Filtration. A method to prepare white cell-poor platelet concentrates with optimal preservation of platelet viability

HLA alloimmunization is a major problem for thrombocytopenic patients receiving long-term platelet support. It is caused by white cells (WBCs) that are present as contaminants in platelet concentrates (PCs). Recent data have shown that filtration is an effective means to reduce WBC contamination, but it has little effect on the recovery of platelets. The present report evaluates two filters, a cellulose acetate (CA) filter requiring the inactivation of platelets with prostacyclin and a cotton wool (CW) filter requiring no platelet inactivation. The results show that, using fresh pooled PCs from six random donors, both filters reduce WBC contamination below 10(7) per PC, the likely threshold below which alloimmunization does not develop. With platelets stored for 2 to 3 days the efficacy of the CW filter decreases. Neither filter inflicts important damage to the platelets, as there is no considerable platelet activation or cell disruption. Moreover, PCs prepared by both filters show normal survival and effectively reduce the bleeding times. Thus, filtration of PCs results in platelets with optimal responsiveness both in vitro and in vivo.     

2种国产血小板滤器滤除白细胞对体外血小板功能的影响

目的考察2种血小板滤器滤除手工法制备的浓缩血小板中的白细胞后血小板功能的变化情况。方法采用富血小板血浆法(PRP法)以400 ml新鲜全血制备浓缩血小板,将6袋ABO同型的浓缩血小板汇集,并用2种国产血小板型去白细胞滤器过滤,各10例(分别以A、B组代之),测定过滤前后的血小板计数、白细胞计数、pH值、血小板CD62p阳性表达率、血小板聚集和低渗休克等指标。结果血小板去白过滤后,A、B 2种滤器(组)的血小板回收率、剩余白细胞数及pH值分别为(87.01±3.47)%vs(87.88±4.77)%、(0.95±0.90)×106vs(0.45±0.58)×106及(7.13±0.13)vs(6.80±0.26)(P>0.05);血小板过滤前后CD62p阳性表达率、血小板最大聚集率和低渗休克,A组分别为(8.06±4.11)%vs(8.21±4.50)%、(70.55±27.21)%vs(71.63±32.24)%和(68.14±10.13)%vs(69.18±9.38)%,B组分别为(10.34±3.26)%vs(10.47±2.42)%、(56.30±18.43)%vs(59.49±19.15)%和(75.73±5.50)vs(73.74±6.52)%(P>0.05)。结论所考察的2种血小板型去白细胞滤器过滤浓缩血小板未增加血小板的活化,对血小板聚集功能及抗低渗休克能力无明显影响,血小板回收率及剩余白细胞数符合相关标准。     

Histologic and immunohistochemical studies on the preparation of white cell-poor red cell concentrates: the filtration process using three different polyester filters

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.     

In vitro characteristics of white cell-reduced single-unit platelet concentrates stored in syringes

BACKGROUND: Platelet concentrates (PCs) for premature infants may be subjected to filtration, centrifugation, and various storage conditions before transfusion. STUDY DESIGN AND METHODS: As there are few data on the cumulative effect of these procedures on PCs, platelet properties (including biochemical and functional in vitro assays) were evaluated after the processing of single units of PCs through a 1-unit-capacity high-efficiency white cell (WBC)-reduction filter followed by syringe storage at either 22 or 37 degrees C for 6 hours. Two- and 5-day-old PCs, volume-reduced PCs, and prestorage WBC-reduced PCs were evaluated. RESULTS: WBC filtration consistently resulted in a 3 to 4 log10 reduction in WBCs, with less than 15-percent platelet loss. No adverse effects of platelet function or evidence of increased platelet activation as determined by the percentage of P-selectin positivity were noted. A decrease in pH associated with increased lactate production and consumption of glucose was observed following syringe storage under all conditions tested. Such changes were most pronounced, however, with volume-reduced PCs stored at 37 degrees C (pH 6.31 +/− 0.15, lactate 23.0 +/− 3.06 mmol/L). All pH levels at the end of storage were above the minimum Food and Drug Administration requirement (pH 6.0). CONCLUSION: The in vitro data suggest that single units of PCs can undergo WBC filtration followed by syringe storage for up to 6 hours and still maintain acceptable storage characteristics. The practice of maintaining volume-reduced PCs in syringes for 6 hours at 37 degrees C in isolettes during transfusion should, however, be avoided.     

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

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

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

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

The evaluation of microbial contamination in platelet concentrates prepared by two different methods.

The microbial contamination of platelet concentrates (PCs) prepared by two different methods both with a high risk of bacterial contamination during preparation and storage were evaluated. For apheresis platelets, the concentrates were obtained using the Haemonetics MCS 3P device. For the random method, platelets were obtained by two phase centrifugation, in the Heraeus Cryofuge 8500 I device using the Kansuk 3-way bags which permit storage for five days. 1620 plateletpheresis units prepared by apheresis, and 9838 units prepared by the random method, were included in the study. Of the 11,458 PCs studied. 32 (0.27%) were false positives and 24 (0.2%) were real positives. All of the positive results occurred in platelets prepared by the random method. C. xerosis and S. epidermidis, S. hominis, Alpha-hemolytic streptococci, all flora of the skin, were isolated in the contaminated concentrates. The risk of microbial contamination of PCs, prepared both by apheresis and from whole blood, continues at a low rate although the products were collected into specific bags following rules including appropriate disinfection of the skin, correct centrifugation collection time and optimal storage conditions including temperature and agitation. These results again emphasize the importance of: obeying phlebotomy rules and hand disinfection of the person who collects the blood as well as the need for careful skin decontamination of the donor, during donation.     

In vitro evaluation of platelet concentrates during storage: Platelet counts and markers of platelet destruction.

BACKGROUND: Differences in platelet counts are observed by use of automated haematology analyzers making interlaboratory comparison difficult. MATERIALS AND METHODS: Twenty-eight single-donor platelet concentrates (PCs) were collected. Platelet concentration and markers of platelet destruction were investigated during storage for 11/12 days. RESULTS: Increasing impedance-immunoplatelet ratio was observed during storage, correlating to platelet fragments, large platelets, platelet density and cell-lysis. High variability was observed for optical-immunoplatelet ratio. CONCLUSION: Immunoplatelet count or correction factor calculated by impedance-immunoplatelet ratio should be used to confirm that platelet unit meets platelet count requirements or to compare results from clinical trials. Optical platelet count is not recommended.     

Improved platelet compatiblity of water vapour glow discharge treated non-woven poly(ethylene terephthalate) leukocyte-reduction filters for different types of platelet concentrates

Non-woven poly[ethylene terephthalate] (NW-PET) filter fabric, usually used for leucocyte removal of red cells, was modified by water vapour glow discharge (WVGD) treatment to improve platelet compatibility. Modified filter material was evaluated with different kinds of platelet concentrates (PCs). In addition, modified filter materials were gamma-sterilized and tested after different time intervals at different storage conditions. Modification of the filter material resulted in an improved platelet recovery after filtration of PC from 57 to about 80%. No significant difference in platelet recovery was observed when filtering either freshly prepared (79 +/- 3.5%, mean +/- SD), overnight-stored single BC-PC (78 +/- 3.3%), overnight-stored single PRP-PC (75 +/- 8.8%) or overnight-stored pooled BC-PC (79 +/- 8.9%). However, freshly prepared pooled BC-PC gave a significantly higher platelet recovery (84 +/- 3.5%). Leukocyte depletion did not differ significantly between the different types of PC. gamma-Sterilization and subsequent storage of the modified filter material for 5, 14 and 26 weeks at 20 degrees C or 37 degrees C had no significant influence on the filtration results of overnight-stored pooled BC-PC. The results of the present study show that WVGD-treated NW-PET is platelet compatible and can be used for leucocyte removal from preferably BC-PC. It can be gamma-sterilized and stored for at least 6 months prior to filtration without affecting the platelet recovery and leucocyte removal.     

In vitro evaluation of prestorage pools consisting of mixed A and O platelet concentrates

BACKGROUND: Current FDA regulations allow the prestorage pooling of whole blood-derived platelet concentrates (PCs) of identical ABO type in a recently cleared platelet (PLT) pooling bag (Acrodose, Pall Medical). It is unclear how ABO-mixed PC pools would store if pooled before storage. STUDY DESIGN AND METHODS: Pools consisting of ABO-identical PLTs and mixed A and O PLTs in varying proportions were evaluated on Days 1, 5, and 7 of storage with measures of the PLT storage lesion, lymphocyte activation, and activation of the complement and coagulation system. Data were analyzed by analysis of variance. RESULTS: Pools did not differ on Day 7 in pH (p = 0.63), hypotonic shock response (p = 0.25), extent of shape change (p = 0.26), morphology score (p = 0.18), or white cell count (p = 0.79), but surface P-selectin expression was more evident in the ABO-mixed pools (p = 0.02). Small microscopic clumps of PLTs were observed in all pools, but were more prominent in the ABO-mixed pools (p < 0.01). PLT counts, however, did not differ between pools (p = 0.93), indicating that only a small proportion of PLTs were clumped. Surface A-antigen expression was proportional to the number of A PCs in each pool and did not vary between study days. Anti-A(1) titers were either unchanged or decreased by one dilution. Complement and coagulation activation markers did not differ between pools. CONCLUSION: Pooling A and O PCs is associated with evidence of increased microscopic PLT clumping and activation, but these differences are not exacerbated with 7-day storage. Other major measures of PLT quality do not differ, and there was no evidence of a mixed lymphocyte reaction.     

In vitro evaluation of metabolic changes and residual platelet responsiveness in photochemical treated and gamma-irradiated single-donor platelet concentrates during long-term storage

BACKGROUND: Photochemical treatment (PCT) prevents replication of pathogens in platelet concentrates (PCs) by cross-linking nucleic acids and thus affects all cells containing DNA or RNA. STUDY DESIGN AND METHODS: Fourteen double-dose single-donor PCs were divided into two study arms. The double-dose PCs were split in two identical units, PCT and conventional control PCs. Study Arm A consisted of seven PCT PCs with corresponding untreated controls, whereas Study Arm B consisted of seven PCT PCs with corresponding gamma-irradiated control. Metabolic changes and agonist-induced platelet (PLT) response were evaluated during storage for up to 12 days. RESULTS: Higher rate of PLT destruction, illustrated by reduced PLT content, increased lactate dehydrogenase levels, and higher CD61+ microparticle formation rate, were observed after PCT. Generally PCT accelerated metabolic changes in PCs and reduced agonist-induced (collagen or thrombin receptor activator peptide [TRAP]) aggregation responses. Flow cytometric analysis of CD62P and CD42b (GPIbalpha) expression showed higher spontaneous PLT activation in PCT PCs from 5 days of storage. Correspondingly, a reduced capacity for up regulation of CD62P expression and down regulation of CD42b was observed in PCT PLTs after stimulation by the agonists ADP or TRAP. CONCLUSION: Generally reduced in vitro PLT quality was observed after PCT during storage for up to 12 days, with marked reduction from 5 days of storage. Compared to conventional PCs, reduced agonist-induced aggregation and glycoprotein expression were observed after PCT during storage, corresponding to significantly higher level of spontaneous PLT activation in PCT PCs. Clinical studies of efficacy and safety of PCT PCs stored for more than 5 days are recommended.     

Preparation and storage of white blood cell-reduced split apheresis platelet concentrates for pediatric use

BACKGROUND: White blood cell (WBC) reduction and bacterial screening induce unacceptable product loss when platelet (PLT) concentrates (PCs) for pediatric transfusion are prepared from whole blood. The aim was to investigate PCs, WBC reduced and bacterially screened, from single-donor apheresis procedures, divided in 3 or 4 pediatric units and stored up to 5 days. STUDY DESIGN AND METHODS: PCs were collected with an apheresis machine and WBC reduced by in-process filtration. The PCs were sampled for bacterial screening and subsequently divided in 70-mL products. Initially, storage characteristics of split units in 400-mL polyvinylchloride (PVC) bags with 17 split PCs originating from five apheresis donations were studied. When a 600-mL container made of the more gas-permeable polyolefin became available, a paired comparison was performed with 9 split PCs from nine donations and with a higher-yield PLT collection procedure. RESULTS: Split PCs contained 69 x 10(9) +/- 14 x 10(9) PLTs in 69 +/- 1 mL of plasma, and storage in the PVC containers gave a pH value of 6.86 +/- 0.10 on Day 6 (mean +/- SD, n = 17). When comparing the containers, the PVC bag contained 98 x 10(9) +/- 15 x 10(9) PLTs in 72 +/- 4 mL versus 102 x 10(9) +/- 18 x 10(9) PLTs in 74 +/- 8 mL for the polyolefin bag (n = 9, not significant). This gave pH values on Day 6 of 6.12 +/- 0.50 in the PVC container, whereas pH remained acceptable in the polyolefin container: 6.85 +/- 0.10 on Day 6 (p < 0.01). CONCLUSION: PCs for pediatric use from split single-donor apheresis concentrates, WBC reduced and bacterially screened, can be stored for up to 5 days in a 600-mL polyolefin container with maintenance of good in vitro storage variables.     

Paired comparison of the in vivo and in vitro results of storage of platelet concentrates in two containers

Both in vitro and in vivo methods are used to test the validity of techniques for storing platelet concentrates for transfusion. In this study, the characteristics of platelet concentrates stored for 5 days at 22 degrees C in two different containers were evaluated by paired comparison using two in vitro measurements and two in vivo measurements. On two occasions, 10 normal subjects donated concentrates that were stored in containers of either the CLX system or the PL-146 system. The first plastic used was chosen at random. If necessary, a concentrate platelet count was reduced to 1,200,000 per microliters by addition of plasma to avoid pH fall. Mean recoveries were 48.2 +/- 10.6 percent (mean +/- 1 SD) and 42.4 +/- 7.8 percent for platelets stored in containers of the CLX and PL-146 systems, respectively. Similarly, survivals (T 1/2 in days) were 3.4 +/- 0.8 and 3.0 +/- 0.7, respectively. Since a paired design was used, the superiority of the CLX system was demonstrable with a one-tailed paired t test. If a paired design had not been used, a pooled t test would have been appropriate and the differences would not have been significant. This result emphasizes the value of the paired design. Furthermore, two in vitro measurements that reflect platelet morphology, dispersion of the size distribution and extent of shape change with adenosine diphosphate, were superior for platelets stored in CLX containers as well, suggesting a relationship between these measurements and in vivo viability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of two detection methods of microorganisms in platelet concentrates

Background: The performance of a bacterial 16S ribosomal DNA real‐time polymerase chain reaction (PCR) assay was evaluated and validated with an automated culture system to determine its use for screening of platelet concentrates (PCs). Study Design and Methods: PCs were spiked with suspensions of Escherichia coli, Serratia marcescens, Staphylococcus epidermidis and St. aureus at 1, 10, and 100 colony‐forming units (CFUs) mL and stored for 5 days. DNA amplification was performed using real‐time PCR. The BacT/ALERT was used as a reference method and samples were inoculated into an aerobic culture bottle; for the PCR assay, aliquots were drawn from all (spiked) PCs on days 0 to 5 of storage. Results: Real‐time PCR detected only the gram‐positive bacteria in PCs spiked with low bacterial titres (1 CFU mL) after 48 h; however, it was able to detect all positive samples in PCs spiked with 10 CFU mL of either gram‐positive or gram‐negative bacteria after 48 h. In addition, real‐time PCR detected all positive samples in PCs spiked with high gram‐positive bacterial titres (100 CFU mL) after 24 h. On the other hand, the BacT/ALERT system showed positive results in all samples within 24 h. Conclusion: The BacT/ALERT method is more sensitive and should continue to be the gold standard for identifying bacterial contaminations in blood samples. The real‐time PCR approach can be used for the screening of PCs for microbial detection before they are released from blood centres or shortly before they are used in blood transfusion, and thus allow an extended shelf life of the platelets.     

Effect of prestorage white cell reduction on bacterial growth in platelet concentrates

Platelet concentrates stored with and without autologous white cells were produced from units of whole blood that had been purposefully contaminated with bacteria immediately after phlebotomy. The blood was inoculated with one of five species of bacterium at either 10 or 50 colony-forming units per mL. The growth of the organisms was quantified throughout the conventional 5-day, 22 degrees C storage period of the platelet concentrates. One species, Klebsiella pneumoniae, failed to grow in any of the components. The remaining species, Staphylococcus epidermidis, S. aureus, Enterococcus faecalis, and Salmonella enteritidis, achieved log growth after 1 day of storage and reached a relative maximum concentration by Day 3. Although the concentration of bacteria immediately after inoculation was lower in the units reduced in white cells by filtration, no significant differences were observed thereafter. Data from this in vitro study support the concept that prestorage white cell reduction of platelet concentrates should not increase the likelihood of transfusion-induced septicemia.