Preparation of antihemophilic factor and fibronectin from human plasma cryoprecipitate

Starting with human plasma cryoprecipitate, fibronectin was separated from antihemophilic factor (AHF) by fractional precipitation under mild conditions resulting in excellent recovery of AHF in the supernatant solution of the cryoprecipitate. Separation of fibronectin enabled accelerated sterile filtration of the supernatant solution containing AHF even after three- to fourfold concentration (by ultrafiltration) to desired potency. The sterile AHF concentrate, dispensed at 1000 u per vial and lyophilized, was completely dissolved within 3 minutes upon addition of 30 ml of pure water. The expected increment in circulating factor VIII and its hemostatic effects were found following intravenous infusion into factor VIII-deficient patients. Yield of AHF of five successive batches, each starting with the cryoprecipitate from some 12,000 units of fresh-frozen plasma, averaged 51 percent. The fibronectin precipitate was purified by affinity on insolubilized gelatin with chaotropic elution at pH 5.5 followed by removal of the chaotrope by diafiltration. Thermal denaturation of adventitious fibrinogen resulted in electrophoretically pure fibronectin which, following lyophilization and reconstitution with pure water, retained biological properties in an in vitro assay designed to reflect opsonic activity. The yield of fibronectin for seven successive batches, each starting with the cryoprecipitate from some 900 units of fresh-frozen plasma, averaged 10 percent.     

In vivo viability of stored red blood cells derived from riboflavin plus ultraviolet light-treated whole blood

BACKGROUND: A novel system using ultraviolet (UV) light and riboflavin (Mirasol System, CaridianBCT Biotechnologies) to fragment nucleic acids has been developed to treat whole blood (WB), aiming at the reduction of potential pathogen load and white blood cell inactivation. We evaluated stored red blood cell (RBC) metabolic status and viability, in vitro and in vivo, of riboflavin/UV light–treated WB (IMPROVE study). STUDY DESIGN AND METHODS: The study compared recovery and survival of RBCs obtained from nonleukoreduced WB treated using three different UV light energies (22, 33, or 44 J/mL_RBC). After treatment, WB from 12 subjects was separated into components and tested at the beginning and end of component storage. After 42 days of storage, an aliquot of RBCs was radiolabeled and autologously reinfused into subjects for analysis of 24‐hour recovery and survival of RBCs. RESULTS: Eleven subjects completed the in vivo study. No device‐related adverse events were observed. By Day 42 of storage, a significant change in the concentrations of sodium and potassium was observed. Five subjects had a 24‐hour RBC recovery of 75% or more with no significant differences among the energy groups. RBC t_1/2 was 24 ± 9 days for the combined three groups. Significant correlations between 24‐hour RBC recovery and survival, hemolysis, adenosine triphosphate (ATP), and CO₂ levels were observed. CONCLUSIONS: This study shows that key RBC quality variables, hemolysis, and ATP concentration may be predictive of their 24‐hour recovery and t_1/2 survival. These variables will now be used to assess modifications to the system including storage duration, storage temperature, and appropriate energy dose for treatment.     

Quality of proteins in riboflavin and UV light-treated FFP during 1 year of storage at −18 °C

Background

The Mirasol® Pathogen Reduction Technology (PRT) System for plasma combines riboflavin (vitamin B₂) and UV light energy to reduce pathogens in plasma products. This study evaluated protein quality of apheresis derived plasma products treated with riboflavin and UV light and stored at −18 °C for up to 1 year. Comparisons were made to paired, untreated controls after 6 months and 1 year of storage.

Methods

This study evaluated the in vitro protein quality of apheresis derived plasma products processed between 8 and 8.5 h from collection and stored at −18 °C for 6 months and 1 year. Plasma (205 ± 5 mL) was combined with 35 ± 5 mL of 500 μM riboflavin, and exposed to UV light (6.24 J/mL). A total of 14 plasma units were used for this study. Plasma units were analyzed using standard coagulation assays immediately following treatment, and after 6 months and 1 year of storage at −18 °C.

Results

Fibrinogen, Total protein, Factors II, VIII, IX, X and XII were modestly affected by storage for 1 year at −18 °C. The average percent protein retention in treated plasma samples after 1 year of storage in comparison to controls held under similar conditions were: Total Protein, 103%, Factor II, 88%, Factors VII and XI, 62%, Factor V 60%, Factor VIII and IX, 77%, Fibrinogen, 75%, Factor X, 85% and Factor XII, 82%.

Conclusion

Results after 6 months (double the EU guidelines recommended storage time) and 1 year (four times longer than the EU guidelines recommended storage time) of storage at −18 °C demonstrated that all proteins were well preserved. Values observed for treated products stored under these conditions are within the same range as those reported for other pathogen inactivated plasma products.     

Long-term frequent plasma exchange donation of cryoprecipitate

Plasma exchange donation accomplishes the selective donation of cryoprecipitate. It facilitates the repeated donation of large quantities of factor VIII by individual donors and reduces donor exposure for recipients. A highly motivated donor is described who has undergone 103 donations between May 1983 and March 1987, producing 359,460 IU of factor VIII and supplying all the factor VIII needed since August 1983 by his severely affected hemophiliac son, now age 14. The donor has remained in good health, and no significant abnormalities have been noted in hematologic, biochemical, immunologic, coagulation, and serum protein testing. Extensive experience with this donor suggests that repeated plasma-exchange donation is safe and can sometimes allow single-donor support of severe hemophiliacs.     

分离白膜后血浆制备冷沉淀的质量评价

目的对采用22℃手工制备富含血小板白膜后分离的血浆制备冷沉淀的质量进行评价。方法 2013年11月—2014年6月,共随机选取22℃手工制备富含血小板白膜后分离的血浆70例,将其作为原料血浆制成冷沉淀后留样(即A组)。同时另随机选取4℃常规制备新鲜冰冻血浆90例,将其作为原料血浆制成冷沉淀后留样(即B组)。用全自动血凝分析仪检测冷沉淀中Ⅷ因子、纤维蛋白原含量,运用统计学方法对比分析2组有无统计学差异。结果 A组、B组Ⅷ因子含量分别为(136.96±59.32)IU、(163.53±77.33)IU(P0.05),合格率分别为97%、99%(P0.05)。A组、B组纤维蛋白原含量为(165.68±25.73)mg/袋、(164.11±17.54)mg/袋(P0.05),合格率分别为91%和98%(P0.05)。结论本文试验数据显示,22℃手工制备富含血小板白膜后分离的血浆可以用于制备冷沉淀。     

Antithrombin III in fresh frozen plasma, cryoprecipitate, and cryoprecipitate-depleted plasma

Antithrombin III (AT III) is a plasma protein that inhibits several activated procoagulants. Hereditary disease or acquired conditions such as severe hepatic dysfunction, nephrotic syndrome and intravascular coagulation may be associated with reduced levels of AT III. Its replacement may be essential in controlling thrombosis. In order to determine the most effective form of replacement, we compared AT III biological activity and antigen levels in conventionally prepared fresh frozen plasma, cryoprecipitate and cryoprecipitate depleted plasma (CDP). Both the activity and antigen levels were comparable in all three products (approximately 100%) and AT III was not concentrated in cryoprecipitate. These results indicate that conventionally prepared CDP, fresh frozen plasma and cryoprecipitate contain equal quantities volume for volume of AT III. On this basis, all products are equally effective as therapy for AT III deficiency, but CDP and fresh frozen plasma are recommended as convenient sources of this factor.     

Preparation of factor VIII concentrates by cryoprecipitate sedimentation

A new method for factor VIII concentrate preparation is proposed. The method is the collection of cryoprecipitate after sedimentation at 4 C in a jacketed glass vessel. The final product is standardized (4.0 +/- 0.3 factor VIII units per ml) and can be infused in hemophilic patients regardless of their ABO blood type. Factor VIII recovery (52.2 +/- 6.4%) is comparable to other processes.     

Evaluation of factor VIII-rich cryoprecipitate and the plasma fibronectin-rich, heparin-precipitable fraction prepared from single- donor plasma units

A plasma fibronectin-rich component was prepared by heparin-induced 4 degrees C precipitation of fresh or stored (21 days at 4 degrees C), single-donor plasma. The recovery of plasma fibronectin was 45 percent at a concentration of 0.05 mg heparin per ml (7.5 units/ml) and 75 percent at 0.1 mg per ml (15 units/ml). The biologic activity of plasma fibronectin, as assessed by the spreading of Chinese hamster ovary cells or attachment of monocytes to gelatin-coated surfaces, was similar to that of plasma fibronectin concentrates made from fresh or stored plasma. Only 20 to 30 percent of the factor VIII activity in fresh plasma was recovered in cryoprecipitate produced after the heparin-induced precipitate containing fibronectin was removed. Cryoprecipitate prepared from the supernatant plasma that remains after heparin-induced cold precipitation in the presence of CaCl2 (5 mM) contained approximately 50 percent less factor VIII. The relatively low recovery of factor VIII in cryoprecipitate prepared from fibronectin-depleted plasma makes cryoprecipitation an unsuitable method of producing fibronectin-rich and factor VIII-rich components effectively from a single unit of fresh plasma. However, heparin-induced cold precipitation provides an efficient method for preparing plasma fibronectin concentrates from small plasma pools or single units of stored or fresh plasma.     

Cryopreservation of apheresis platelets treated with riboflavin and UV light

BackgroundPathogen reduction technology (PRT) is increasingly used in the preparation of platelets for therapeutic transfusion. As the Czech Republic considers PRT, we asked what effects PRT may have on the recovery and function of platelets after cryopreservation (CP), which we use in both military and civilian blood settings.Study design and methods16 Group O apheresis platelets units were treated with PRT (Mirasol, Terumo BCT, USA) before freezing; 15 similarly collected units were frozen without PRT as controls. All units were processed with 5–6% DMSO, frozen at − 80 °C, stored > 14 days, and reconstituted in thawed AB plasma. After reconstitution, all units were assessed for: platelet count, mean platelet volume (MPV), platelet recovery, thromboelastography, thrombin generation time, endogenous thrombin potential (ETP), glucose, lactate, pH, pO2, pCO2, HCO3, CD41, CD42b, CD62, Annexin V, CCL5, CD62P, and aggregates > 2 mm and selected units for Kunicki score.ResultsPRT treated platelet units had lower platelet number (247 vs 278 ×10⁹/U), reduced thromboelastographic MA (38 vs 62 mm) and demonstrated aggregates compared to untreated platelets. Plasma coagulation functions were largely unchanged.ConclusionsSamples from PRT units showed reduced platelet number, reduced function greater than the reduced number would cause, and aggregates. While the platelet numbers are sufficient to meet the European standard, marked platelets activation with weak clot strength suggest reduced effectiveness.     

2种方法制备冷沉淀的质量分析

目的通过对冷藏融化法和水浴融化法制备冷沉淀的质量分析,选择合适的制备冷沉淀的方法。方法随机抽取2006年前后采用冷藏融化法和水浴融化法制备的冷沉淀各60份;利用血凝仪检测冷沉淀中FⅧ和Fg的含量,并计算FⅧ和Fg的合格率。结果冷藏融化法和水浴融化法的FⅧ含量(IU/袋)分别为:70.30±23.68,161.62±39.45;FⅧ合格率分别为:43.33%(26/60),93.33%(56/60),两者比较均有统计学差异(P<0.001)。Fg的含量(mg/袋)分别为:222.72±63.75,228.12±66.83;Fg合格率分别为:88.33%(53/60),91.67%(55/60),两者比较均无统计学差异(P>0.05)。结论水浴融化法制备冷沉淀简便、快速,Ⅷ合格率高,明显优于冷藏融化法,值得推广应用。     

全血手工制备浓缩血小板后的血浆再制备冷沉淀的质量评价

目的评价室温新鲜全血白膜法制备浓缩血小板后的血浆再制备冷沉淀的质量。方法实验组为24例,新鲜全血(400 mL)置室温于<8 h用白膜法制备浓缩血小板后所得的血浆,冰冻保存。对照组1为12例,常规制备新鲜冰冻血浆,冰冻保存。对照组2为12例,新鲜冰冻单采血浆,血浆单采完毕分装为200 mL/袋并立即冰冻保存。3组血浆按常规制备冷沉淀,评价其质量:外观、凝血因子FⅧ及Fib的含量;血细胞残留量。结果 3组冷沉淀外观均正常;WBC含量在3组间无统计学意义。与对照组1比较:实验组凝血因子FⅧ(81.76±34.07)IU较低,Fib(202.63±48.58)mg及Plt(7.81±5.81)×109均较高。与对照组2比较:实验组凝血因子FⅧ含量相当,Fib(202.63±48.58)mg较高、Plt(7.81±5.81)×109较低。结论全血来源的制备浓缩血小板后的冰冻血浆还可以用于冷沉淀的制备,其质量符合国家标准。     

冷上清做置换液血浆置换治疗血栓性血小板减少性紫癜

目的　观察新鲜冰冻血浆制作冷沉淀后的上清液 (冷上清 )治疗血栓性血小板减少性紫癜 (TTP)的临床效果。方法　用冷上清作置换液 ,对患者作血浆置换 ,置换后症状缓解 ,继续以冷上清输注 ,配合激素、免疫抑制剂、抗血小板聚集药物治疗。结果　 1 0名患者中 ,9名各置换 3～ 6次 ,1名因反复发作 3次 ,共置换 1 0次 ;8名治愈 ,2名死亡。结论　应用冷上清作置换液血浆置换治疗TTP ,与用新鲜冰冻血浆作置换液有同样的效果。且当新鲜冰冻血浆输注无效时 ,应用冷上清后也可获得缓解     

Comparison of coagulation factor XIII content and concentration in cryoprecipitate and fresh-frozen plasma

BACKGROUND: For patients with plasma coagulation factor XIII (pFXIII) deficiency, recommended means of replacement include infusions of fresh‐frozen plasma (FFP), cryoprecipitate, or (where available) factor (F)XIII concentrates. Quantitative differences in pFXIII concentration in FFP and cryoprecipitate are not well defined and were, therefore, the subject of this study. STUDY DESIGN AND METHODS: FFP and cryoprecipitate (10 bags each from blood group O donors) were analyzed to quantify pFXIII activity and antigen. Coagulation FVIII, fibrinogen, and von Willebrand factor (VWF) were also quantitated. RESULTS: Mean (±SD) pFXIII activity in cryoprecipitate and FFP bags was 60 ± 30 and 288 ± 77 U per bag, respectively, and pFXIII antigen and activity levels were concordant. Other comparisons (mean ± SD) between cryoprecipitate and FFP, respectively, were as follows: coagulation FVIII activity, 133 ± 37 and 265 ± 83 U per bag; fibrinogen content (Clauss kinetic assay), 183 ± 44 and 725 ± 199 mg per bag; VWF antigen content, 181 ± 53 and 218 ± 70 U per bag; VWF ristocetin cofactor activity, 168 ± 34 and 221 ± 65 U per bag; VWF collagen‐binding activity, 164 ± 40 and 208 ± 71 U per bag; and fluid (plasma) volumes per bag, 21.3 ± 2.7 and 245 ± 29 mL. CONCLUSION: In contrast to other cryoprecipitable coagulation proteins, pFXIII is only mildly enriched in cryoprecipitate when compared with FFP (approx. two‐ to threefold). Although both products can provide effective pFXIII replacement, FFP may be preferred when infusion volume is not a major consideration and pFXIII concentrates are not available. VWF is substantially enriched in cryoprecipitate (approx. ninefold compared with its concentration in FFP), with VWF activity content exceeding that of FVIII by approximately 26 percent on average.     

Transfusing methylene blue-photoinactivated plasma instead of FFP is associated with an increased demand for plasma and cryoprecipitate

BACKGROUND: Photodynamic virus inactivation of plasma with methylene blue significantly decreases the recovery of fibrinogen and coagulation factors V and VIII. Because an adequate supply of fibrinogen is essential for the therapeutic efficacy of transfused plasma in many clinical settings, it was plausible that transfusing photoinactivated plasma (PIP) instead of FFP would result in an increased demand for plasma and cryoprecipitate. STUDY DESIGN AND METHODS: The study involved a retrospective analysis of the use of plasma at a university hospital (Barcelona, Spain) over three 1-year periods: one before the implementation of PIP therapy and two after. Blood components transfused to plasma recipients were listed by broad diagnostic categories based on the Diagnosis-Related Group classification. RESULTS: During the period under study, 2,967 patients were given plasma in this hospital. They received 27,434 units of plasma, 1,660 of cryoprecipitate, 10,079 of platelets, and 24,607 of packed RBCs. Patients undergoing surgical procedures accounted for 74 percent of all transfused plasma. In 71 percent of patient admissions, packed RBCs were transfused in addition to plasma. Diagnostic categories with the greatest requirement for plasma were cardiac valve surgery, liver transplant, wounds and traumatic injuries, and bowel surgery. The use of PIP was associated with a 56-percent increase in the aggregated demand for plasma, whereas the transfusion of non-virus-inactivated cryoprecipitate increased twofold the first year and threefold the second year. The growth in the use of plasma took place in all the diagnostic categories. In those categories that include patients with liver disease, a partial substitution of PIP for cryoprecipitate was observed during the second year after PIP therapy implementation. CONCLUSION: The use of PIP was associated with a marked increase in the demand for plasma and cryoprecipitate, which probably was due to the low hemostatic quality of the new component. It is possible that such an increase overrode the potential health benefits derived from transfusing virus-inactivated plasma.     

病毒灭活新鲜冰冻血浆制备冷沉淀的可行性探讨

目的探讨亚甲蓝光化学法(MB-P)病毒灭活新鲜冰冻血浆制备冷沉淀凝血因子的可行性。方法选取2014年1~6月无偿献血者捐献的400 mL全血93袋,分离新鲜冰冻血浆200 mL后再均分为2袋,各100mL,分别作为对照组和试验组。对照组直接制备冷沉淀凝血因子,试验组经MB-P法病毒灭活后再制备冷沉淀凝血因子。检测每袋冷沉淀凝血因子中FⅧ和Fbg水平。结果对照组FⅧ和Fbg水平分别为(82.9±7.1)IU/100mL、(102.4±8.5)mg/100mL,试验组分别为(56.6±5.3)IU/100mL、(83.3±5.6)mg/100mL,试验组FⅧ和Fbg水平均低于对照组,组间比较差异均有统计学意义(P0.05),且均符合相关国家标准。结论加强血液采集、储存、运输和制备过程中的质量控制,使用经MB-P法病毒灭活新鲜冰冻血浆制备的冷沉淀凝血因子符合相关国家标准。     

Thrombin generation and clot formation in methylene blue–treated plasma and cryoprecipitate

BACKGROUND: Methylene blue (MB) treatment of plasma is known to reduce the activity of clotting factors, but its effect on thrombin generation and clot formation is not well documented.
STUDY DESIGN AND METHODS: Individual clotting factors and inhibitors and global tests of thrombin generation and clot formation using rotational thrombelastometry (ROTEM) were assessed in a paired study of standard or MB plasma and cryoprecipitate (n = 20 each).
RESULTS: MB treatment resulted in a 10 percent reduction in endogenous thrombin potential and 30 percent decrease in peak thrombin as well as the expected 20 to 35 percent loss of Factor (F)VIII, fibrinogen, and FXI activity. MB treatment had no effect on the rate of clot formation and increased the clot firmness by 20 percent as assessed by ROTEM. There were minimal further changes in either coagulation factor levels or thrombin generation when thawed plasma was stored for an additional 24 hours. FVIII and fibrinogen content of MB cryoprecipitate was reduced by 30 and 40 percent, respectively, but this was not associated with altered clot time or rate of clot formation by ROTEM and only an 8 percent decrease in clot firmness.
CONCLUSIONS: It is concluded that MB treatment is associated with a reduction in the thrombin-generating capacity of plasma, but has very little effect on the strength of clot formation as assessed by thrombelastometry. The thrombin-generating capacity of standard and MB plasma is relatively unaltered by subsequent storage of thawed plasma at 4°C for 24 hours.     

Modulation of fibrinogen content in cryoprecipitate by temperature manipulation during plasma processing

In routine blood bank production of single-donation cryoprecipitate, the introduction of a 16-hour hold at 4 degrees C, with the frozen plasma units packed into polystyrene containers, resulted in plasma prethaw temperatures of -4 degrees C to -8 degrees C. This in turn resulted in cryoprecipitate fibrinogen levels that were 214 percent of those obtained when units were thawed immediately after removal from -30 degrees C storage. In scale-model production of factor VIII concentrate, plasma warmed from -30 to -10 to -15 degrees C over 18 hours before pooling and thawing yielded cryoprecipitate fibrinogen levels that were 66 percent of those found in plasma warmed to -2 to -5 degrees C over the same period. Processing -30 degrees C plasma without a warming period led to cryoprecipitate fibrinogen levels that were 40 percent of those obtained from plasma warmed to -2 to -5 degrees C. These differences were accentuated after purification of the cryoprecipitates to an intermediate-purity factor VIII concentrate. These results suggest that simple modifications in production methods allow the fibrinogen content of cryoprecipitate to be tailored to specific uses.     

The influence of riboflavin photochemistry on plasma coagulation factors

Studies with riboflavin in the 1960s showed that it could be effective at inactivating pathogens when exposed to light. The principal mode of action is through electron transfer reactions, most importantly in nucleic acids. This suggested that it could act as a photosensitizer useful in the inactivation of pathogens found in blood products.

Objective

To study the influence of photo-inactivation with riboflavin on the coagulation factors of plasma.

Methods

The photo-inactivation procedure of riboflavin plus light was applied. Fifty isogroup pools of two plasmas were made from 100 U of plasma that were derived from whole blood products that had previously been held overnight. Pools were split into two bags. One of them was photo-inactivated, and post inactivation samples were obtained. The second bag was not photo-inactivated and samples were taken. Total protein, fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, antithrombin III, PC, PS, α-2 antiplasmin and vWF:Ag, the multimeric structure of vWF and ADAMTS-13 were analyzed.

Results

In plasma, the proteins most sensitive to photo-inactivation were fibrinogen, FXI, FVIII, FV, and FIX (33%, 32%, 30%, 18% and 18% loss, respectively). Coagulation inhibitors, PS, antithrombin III and PC showed little decrease (all 2%). Retention of vWF and ADAMTS-13 were 99% and 88%, respectively.

Conclusions

As with other pathogen reduction procedures for plasma products, treatment with riboflavin and UV light resulted in reduction in the activity levels of several pro-coagulant factors. Coagulation inhibitors are well preserved.     

核黄素联合紫外线A照射对血浆中伪狂犬病毒的灭活效果

目的考察核黄素联合紫外线A对血浆中伪狂犬病毒的灭活效果。方法以伪狂犬病毒为模拟病毒,以Vero细胞为培养细胞,用病毒感染细胞,制备病毒增殖液;分别采用5、10、15及20J/cm2联合核黄素处理血浆,观察处理前后血浆病毒灭活的效果,筛选灭活病毒合适的紫外线剂量;将含有伪狂犬病毒血浆的样本分为实验组:采用上述筛选的紫外线强度照射联合核黄素处理;对照组1:单独采用紫外线A照射;对照组2:单独采用核黄素处理;阴性对照组:未采用紫外线照射和核黄素处理;分别在实验前后采用96孔细胞病变法,对照细胞病变效应,根据Reed-Muench公式计算病毒滴度。结果采用不同强度的紫外线联合核黄素处理血浆,紫外线强度为15及20J/cm2的灭活血浆病毒的效果明显,处理后病毒滴度分别下降4.55和4.39logs;实验组和对照组1病毒滴度分别下降4.55和4.28logs,有统计学意义(P<0.05);对照组2病毒滴度降低1.93logs,没有病毒灭活效果。结论核黄素联合紫外线可灭活血浆中伪狂犬病毒,单独紫外线照射也具有灭活血浆伪狂犬病毒的效果;而仅单独采用核黄素处理而未联合紫外线照射,对血浆中伪狂犬病毒灭活效果不明显。