Filtration through a polyester white cell-reduction filter of plasma- poor platelet concentrates prepared with an acetate-containing additive solution

It is of practical importance to known whether the adsorption of platelets and contaminating white cells (WBCs) by the WBC-reduction filter is altered when platelet concentrates (PCs) are prepared in a plasma-poor condition with an acetate-containing additive solution (Seto sol). Plasma-poor PCs with 11-percent residual plasma were prepared from apheresis platelet-rich plasma by using a sterile docking device with steam-sterilized Seto sol. Seto sol contains 115 mM (115 mmol/L) NaCl, 4 mM (4 mmol/L) KCl, 3 mM (3 mmol/L) MgCl2, 10 mM (10 mmol/L) Na3PO4, 15 mM (15 mmol/L) acetate, 3 mM (3 mmol/L) Na3 citrate, and 10 mM (10 mmol/L) glucose (pH 7.1). The solution was steam- sterilized under nitrogen gas. On Days 1 and 5, pooled Seto sol PCs (2.4 × 10(11) platelets) were filtered with a polyester filter at a flow rate of 10 mL per minute. The WBC-removal rate was over 99.9 percent with a platelet recovery of 88 percent following Day 1 filtration. These values were very similar to those of plasma PCs, and 84-percent recovery was achieved following Day 5 filtration. However, when 1 unit of Seto sol PCs with half the number of platelets was filtered with the polyester filter, platelet recovery was about 16 to 17 percent less than that of plasma PCs. Platelet quality was maintained if pooled Seto sol PCs were filtered on Day 1 and stored for over 4 days. Filtration did not alter platelet function in 1-day-old or 5-day-old Seto sol PCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Removal of white cells from red cells by transfusion through a new filter

The effectiveness of a new filter (RC100) for the preparation of white cell-depleted red cells (RBCs) at the bedside was evaluated in vitro and in vivo using three RBC products: standard RBC concentrate (CPDA units), RBCs suspended in saline-adenine-glucose-mannitol additive solution after the removal of plasma (SAGM units), and RBCs suspended in SAGM after the removal of plasma and buffy coat (SAGM-BC units). Median RBC recovery was at least 92 percent when 2 units were administered through one filter; median values for residual white cells and platelets were less than or equal to 20 × 10(6) and less than or equal to 2.5 × 10(9) per 2 units, respectively. The in vivo study was carried out in 80 multiply transfused patients with thalassemia, 35 of whom had experienced frequent nonhemolytic transfusion reactions when given standard or buffy coat-free RBCs. During the 6-month study, each patient was given two transfusions of each type of RBC product One febrile nonhemolytic transfusion reaction occurred in each of two patients receiving SAGM-BC units, but in no other case. If the flow rate is not reduced, the median transfusion time is 35 minutes per CPDA unit and 15 minutes per SAGM and SAGM-BC unit. It is concluded that the transfusion of RBCs through the RC100 is a simple and effective procedure to administer white cell-depleted RBCs prepared at the bedside.     

White cell depletion of single-donor platelet preparations by a new adsorption filter

A newly developed adsorption filter has been evaluated for the removal of white cells (WBCs) from platelet concentrates. It consists of surface-modified polyester fibers and can be used at the patient's bedside. WBC mean removal rate was 98.8 +/- 0.9 percent, and platelet recovery was 99.0 +/- 0.7 percent. The aggregation curves (ADP, collagen) and hypotonic shock response of platelets were not influenced by the filtration, and the plasma electrolyte and lactate dehydrogenase concentrations were unaffected. Transmission and scanning electron micrographs showed no difference in platelet ultrastructure before and after filtration. Determination of posttransfusion platelet recovery did not reveal any significant difference from that of nonfiltered platelets. The tested filter seems to be a suitable device for WBC depletion of single-donor platelet concentrates at the bedside.     

流式细胞术检测浓缩血小板中微量白细胞方法的探讨

目的探讨流式细胞术(FCM)荧光微球绝对计数法在浓缩血小板微量白细胞检测中的应用价值。方法对两种FCM方法(LeucoCOUNT和Leuco-Finder)计数浓缩血小板样本中微量白细胞的结果进行比较分析,并探讨了样本量与试剂量的最适比例。结果两种FCM法白细胞检测值与期望值均具高度相关性(P<0.001),相关系数(r)分别为0.9988(LeucoCOUNT)和0.9986(Leuco-Finder)。微量白细胞计数<70/μl时,样本量增加到总量的64%(700μl样本+400μl LeucoCOUNT试剂)为最适比例。结论流式细胞术荧光微球绝对计数法是一种快速、稳定、客观、精确的检测微量细胞数的方法,FCM法LeucoCOUNT和Leuco-Finder均适合于作为去白细胞浓缩血小板制剂的质控方法。     

Evaluation of a new whole-blood filter that allows preparation of platelet concentrates by platelet-rich plasma methods

BACKGROUND: A novel WBC-reduction in-line whole-blood (WB) filter that does not retain platelets was evaluated to assess the filtration performance and, after processing WB by the platelet-rich plasma (PRP) method, to analyze the storage quality of filtered platelet concentrate (PC) units. STUDY DESIGN AND METHODS: To analyze the filter retention, blood was collected from random donors into quadruple blood packs with an integral in-line filter (Imuflex WB-SP, Terumo; n = 25) or in standard triple bag systems (n = 30). To assess the in vitro storage characteristics of platelets, 26 WB units were pooled in pairs and redistributed into 13 units that underwent WBC reduction and 13 units that were not WBC reduced. In all cases, WB was separated into RBCs, PCs, and plasma by the PRP method and platelet function was compared. RESULTS: The filtration procedure led to RBC and PC WBC-reduced products that met the AABB and European requirements. The average filtration time was 30 minutes, the filter retained about 45 mL of WB, and there was no further loss of RBCs during the fractionation procedure. In vitro PC storage characteristics of the filtered units were similar to those of the nonfiltered units. CONCLUSION: A 4- and 3-log WBC reduction was observed in RBC and PC units that were produced by the PRP method, with a mean residual WBC content of 0.24 +/- 0.38 x 106 and 0.02 +/- 0.03 x 106 per unit, respectively. The procedure, performed under relatively simple logistics, results in good-quality, standard components that may reduce costs and ease the process of WBC reduction in transfusion services.     

Evaluation of a new citrate-acetate-NaCl platelet additive solution for the storage of white cell-reduced platelet concentrates obtained from half-strength CPD pooled buffy coats

BACKGROUND: A new citrate-acetate-NaCl platelet additive solution, identified as PAS 2, was developed to prepare platelet concentrates (PCs) from pooled 0.5 CPD buffy coats (BCs). STUDY DESIGN AND METHODS: A study was undertaken to evaluate PAS 2 in vitro (n = 8) and in vivo (n = 9) against a commercially available solution (Plasma-Lyte A). In a paired in vitro study, a comparison was made of platelet and white cell concentration; blood gases and bicarbonate; glucose and lactate concentration; total intracellular concentration of adenine nucleotides and beta-thromboglobulin release. RESULTS: A lower platelet yield (p < 0.0001) and a higher beta-thromboglobulin release (p < 0.01) are observed with Plasma-Lyte A. For this reason, half-strength (0.5) CPD was changed to full-strength CPD in the clinical study with Plasma-Lyte A. In a clinical evaluation of nine patients with bone marrow failure, all received PCs with both PAS 2 and Plasma-Lyte A that had a shelf life of at least 4 days. Corrected count increments (CCls) were as follows, on average (95% Cl): the CCl at 1 to 4 hours was 22.4 (95% Cl, 15.2-29.4) for PAS 2 and 24.0 (95% Cl, 16.9-31.2) for Plasma-Lyte A; that at 12 to 24 hours was 11.3 (95% Cl, 4.1-18.4) for PAS 2 and 14.2 (95% Cl, 7.1-21.3) for Plasma-Lyte A; and that at 36 to 48 hours was 4.2 (95% Cl, -3.0-11.3) for PAS 2 and 8.7 (95% Cl, 1.1-16.2) for Plasma- Lyte A. No significant difference between the two solutions was found. CONCLUSIONS: PAS 2 and Plasma-Lyte A make important contributions to platelet transfusion quality improvement and give an excellent CCl even after 4 days of storage.     

Preparation of white cell-reduced platelet concentrates from whole blood during component preparation

This article introduces a new method of component preparation that is capable of producing white cell (WBC)-reduced platelet concentrates (PCs) from whole blood. Whole blood is separated into packed red cells (RBCs) and platelet-rich plasma (PRP) by centrifugation, and the PRP is expressed through a newly designed WBC removal filter into the platelet storage bag. The filtered PRP is then centrifuged and yields WBC-reduced PCs and plasma for freezing as fresh-frozen plasma (FFP). The method uses standard triple-pack blood bags and centrifugation protocols. Fifteen WBC-reduced PCs prepared with this technique had an average volume of 56.7 mL, an average Day 5 platelet content of 8.6 x 10(10) per unit, and an average Day 5 WBC content of 0.83 +/- 0.7 x 10(4) per unit (0.14 WBCs/microL). This represents WBC removal equal to at least 99.9 percent (3 log10) of the WBCs found in standard PCs prepared in our laboratory by an identical centrifugation protocol. Paired studies documented a 4.5-percent platelet loss by filtration. Filtration had no effect on the plasma prepared for FFP as measured by prothrombin time; activated partial thromboplastin time; factors I, V, VIII:C, and VIII:von Willebrand factor; antithrombin-III; albumin; globulin; or total protein. This method holds promise as a simple and highly effective technique for the production of WBC-reduced PCs by filtration during component preparation.     

Factors affecting white cell content in platelet concentrates

In this study, we investigated the factors affecting white cell content in platelet concentrates. White cell yields can be reduced 50 percent by stopping platelet-rich plasma expression when the interface is 1 cm from the top of the blood bag as compared to stopping expression when the interface reaches the top of the bag. Further reductions can be achieved by careful handling during transfer of units from the centrifuge cups to expressors (after the first spin) and by carefully balancing units against each other to ensure proper rotor balance during the first spin. Following these suggestions, blood banks should be able to produce platelet concentrates with white cell yields between 2 and 6 X 10(7) and with platelet yields between 7.5 and 8 X 10(10). Transfusion of this product may reduce febrile reactions and lower the incidence of alloimmunizations.     

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.     

Real-time amplification of HLA-DQA1 for counting residual white blood cells in filtered platelet concentrates

BACKGROUND A real-time polymerase chain reaction (PCR) assay based on amplification of a conserved region of the HLA-DQA1 locus was developed and validated to assess its suitability in quantitating low levels of white blood cells (WBCs) in filtered platelet (PLT) concentrates (PCs). STUDY DESIGN AND METHODS: To determine the detection limit, serial dilutions of nonfiltered PCs with known quantities of WBCs were prepared. The analytical sensitivity and accuracy of the assay was tested with WBC concentrations ranging from 300 to 0.03 per microL with real-time PCR and flow cytometry. In addition, 126 random PCs were investigated to assess the capacity of the PCR method to quantify residual WBCs in clinical specimens. RESULTS: A sensitivity of 0.2 WBC equivalent per micro L (1.5 x 10(4) WBC equivalents/unit) was achieved. The assay was shown to be accurate and the HLA-DQA1 gene was reproducibly and consistently amplified in all tested samples (coefficient of variance of < 5%). Overall, the results of the PCR assay correlated well with those of the flow cytometry. The PCR assay detected a concentration of 3 WBCs per micro L (approximately 1 x 10(6) WBCs/unit) with 100 percent accuracy. CONCLUSION: Real-time PCR is rapid, sensitive, accurate, and reproducible. Hence this approach may prove suitable in routine monitoring of residual WBCs in PCs.     

White cells in fresh-frozen plasma: evaluation of a new white cell- reduction filter

BACKGROUND: Fresh-frozen plasma (FFP) has generally been regarded as an acellular component. Recently, viable lymphocytes have been detected in this component and the question of irradiation of FFP for certain patients has been raised. Whether the numbers of white cells (WBCs) in FFP are sufficient to require WBC-reduction of acellular components for patients receiving WBC-reduced cellular components has not been determined. WBC numbers in FFP were examined, and the performance of a new commercial WBC-reduction filter for FFP was assessed. STUDY DESIGN AND METHODS: WBC numbers in plasma processed for use as FFP and in thawed FFP were counted before and after WBC-reduction filtration by the use of flow cytometry Fast and slow filtration was used to simulate laboratory and bedside filtration, respectively. Three different methods for plasma harvesting (soft-spin, hard-spin, and second-spin methods) were assessed. The filter capacity was also examined. RESULTS: The numbers of WBCs in plasma covered a three-log10 range (soft-spin method, 0.04-3.6 × 10(6); hard-spin method, 0.47-45.4 × 10(6); second- spin method, 0.4–37.2 × 10(6)). For the hard-spin and second-spin methods which produced the greatest plasma yields, 92 percent and 85.7 percent of bags, respectively, had counts>1 × 10(6) and 43 percent (hard-spin method) and 45.7 percent (second-spin method) had counts>5 × 10(6). There was no significant difference between the counts obtained in plasma and thawed FFP. The filter reduced WBC numbers to <1 × 10(5) in all but 3 of 49 bags. In the remaining three, there were <2 × 10(5) WBCs. Five bags of plasma could be processed effectively through each filter. CONCLUSION: FFP may contain WBC numbers above the threshold at which the use of WBC-reduction filters for cellular components in some patients is necessary. Confirmation of these findings and similar investigations on plasma prepared by other methods may help in defining a role for the use of WBC-reduction filters for FFP     

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.     

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.     

Histamine levels in stored platelet concentrates. Relationship to white cell content

The histamine levels in samples from platelet concentrates (PC) were measured at various storage times by a radioenzymatic assay. Elevated histamine levels were detected in 5 of 14 PC after 3 days of storage (range, less than 1 to 13.3 ng/ml) and in 9 of 14 PC after 5 days (range, less than 1 to 22.2 ng/ml). A very good linear correlation (r = 0.913) was found between the initial white cell content of the PC and the histamine level at 5 days of storage. The rise in histamine content was not influenced by the type of plastic container. The results indicate a process of histamine release by the white cells during storage. Although histamine is metabolized rapidly in vivo, a critical histamine threshold could be reached in man by the rapid infusion of stored PC containing high levels of histamine. This could explain some unexpected transfusion reactions in patients receiving PC.     

Storage of platelet concentrates

The American National Red Cross has initiated a cooperative clinical study designed to evaluate the therapeutic effectiveness of fresh platelets and platelets stored at 4 or 22 C for up to 72 hours. Preliminary results, in confirmation of earlier work, indicate an inverse relationship between platelet count and bleeding time in patients with thrombocytopenia transfused with fresh platelets. Also, the observed rise in platelet count immediately following transfusion of fresh platelets is generally maintained for four to eight hours after which time it declines significantly.