Evaluation of the tolerability and immunogenicity of ultraviolet C‐irradiated autologous platelets in a dog model

BACKGROUND: The THERAFLEX ultraviolet (UV) platelets (PLTs) pathogen reduction system for PLT concentrates (PCs) operates using ultraviolet C (UVC) light at a wavelength of 254 nm. UVC treatment can potentially alter proteins, which may affect drug tolerance in humans and influence the immunogenicity of blood products. This preclinical study in beagle dogs was designed to evaluate the safety pharmacology of UVC‐irradiated PCs after intravenous administration and to determine whether they are capable of eliciting humoral responses to PLTs and plasma proteins. STUDY DESIGN AND METHODS: Six beagle dogs each were transfused once every other week for 10 weeks with UVC‐irradiated or nonirradiated PCs. All PCs were autologous canine single‐donor products prepared from whole blood. Safety pharmacology variables were regularly assessed. The impact of UVC irradiation on PLT and plasma proteomes was analyzed by one‐ and two‐dimensional gel electrophoresis. Serum samples were tested for UVC‐induced antibodies by Western blot and flow cytometry. RESULTS: Dogs transfused with UVC‐irradiated PCs showed no signs of local or systemic intolerance. Few but significant changes in PLT protein integrity were observed after UVC irradiation. Even after repeated administration of UVC‐irradiated PCs, no antibodies against UVC‐exposed plasma or PLT proteins were detected. CONCLUSIONS: Repeated transfusions of autologous UVC‐treated PCs were well tolerated in all dogs studied. UVC irradiation did not cause significant plasma or PLT protein modifications capable of inducing specific antibody responses in the dogs. High‐resolution proteomics combined with antibody analysis introduces a comprehensive and sensitive method for screening of protein modifications and antibodies specific for pathogen reduction treatment.     

Pathogen inactivation of platelets with a photochemical treatment with amotosalen HCl and ultraviolet light: process used in the SPRINT trial

BACKGROUND: A photochemical treatment (PCT) system has been developed to inactivate a broad spectrum of pathogens and white blood cells in platelet (PLT) products. The system comprises PLT additive solution (PAS III), amotosalen HCl, a compound adsorption device (CAD), a microprocessor-controlled ultraviolet A light source, and a commercially assembled system of interconnected plastic containers. STUDY DESIGN AND METHODS: A clinical prototype of the PCT system was used in a large, randomized, controlled, double-blind, Phase III clinical trial (SPRINT) that compared the efficacy and safety of PCT apheresis PLTs to untreated apheresis PLTs. The ability of multiple users was assessed in a blood center setting to perform the PCT and meet target process specifications. RESULTS: Each parameter was evaluated for 2237 to 2855 PCT PLT products. PCT requirements with respect to mean PLT dose, volume, and plasma content were met. Transfused PCT PLT products contained a mean of 3.6 x 10(11) +/- 0.7 x 10(11) PLTs. The clinical process, which included trial-specific samples, resulted in a mean PLT loss of 0.8 x 10(11) +/- 0.6 x 10(11) PLTs per product. CAD treatment effectively reduced the amotosalen concentration from a mean of 31.9 +/- 5.3 micromol per L after illumination to a mean of 0.41 +/- 0.56 micromol per L after CAD. In general, there was little variation between sites for any parameter. CONCLUSIONS: The PCT process was successfully implemented by 12 blood centers in the United States to produce PCT PLTs used in a prospective, randomized trial where therapeutic efficacy of PCT PLTs was demonstrated. Process control was achieved under blood bank operating conditions.     

Pathogen inactivation of Trypanosoma cruzi in plasma and platelet concentrates using riboflavin and ultraviolet light.

BACKGROUND: Emigration of people infected with Trypanosoma cruzi to non-endemic areas has resulted in transfusion transmission to immunocompromised recipients. We studied the feasibility of inactivating T. cruzi using a new technology which utilizes riboflavin as a photosensitizer in combination with UV light, Mirasol PRT. METHODS: One billion T. cruzi organisms and 30 mL of 500 microM riboflavin were added to each of six units of human plasma and six units of platelets. To determine the level of detection of organism, a sample of each unit was cultured in tenfold serial dilutions beginning with 100 billion/250 mL as the starting culture. After 30 min, each unit was illuminated with 5.9 J/cm(2) of UV light (6.24 J/mL). The units were then cultured again in tenfold serial dilutions post-treatment. RESULTS: A 6 log reduction of pathogens was demonstrated in 5 of 6 units of plasma, and a 7 log reduction of pathogens was demonstrated in one unit. A 6 log reduction of pathogens was demonstrated in 3 of 6 units of platelets, a 7 log reduction was demonstrated in 2 of 6 units of platelets, and an 8 log reduction of pathogens was demonstrated in 1 of 6 units. CONCLUSIONS: Mirasol PRT treatment demonstrated an ability to inactivate 5-7 logs of T. cruzi in plasma and platelets.     

In vitro pH effects on in vivo recovery and survival of platelets: an analysis by the BEST Collaborative

BACKGROUND: The pH environment of stored platelet (PLT) products is recognized as an important factor and is generally used as a key surrogate measure of PLT viability. It is the only in vitro measurement that has been translated into industry standards and regulatory rules or specifications for storage of PLT products. The objective of this study was to evaluate the effect of in vitro pH on the in vivo recovery and survival of autologous PLT products.
STUDY DESIGN AND METHODS: Data from individual autologous radiolabeled PLT kinetic studies were solicited from independent laboratories. PLTs stored for at least 5 days in 100 percent autologous plasma with a pH_22°C of at least 6.2 were analyzed. Data were fit to a mixed-effects regression model with fixed effects of pH_22°C, time of storage, and preparation method-storage bag combination.
RESULTS: Eight research laboratories reported 476 individual recovery and survival results with associated pH before labeling from a variety of autologous, radiolabeled PLT kinetic studies from September 1999 to March 2005. These results are from 254 individual subjects who donated a total of 386 PLT units, with up to nine collections per subject reported. The effect of pH on either PLT recovery (p = 0.86) or survival (p = 0.55) was not significant. Time of storage and the method-bag combination both had significant effects on these outcomes (p < 0.0001).
CONCLUSION: These data suggest that there is no relationship between in vitro pH at a pH_22°C of at least 6.2 and in vivo PLT viability as measured by radiolabeled recovery and survival of autologous PLTs.     

Transfusing platelets 2 h after the completion of amphotericin-B decreases its detrimental effect on transfused platelet recovery and survival

Platelet transfusion support is required during bone marrow aplasia following ablative chemotherapy and bone marrow progenitor cell transplantation (BMT). Amphotericin-B is frequently given to these patients, both therapeutically and prophylactically, and has been described to have a negative impact on the results of platelet transfusions. We conducted a prospective study of the effect of amphotericin-B on transfused platelet recovery and survival in 81 BMT or acute leukaemia patients. One hundred and ninety-five platelet transfusions administered to 81 consecutive patients were analysed. The platelets were transfused 2 h after the completion of amphotericin-B. Using this schedule resulted in no effect of amphotericin-B on platelet recovery or survival, although platelet increments were modestly depressed in patients receiving high- vs. low-dose amphotericin-B. We conclude that the timing of amphotericin-B infusion be evaluated in patients demonstrating poor platelet recovery and survival. Transfusing platelets at least 2 h after the completion of amphotericin-B decreases the detrimental effect of this antifungal agent on transfused platelet recovery and survival.     

Seven-day storage of single-donor platelets: recovery and survival in an autologous transfusion study

BACKGROUND: Bacterial screening may effectively reduce the morbidity and mortality risk associated with extended storage of platelets. Platelet viability then becomes the primary determinant of acceptable storage time. This study evaluates the effectiveness of platelets stored in plasma for 7 days. STUDY DESIGN AND METHODS: WBC-reduced, single-donor platelets (n = 24) were collected and stored by standard methods at two sites. Standard in vitro platelet biochemical and functional parameters were monitored over the storage period. On Days 5 and 7 of storage, platelets were alternately labeled with 51Cr and (111)In and returned to the subject, and recovery and survival were determined. RESULTS: Component pH(22 degrees C) was maintained in the range 6.2 to 7.61 through 7 days and did not detrimentally affect either in vitro or in vivo outcomes. In vitro platelet characteristics were adequately maintained over 7 days. Day 5 platelets had better recovery (63.0 +/- 4.36 vs. 53.9 +/- 4.36%, p < 0.0001) and survival (161 +/- 8.1 vs. 133 +/- 8.1 hr, p = 0.006) than Day 7 platelets adjusting for radioisotope, center, and donor effects. CONCLUSION: Although declines in recovery and survival were noted, these are less than used previously to gain licensure of 7-day storage and are unlikely to be clinically significant. Extension of storage to 7 days could be implemented with bacterial screening methods to select out contaminated components without a significant effect on the platelet efficacy compared to 5-day components.     

Quantitative and qualitative analysis of coagulation factors in cryoprecipitate prepared from fresh‐frozen plasma inactivated with amotosalen and ultraviolet A light

BACKGROUND: There were no previous studies about the quality of cryoprecipitate prepared from fresh‐frozen plasma (FFP) inactivated with amotosalen and ultraviolet A (UVA) light. The aim of this study was to analyze the quantity and quality of coagulation factors in cryoprecipitate prepared from FFP treated with amotosalen and UVA light. STUDY DESIGN AND METHODS: FFP was obtained from whole blood donations and inactivated with amotosalen and UVA light according to the manufacturer's instructions. Fibrinogen, factor VIII (FVIII), von Willebrand factor antigen (VWF : Ag) and activity (VWF : RCo), the von Willebrand factor cleavage protease activity (ADAMTS‐13), and the multimeric structure of VWF were analyzed. RESULTS: The content of fibrinogen, FVIII, and ADAMTS‐13 was lower in cryoprecipitates prepared from amotosalen‐treated plasma when compared with cryoprecipitates prepared from nontreated plasma (35, 40, and 18% loss, respectively). The quantity and quality of VWF as well as VWF multimer patterns were not affected by the inactivation method. CONCLUSION: Cryoprecipitates prepared from amotosalen‐treated FFP contained significantly reduced levels of fibrinogen, FVIII, and ADAMTS‐13. However, the VWF quantity and quality was well preserved.     

In vivo recovery and survival of apheresis and whole blood-derived platelets: a paired comparison in healthy volunteers

BACKGROUND: Methods of platelet preparation may alter the recovery and survival characteristics of platelets following transfusion. As suggested by a recent clinical trial, platelet recovery may be better preserved with apheresis platelet preparations than with platelets prepared from whole blood by the platelet-rich plasma (PRP) method. STUDY DESIGN AND METHODS: In vivo platelet recovery and survival of autologous leukoreduced (LR) apheresis platelets and autologous filter-LR PRP platelets were compared in 22 healthy volunteers using a paired crossover design. On the same day, each participant gave one apheresis platelet donation and one whole blood donation from which platelets were recovered from the PRP. The sequence of donations was randomly assigned for each participant. Following 5 days of storage and bacterial screening, a sample from each platelet product was labeled with either (51)chromium or (111)indium (randomly assigned) and both samples were simultaneously re-infused into the original donor. Recovery and gamma-function platelet survival were calculated for each platelet product using the multiple hit mathematical model. RESULTS: Five day stored LR-apheresis platelets had 18.8 percent better recovery, and 32.9 percent longer gamma-survival than filter-LR PRP platelets. Stored apheresis platelets had lower p-selectin expression and higher morphology scores than stored PRP platelets. CONCLUSIONS: Filter-LR PRP platelet preparation appears to adversely affect platelet recovery and survival characteristics. The reasons for this effect are not clear. These results may not apply to all apheresis and PRP methods of platelet preparation.     

Pathogen reduction of fresh plasma using riboflavin and ultraviolet light: effects on plasma coagulation proteins

BACKGROUND: Treatment with riboflavin and ultraviolet (UV) light reduces the pathogens present in blood components. This study assessed changes to the coagulation proteins that had occurred during this treatment of fresh plasma units before freezing.
STUDY DESIGN AND METHODS: Twenty fresh plasma units (230 ± 30 mL) were treated by the Mirasol process (CaridianBCT Biotechnologies) and frozen within 8 hours of donation. Plasma units were combined with 35 mL of a 500 µmol/L riboflavin solution in an illumination bag to achieve a final concentration of approximately 60 µmol/L riboflavin. The bag was placed in the Mirasol illuminator and exposed to UV light (6.24 J/mL). Samples were frozen before and after treatment.
RESULTS: Recoveries observed were 67.7 ± 3.9% Factor (F)XI, 68.5 ± 3.3% FVIII:C, 78.8 ± 4.5% fibrinogen, 78.9 ± 4.1% FV, 79.0 ± 4.2% FVII, 79.0 ± 8.6% F IX, 79.7 ± 2.6% FX, and 85.0 ± 3.7% FII. Von Willebrand factor (VWF) antigen, VWF:ristocetin cofactor, and ADAMTS13 recoveries were 87.0 ± 7.1, 85.5 ± 6.6, and 73.3 ± 15.2%, respectively, while that of protein C was 83.6 ± 2.6%. A loss of high-molecular-weight VWF multimers was observed in most units. Recoveries for protein S, antithrombin, and plasmin inhibitor were greater than 90%. The mean FVIII:C concentration, after treatment, was 0.76 ± 0.17 IU/mL.
CONCLUSIONS: As with other pathogen reduction technologies, the Mirasol process resulted in some loss of coagulation factor activity. For most Mirasol-treated units and for most of the tested factors this is unlikely to have clinical impact, but trials are required to demonstrate this.     

汕头市2003年流感病毒感染的病原及血清流行病学调查

流感是由流感病毒引起的急性呼吸道传染病。其传染速度快,发病率高。由于流感病毒抗原易发生变异,所以它是人类至今尚不能有效控制的世界性传染病,并被世界卫生组织列为21世纪重点防治的传染病之一。20世纪,全球共发生4次流感大流行,其中3次起源于中国。1998年广东省首次发现禽H9N2流感病毒能感染人,许多流感专家推测下次世界性流感大流行即将到来,其首发地仍很可能在中国。近年来,广东省大部分地区,特别     

白膜法汇集浓缩血小板体外质量和功能特性研究

目的研究白膜法汇集浓缩血小板体外质量和功能特性,为国家制定统一的白膜法血小板质量标准提供依据。方法应用白膜法制备汇集浓缩血小板并检测分析各项质量指标,与机采血小板国家标准(GB18469-2001)比对。分别用血小板聚集仪和流式细胞仪测定白膜法汇集浓缩血小板、机采血小板对诱导剂ADP的最大聚集率与血小板膜表面糖蛋白分子CD62P和PAC-1的表达率。结果白膜法制备的汇集浓缩血小板质量指标分别为:容积为(270±14)m l,含量为(2.58±0.51)×1011,pH值为7.24±0.09,红细胞混入量为(6.61±0.16)×109,白细胞混入量为(0.011±0.008)×109。两组血小板对ADP的最大聚集率分别为(85.1±9.0)%和(87.9±11.1)%,P>0.05;两组血小板膜表面糖蛋白分子CD62P表达率分别为(12.3±1.7)%和(11.7±2.1)%,P>0.05;PAC-1表达率分别为(9.3±1.7)%和(8.9±1.4)%,P>0.05。结论白膜法汇集浓缩血小板体外质量指标均达到机采血小板国家标准,血小板功能活性保持良好。     

The effect of phospholipase C on human blood platelets.

The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C. A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity. The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.