after collection of peripheral blood stem cells (Day 20), platelet production was increased, but the production of neutrophils and red cells was decreased. If two or more peripheral blood stem cell components are collected, then the platelet count should be measured after the second and subsequent collections. Further studies on the long-term effect of G-CSF on blood counts are needed.     

Automated blood component collection with the MCS 3p cell separator: evaluation of three protocols for buffy coat-poor and white cell- reduced packed red cells and plasma

BACKGROUND: Automated collection of blood components with a cell separator (MCS 3p, Haemonetics), was performed according to three protocols. STUDY DESIGN AND METHODS: The first protocol provided 2 units of fresh-frozen plasma (FFP); and one buffy coat-poor red cell (RBC) concentrate in additive solution. The second protocol included an additional in-line filtration of the RBC in a closed system after storage at 4 degrees C for 24 hours. In the third protocol, an additional platelet concentrate (PC) was recovered from the buffy coat. Cell counts and biochemical characterization of the RBCs (n=20 each) were determined on Days 0, 1, 14, 28, and 49. RESULTS: The RBC volume was 336 +/? 9 mL (first protocol), 337 +/? 7 mL (second protocol) and 293 +/? 12 mL (third protocol) with a hematocrit of 59 +/? 2, 53 +/? 3, and 61 +/? 5, percent respectively. On Day 49, hemolysis was 0.24 +/? 0.1 percent (first protocol), 0.33 +/? 0.32 percent (second protocol), and 0.38 +/? 0.1 percent (third protocol). The filtered RBC concentrate met the international standards for white cell-reduced RBCs. Filtration resulted in a clinically irrelevant increase of hemolysis. The in vitro RBC values (lactate dehydrogenase, 2-hydroxybutyrate dehydrogenase, hemolysis, potassium, 2,3 DPG, ATP) were at least equal to those in RBCs collected by conventional whole-blood donation. There is a trend toward extended preservation of 2,3 DPG in RBCs collected by apheresis. Two units of FFP could be collected with each donation (first protocol: 420 +/? 55 mL, 5.4 +/? 7 WBCs/microL, 6.5 +/? 5 × 10(3) platelets/microL; second protocol: 440 +/? 33 mL, 3 +/? 5.2 WBCs/microL, 32 +/? 12 × 10(3) platelets/microL; third protocol: 398 +/? 32 mL, 5 +/? 12 WBCs/microL; 3.4 +/? 3.5 × 10(3) platelets/microL). PCs prepared from the buffy coat collected by the third protocol contained 90 +/? 30 × 10(9) platelets in 88 +/? 14 mL of plasma. In vitro test results in these PCs were superior to those in PCs collected by conventional whole-blood donation. The procedure was well tolerated by all donors. No adverse reactions appeared. CONCLUSION: Erythroplasmapheresis with the MCS 3p cell separator is a useful alternative to conventional whole-blood donation and separation.     

Improved removal of white cells with minimal platelet loss by filtration of apheresis platelets during collection

BACKGROUND: Filtration of apheresis platelets to remove white cells (WBCs) requires operator intervention after the collection procedure (postcollection filtration), which may cause variable and unsatisfactory filter performance (WBC removal and platelet loss). The MCS+ LN9000 apheresis system filters platelets through a WBC-reduction filter during each collection cycle (continuous filtration) at a flow rate of 15 to 25 mL per minute. Apheresis platelets obtained by continuous filtration were evaluated in terms of platelet loss, WBC removal, and platelet storage properties and then were compared to unfiltered apheresis platelets and to apheresis platelets that underwent postcollection filtration. Two WBC-reduction filters were tested (LRF6 and LRFXL). STUDY DESIGN AND METHODS: In 70 apheresis platelets, postcollection filtration was performed by using the LRF6 at flow rates of 80 mL per minute (n = 30) and 50 mL per minute (n = 30) and the LRFXL at 50 mL per minute (n = 10). One hundred fifty-eight apheresis platelets underwent continuous filtration through the LRF6 (n = 58) or the LRFXL (n = 100). Unfiltered apheresis platelets (controls) (n = 30) were obtained by the same collection protocol. RESULTS: Estimated platelet loss with continuous filtration was 7 percent for the LRFXL and 3 percent for the LRF6. A reduction in the filtration flow rate from 80 to 50 mL per minute with postcollection filtration through the LRF6 resulted in markedly lower WBC levels, with 10 percent versus 57 percent of the apheresis platelets having WBC counts <1 × 10⁵, respectively. Additional improvements in WBC removal were found with continuous filtration; 85 percent of the apheresis platelets filtered with the LRF6 and 100 percent of the apheresis platelets filtered with the LRFXL had WBC counts <1 × 10⁵. CONCLUSIONS: Continuous or postcollection filtration of freshly collected apheresis platelets resulted in minimal platelet loss. Better WBC removal from apheresis platelets was obtained with continuous filtration than with postcollection filtration, likely because of the slower flow rate. Platelet storage quality was not affected by filtration.     

White cell subsets in apheresis and filtered platelet concentrates

BACKGROUND: White cell (WBC)-reduced platelet concentrates (PCs) are defined by their absolute WBC count, a criterion which provides no information regarding the various WBC subsets contained in the PC. These heterogeneous cells are known to mediate different physiologic and pathophysiologic functions and account for distinct adverse transfusion responses. This study describes a method which allows the detection and quantification of these subsets and characterizes their presence in a variety of platelet components. STUDY DESIGN AND METHODS: Random-donor pooled PCs (RD PCs) and single-donor apheresis PCs (SD PCs) were studied. RD PCs consisting of 6 units of 2- to 3-day old PCs were randomly assigned to be filtered with one of four WBC-reduction filters from three different manufacturers (n=34). The residual WBCs were pelleted by centrifugation and isolated on a density gradient. The various WBC subsets were quantified by flow cytometry in unfiltered and filtered PCs using fluorescence and two-angle light scatter. SD PCs obtained with two manufacturer's systems and three processing protocols (n=30) were studied in like manner. RESULTS: WBC counts for non-WBC- reduced PCs averaged 3 × 10(8) in RD PCs and ranged from 8.6 to 9.6 × 10(6) per SD PC. Residual WBC counts in filtered PCs ranged from 2.3 × 10(4) to 2.2 × 10(5) and those in WBC-reduced SD PCs averaged 2.2 × 10(5) per unit. The data demonstrate significant phenotypic differences among PCs produced with various procedures. All SD PCs and two of four filtered RD PCs contained five WBC populations including granulocytes and monocytes, while RD PCs filtered with the remaining manufacturer's devices contained only lymphocytes. CONCLUSION: The data confirm that distinct phenotypic differences exist among PCs prepared with different devices and/or procedures. It is suggested that as for non-generic pharmaceuticals, the clinical benefits of these various PCs should be individually proved.     

Chemokines in stored platelet concentrates

BACKGROUND: Platelets contain several mediators, belonging to a family of proinflammatory cytokines named chemokines, that are stored in the organelles. Release and accumulation of these chemokines during storage of platelet concentrates (PCs) might be responsible for nonhemolytic transfusion reactions. STUDY DESIGN AND METHODS: Analysis was done of pH and the levels of platelet factor 4, beta-thromboglobulin, interleukin 8, RANTES, macrophage-inflammatory protein-1 alpha, lactate dehydrogenase, and serotonin in the supernatant of stored PCs on Days 1, 3, 5, and 8. PCs were prepared by apheresis or from pools of four buffy coats. Buffy coat PCs were filtered before storage. RESULTS: Nonfiltered apheresis PCs, which had a higher white cell contamination (p < 0.01), contained significantly more platelets than did buffy coat PCs (p = 0.02). The pH decreased significantly in apheresis PCs (p = 0.01), whereas there was a significant increase in lactate dehydrogenase (p < 0.001). In buffy coat PCs, pH remained stable and lactate dehydrogenase increased moderately. Concentrations of platelet factor 4 and beta-thromboglobulin increased steadily in both preparations over the storage period (p < 0.001). Macrophage- inflammatory protein-1 alpha was hardly detectable in the supernatant of both PCs, while RANTES levels increased significantly with storage time (p < 0.001). Interleukin 8 was not found in the supernatant of any PCs, with the exception of one apheresis PC with high white cell contamination (> 10(9)/ L). Serotonin levels were higher in apheresis PCs (p = 0.01), but the levels did not correlate with storage time. CONCLUSION: Platelet factor 4, beta-thromboglobulin, and RANTES were released from platelets during storage and accumulated over time in the PCs. These chemokines might play a causative role in nonhemolytic transfusion reactions because of their inflammatory potential, but the clinical effects of the transfusion of PCs with high chemokine contents remain to be investigated.     

Composition of peripheral blood progenitor cell components collected from healthy donors

BACKGROUND: Peripheral blood progenitor cell (PBPC) components are being collected from healthy donors for allogeneic transplantation, but the quantity, quality, composition, and variability of PBPCs collected from healthy people given granulocyte-colony-stimulating factor (G-CSF) have not been evaluated. STUDY DESIGN AND METHODS: PBPC components were collected from 150 healthy people who were given G-CSF (5, 7.5, or 10 microg/kg/day) for 5 days. The components were evaluated for white cell (WBC), mononuclear cell, CD34+ cell, neutrophil, platelet, and red cell (RBC) composition. RESULTS: The quantities collected were: WBCs, 35.0 +/? 16.4 × 10(9) (range, 11.9–163.3 × 10(9)); mononuclear cells, 33.3 +/? 14.4 × 10(9) (range, 11.9–139.6 × 10(9)); CD34+ cells, 412 +/? 287 × 10(6) (range, 70–1658 × 10(6)); neutrophils, 1.71 +/? 3.59 × 10(9) (range, 0–27.6 × 10(9)); RBCs, 7.2 +/? 4.0 mL (range, 0–22.1 mL); and platelets, 480 +/? 110 × 10(9) (range, 250–920 × 10(9)). PBPC components collected from people given G-CSF at 7.5 or 10 microg per kg per day contained significantly more CD34+ cells (respectively, 428 +/? 300 × 10(6); range, 70–1658 × 10(6) and 452 +/? 294 × 10(6); range, 78- 1380 × 10(6)) than those from people given G-CSF at 5 microg per kg per day (276 +/? 186 × 10(6); range, 91–767 × 10(6)) (p = 0.007 and p = 0.002). When 10 microg per kg per day of G-CSF was given, 50 percent of the components contained enough CD34+ cells for transplantation to a 75- kg recipient (375 × 10(6) CD34+ cells), but 10.6 percent of the components contained less than 150 × 10(6) CD34+ cells and thus would provide a transplantable dose only for a 30-kg patient. CONCLUSION: One PBPC component collected from a healthy donor given 7.5 or 10 microg per kg per day of G-CSF should contain 70 to 1660 × 10(6) CD34+ cells, with 0 to 22 mL of RBCs. Because of the great variability in the number of CD34+ cells collected, the quantity of CD34+ cells in each component should be measured after each procedure to ensure that sufficient quantities of cells are present for a successful transplant.     

Treatment of normal individuals with granulocyte-colony-stimulating factor: donor experiences and the effects on peripheral blood CD34+ cell counts and on the collection of peripheral blood stem cells

BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF) has been used in patients to increase the level of circulating hematopoietic progenitors. Although G-CSF has been administered to some healthy individuals, the kinetics of mobilization of peripheral blood stem cells (PBSCs), the optimum dose schedule and the incidence and nature of adverse reactions in normal individuals are not completely defined. STUDY DESIGN AND METHODS: Normal individuals (n = 102) who received G- CSF for 5 or 10 days at doses of 2, 5, 7.5, or 10 micrograms per kg per day were studied. The subjects were observed for symptoms and physical changes, and blood samples were obtained for a variety of laboratory tests. After 5 or 10 days of G-CSF treatment, PBSCs were collected by apheresis and analyzed. RESULTS: Overall, 89 percent of the individuals completed the 5-day treatment protocol and 88 percent completed the 10- day protocol without modification of the dose of G-CSF administered. Ninety percent of donors experienced some side effect of G-CSF. The most frequent effects noted were bone pain (83%), headache (39%), body aches (23%), fatigue (14%), and nausea and/or vomiting (12%). The dose of G-CSF administered directly affected the proportion of people with bone pain (p = 0.025) or body aches (p = 0.045) or who were feeling hot or having night sweats (p = 0.02) or taking analgesics (p = 0.01). With the 5-day dose schedule, several changes in serum chemistries occurred, including increases in alkaline phosphatase (p = 0.001), alanine aminotransferase (p = 0.0013), lactate dehydrogenase (p = 0.0001), and sodium (p = 0.0001). Decreases occurred in glucose (p = 0.045), potassium (p = 0.0004), bilirubin (p = 0.001), and blood urea nitrogen (p = 0.0017). In donors who received G-CSF for 5 days, the absolute neutrophil count was increased after one G-CSF dose, and it reached a maximum on Day 6, as did the number of CD34+ cells (64.6 +/? 55.9 × 10(6) cells/L). In those same donors, the platelet count after apheresis on Day 6 was 32 +/? 13 percent lower than pretreatment values (250 +/? 42 × 10(9) cells/L). In donors receiving G-CSF for 10 days, the neutrophil count reached a maximum on Day 8, but the number of CD34+ cells peaked on Day 6 (58.3 +/? 52.1 × 10(5) cells/L) and then declined. The platelet count decreased from pretreatment values by 28 +/? 12 percent prior to apheresis on Day 11. When individuals were treated for 5 days with G-CSF, the quantity of CD34+ cells collected was directly related to the G-CSF dose. When 5 micrograms per kg per day was given, 2.80 +/? 1.81 × 10(8) cells were collected, compared with collection of 4.67 +/? 3.11 × 10(8) cells when 10 micrograms per kg per day was given (p = 0.04). More important, PBSCs collected after 10 days of G-CSF administration (5 micrograms/kg/day) had significantly fewer CD34+ cells (0.82 +/? 0.37 × 10(8) cells, p = 0.01) than did PBSCs collected after 5 days of G-CSF (5 micrograms/kg/day). CONCLUSION: Most normal donors receiving G-CSF experience side effects, but these are mild to moderate in degree. Some alterations in blood chemistries occur, but none were clinically serious. Because of the symptoms associated with G-CSF, these individuals must be monitored closely. The treatment of normal donors with G-CSF for more than 5 days significantly decreased the number of circulating CD34+ cells and the quantity collected by apheresis.     

Effect of filtration and storage of platelet concentrates on the production of the chemotaxins C5a, interleukin 8, tumor necrosis factor alpha, and leukotriene B4

BACKGROUND: The production in platelet concentrates (PCs) of C3 activation products (C3bc), terminal complement complex (TCC), and chemotaxins C5a, interleukin (IL)-8, tumor necrosis factor alpha (TNFalpha), and leukotriene B4 (LTB4) and the proposed reduction in concentration of the chemotaxins by white cell reduction were examined. STUDY DESIGN AND METHODS: Samples were collected from supernatants of PCs produced by apheresis (apheresis PCs) or from buffy coats (BC PCs) immediately after the production, after white cell-reduction filtration on Day 1, and after 5-day storage, and examined by enzyme immunoassays. RESULTS: Complement was activated in all PCs during storage, and the concentration of activation products was not influenced by prestorage filtration. In prestorage white cell-reduced BC PCs, only C3bc levels increased. Levels of IL-8, TNFalpha, and LTB4 increased during storage of apheresis PCs, but not in filtered units, except for LTB4. In contrast, levels of IL-8 decreased after storage of filtered BC PCs. C5a correlated significantly with IL-8, which also correlated with TNFalpha and LTB4. CONCLUSION: Both C5a and TNFalpha generation in apheresis PCs seem to induce white cell IL-8 production, which mediates cellular LTB4 release. Prestorage white cell reduction is recommended for reducing chemotactic cytokine and leukotriene levels in all PCs. Production of BC PCs is recommended to achieve less complement activation, which is not affected by filtration.     

The in vitro evaluation of two filters (Erypur and Imugard IG 500) for white cell-poor platelet concentrates

White cell-poor blood components are useful in patients with white cell antibodies. White cells are efficiently removed by two different filters, Imugard and Erypur, which have used saline as the filter solution. This study evaluated these filters as to their production of white cell-poor platelets. Pools of random-donor platelet concentrates were filtered. Prefiltration and postfiltration samples were evaluated for percentages of platelet recovery, white cell (WBC) removal, and platelet function. The two filter solutions tested were normal-strength saline (NSS) and fresh-frozen plasma (FFP). Postfiltration samples using NSS showed no measurable platelet aggregation with ADP, epinephrine, or collagen. However, with FFP, both filters showed 100 percent platelet aggregation with ADP, epinephrine, and collagen. The FFP filter solution provided excellent white cell removal in both filters (Imugard: 100% WBC removal or less than 1.0 X 10(6) residual WBC; Erypur: 99.5% removal or greater than 1.0 X 10(7) residual WBC); however, platelet recovery was better with Imugard (95%) than with Erypur (55%). The filtration procedure is an excellent method for the preparation of white cell-poor platelets; however, the quantity of the saline solution recommended for the filtering of red cells must be minimized for platelets.     

In vitro and in vivo evaluation of cotton wool filtration of platelet concentrates obtained by automated and manual apheresis

The effect of cotton wool filtration of apheresis platelet concentrates (PCs) on platelet viability and complement activation was evaluated by two laboratories. PCs were prepared by automated (Lab A, n = 5) or manual (Lab B, n = 5) apheresis. After storage for 1 day, the PC was filtered through cotton wool before transfusion on one occasion and, on the other occasion, filtered through a standard screen filter before transfusion to the same donor. Five paired studies were performed by each laboratory. Except for a small, but significant reduction in mean platelet size, from 7.3 +/- 1.1 to 6.6 +/- 0.9 microns 3, after cotton wool filtration, no effect of filtration on various tests of in vitro platelet function and morphologic integrity was found. As demonstrated by autologous radiolabeled studies, no effect of cotton wool filtration on platelet viability was found by Laboratory B, while Laboratory A found a slight increase in the percentage of recovery from 59 +/- 4 to 68 +/- 13 percent, and a small reduction in survival, from 8.2 +/- 0.9 to 7.7 +/- 0.5 days after cotton wool filtration (p less than 0.05). Cotton wool filtration was associated with a slight increase in C3a levels found in manual apheresis PCs. Neither laboratory found any effect of cotton wool filtration per se on the recipients' white cell (WBC) counts or C3a and C5a levels after transfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of a 12-hour, 22 degrees C holding period for buffy coats on the preparation of platelet concentrates stored in plasma

BACKGROUND: The preparation of platelet concentrates (PCs) from buffy coats (BCs) stored at room temperature is controversial, because of the strong metabolic activity of cells in BCs and the possible detrimental effect of neutrophil enzymes on platelets when the holding time before separation is prolonged. Despite good in vitro and in vivo behavior of BC-PCs stored in synthetic solution, little is known of the quality of BC-PCs stored in plasma. STUDY DESIGN AND METHODS: Comparison was made of PCs prepared from BCs held at 22 degrees C for 3 hours (3-hour BC- PCs) or overnight (12-hour BC-PCs) and stored in plasma. Platelet and white cell counts, pH, response to osmotic shock, and morphologic scores were determined on 20 PCs of each type. The decrease in dense granule and alpha granule content, a marker of platelet activation, were estimated by mepacrine counting and beta-thromboglobulin measurement, respectively (n = 8–10). Platelet function was studied in terms of aggregation and thromboxane production in response to various concentrations of collagen and thrombin (n = 8–17). PCs prepared from unstored BCs (n = 15) and from BCs held for 90 minutes (n = 15) were used as controls. RESULTS: Platelet yield was increased from 53 +/? 10 percent of donated platelets to 73 +/? 4 percent by increasing the BC holding time from 0 to 90 minutes to 3 hours (p < 0.001). Similar yields (7.8 +/? 1.8 vs. 7.9 +/? 2 × 10(10) platelets) and white cell contamination (0.9 +/? 0.8 vs. 1.0 +/? 0.9 × 10(7)) were obtained with 3-hour and 12-hour BC-PCs. At the end of the storage period (Day 5), all variables known to correlate with platelet survival in vivo were well maintained in both 3-hour and 12-hour BC-PCs: pH > or = 6.9, response to osmotic shock > or = 70 percent, and morphology scores always > or = 240. During storage, the dense granule content decreased moderately (30% after 5 days), whatever the conditions. By contrast, the total platelet beta-thromboglobulin content was better preserved in 12-hour BC-PCs than in 3-hour BC-PCs (p < 0.04). No significant differences were observed in collagen-induced aggregation and thromboxane production in the two PC preparations. However, aggregation responses to thrombin were higher in 12-hour BC-PCs on Day 5 of storage (p < 0.01). CONCLUSION: BCs can be held at 22 degrees C for up to 12 hours, with no detrimental effect on the quality of PCs stored for up to 5 days in plasma. Such a holding time might help overcome logistic problems in blood banks     

Loss of high-affinity thrombin receptors during platelet concentrate storage impairs the reactivity of platelets to thrombin

BACKGROUND: The storage of platelet concentrates (PCs) induces a reduction in the platelet surface expression of glycoprotein (GP) Ib alpha. The location of the platelets' high-affinity binding site for thrombin has been postulated as being located on GPIb alpha. This study attempts to determine whether loss or alteration of GPIb alpha during storage of PCs is related to impairment in the reactivity of platelets to thrombin. STUDY DESIGN AND METHODS: In this study, platelet surface expression of GPIb alpha was monitored by means of flow cytometry, throughout standard storage of PCs for up to 10 days. Two thrombin- induced platelet responses, the binding of radiolabeled fibrinogen and the platelet surface expression of P-selectin, were evaluated. Thrombin- binding assays were also performed to assess the number of thrombin receptors in platelets. RESULTS: The surface expression of the GPIb/IX complex declines during storage of PCs. The thrombin-induced maximal binding of fibrinogen in platelets stored for 3, 7, and 10 days was 77 +/? 7 percent, 60 +/? 20 percent, and 34 +/? 25 percent, respectively, of that found in fresh platelets. Moreover, the concentration of thrombin needed for 50 percent of platelets to express the CD62 antigen P-selectin at the surface increased from 0.05 U per mL in fresh platelets to 0.11, 0.56, and 1.2 U per mL in platelets stored for 3, 7, and 10 days, respectively. Thrombin-binding experiments demonstrated a significant reduction in the number of high-affinity binding sites throughout storage of PCs (55 +/? 21 sites/platelet in 10-day-stored platelets vs. 73 +/? 25 in fresh platelets). A significant correlation was also observed between the number of high-affinity thrombin-binding sites and surface expression of GPIb alpha. Selective blockage of the thrombin-binding site on GPIb alpha with monoclonal antibody LJ-Ib10 also inhibited the response of fresh platelets to thrombin, up to a level equivalent to that found in 3-day-stored platelets. CONCLUSION: The loss of the GPIb alpha-located high-affinity thrombin-binding site may impair the ability of platelets to become activated by thrombin as storage time increases.     

Effects of prestorage white cell reduction on platelet aggregate formation and the activation state of platelets and plasma enzyme systems

BACKGROUND: The introduction of prestorage white cell (WBC) reduction in random-donor platelet concentrates in Canada has increased the occurrence of particulate material in PCs. The effects of filtration on platelet activation state and the activation of plasma enzyme systems were assessed. STUDY DESIGN AND METHODS: Particulate material was examined by light microscopy, electron microscopy, protein electrophoresis, and biochemical analysis. Thirty PCs (10 unfiltered, 20 filtered) were examined during processing and 5-day storage for pH, platelet count and mean volume, morphology, activation marker expression, and hypotonic shock response. Complement activation, thrombin generation, and fibrinolysis were assessed by using specific enzyme immunoassays or chromogenic assays. RESULTS: By all analyses, the particulate material appeared to be platelet aggregates. Platelets exposed to WBC-reduction filters expressed a significantly higher level of activation markers CD62 and CD63, altered morphology, and increased platelet microparticles throughout the storage period than did unfiltered platelets. Complement activation at the C3 level was significantly increased in filtered units with little evidence of coagulation or fibrinolytic system activation. CONCLUSION: Exposure of platelets to filters during prestorage WBC reduction increased platelet activation and mildly increased complement activation over the levels during the storage period. These alterations can contribute to the formation of irreversible platelet aggregates during processing.     

Clinical importance of platelet antigens

HLA antibodies are the most important cause for immunological refractorines to platelet transfusions, and their formation can be avoided in the majority of patients by exclusive use of leukocyte filtered blood products with a residual white cell count < 1 × 10⁶ per unit.Chemotherapy reduces the risk of platelet antibody formation; patients under cytostatic treatment for malignancies often demonstrate transient immunization and they can also lose antibodies present at start of the treatment.In the management of patients with immunologic refractoriness, treatment should first be attempted with HLA compatible platelets from single donors. If a transfusion effect is not obtained, cross match (lymphocytotoxicity test + platelet radioactive antiglobulin test) negative platelets from single donors should be attempted. As the next step related donors can be evaluated, and finally treatment with plasma apheresis or high dose intravenous IgG therapy should be considered.     

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.     

Comparison of platelet loss during leukocyte reduction at 4, 24, and 48 hours postcollection using a closed system apheresis kit with integral filter

Prestorage leukocyte reduction of platelet concentrates may reduce adverse effects of transfusion while affording better quality control. Platelets and leukocytes may undergo activation during storage, which could affect the performance of leukocyte reduction filters. The purpose of this study was to evaluate the efficiency of leukocyte reduction and concomitant platelet loss with a new apheresis kit with an integral leukocyte reduction filter. Twelve donors underwent plateletpheresis on three occasions using the CS-3000 PLUS Blood Cell Separator with the Access™ Management System and the Access™ Closed System Apheresis Kit with Integral Sepacell® Leukocyte Reduction Filter and Double Return Line Needle (Baxter-Fenwal Division, Deerfield, IL). Of the three products from each donor, one each was filtered at 4, 24, and 48 hours completion of the plateletpheresis. Mean prefiltration platelet count was 4.43 × 10¹¹ and mean postfiltration platelet count was 3.56 × 10¹¹. Mean platelet recovery at 4, 24, and 48 hours filtration was 75%, 83%, and 84%, respectively. Analysis of variance (ANOVA) demonstrated that platelet recovery with filtration at four hours was significantly less than with filtration at 24 hours (P = 0.0236) and filtration at 48 hours (P = 0.0122). Platelet recovery with filtration at 24 hour did not differ significantly from filtration at 48 hours (P = 0.7684). Mean prefiltration WBC count was 0.93 × 10⁶ and mean postfiltration WBC count was 0.12 × 10⁶. Efficiency of leukocyte reduction was not significantly related to when filtration was performed. There was no significant variation from donor to donor in platelet recovery or in leukocyte reduction efficiency. This method of prestorage leukocyte reduction demonstrated slightly but statistically significantly better platelet recovery with filtration at 24 or 48 hours after platelet collection compared to four hours. All filtration times provided acceptable platelet yields with very low residual WBC. J. Clin. Apheresis 12:14–17, 1997 © 1997 Wiley-Liss, Inc.     

Effect of filtration of platelet concentrates on the accumulation of cytokines and platelet release factors during storage

BACKGROUND: Platelet transfusions are frequently accompanied by febrile nonhemolytic transfusion reactions. These may be due, in part, to the release of cytokines interleukin 1 beta (IL-1 beta), interleukin 6 (IL- 6), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-alpha) by white cells (WBCs) into the plasma during storage of platelet concentrates (PCs). Acting as endogenous pyrogens, these agents may induce inflammatory responses. STUDY DESIGN AND METHODS: This study proposed to determine if WBC reduction in PCs by filtration significantly reduced the levels of cytokines normally generated during storage of unfiltered PCs up to 5 days. Serotonin, platelet-derived growth factor (PDGF-AB), and von Willebrand factor levels were also assessed to establish whether or not filtration or storage elicited significant platelet activation and granule release. RESULTS: Filtration significantly reduced total WBC counts by 99.1 percent before storage (p < 0.001) without affecting total platelet counts. Compared to unfiltered PCs, filtration prevented a rise in the levels of each cytokine by Day 3 for IL-1 beta (27.7 vs. 0.6 pg/mL; p < 0.05), IL-6 (114.2 vs. 0.4 pg/mL; p < 0.001), and IL-8 (4.2 vs. 0.02 ng/mL; p < 0.001). By Day 5, further increases in the levels of all cytokines were noted in unfiltered PCs, but Day 0 levels remained in filtered PCs (IL-1 beta: 105.4 vs. 0.4 pg/mL, p < 0.001; TNF-alpha: 42.2 vs. 7.5 pg/mL, p < 0.025; IL-6: 268.8 vs. 0.4 pg/mL, p < 0.001; and IL-8: 7.6 vs. 0.02 ng/mL, p < 0.001). From Day 0 to Day 5, there were significant increases in serotonin (21.3 vs. 6.3 ng/mL, p < 0.05), PDGF-AB (72.6 vs. 25.8 ng/mL, p < 0.001), and von Willebrand factor (4.7 vs. 2.7 IU/mL, p < 0.05) in unfiltered PCs, with similar increased levels being observed in filtered PCs during storage. CONCLUSION: These data indicate that the accumulation of high levels of cytokines in stored PCs could be prevented by WBC-reduction filtration of PCs without the induction of significant platelet activation or granule release. As cytokines have the potential to induce febrile nonhemolytic transfusion reactions in patients, the transfusion of WBC-reduced PCs would be expected to reduce the frequency and severity of such reactions.