Postthaw stability of fibrinogen in cryoprecipitate stored between 1 and 6 degrees C

The fibrinogen activity in thawed cryoprecipitate stored between 1 and 6 degrees C is maintained essentially unchanged in most bags for a month. Occasionally, a bag will have a reduction in fibrinogen. If pooling has not occurred, thawed cryoprecipitate should be useful as a source of fibrinogen for a period of time considerably in excess of the 6 hours allowed for its use as a source of factor VIII or von Willebrand factor.     

Comparison of cryoprecipitate with fibrinogen concentrate for acquired hypofibrinogenaemia

BackgroundThe benefits of fibrinogen concentrate in hypofibrinogenaemia have been established in congenital and has been used in acquired disorders. Most European countries have already changed their practice, using fibrinogen concentrate.MethodsWe compared the use of fibrinogen concentrate in acquired hypofibrinogenaemia to cryoprecipitate, which continues to be the standard of care in the UK. We undertook a retrospective analysis of fibrinogen increment in patients treated for acquired hypofibrinogenaemia.ResultsSixty four transfusion episodes receiving cryoprecipitate and 36 episodes receiving fibrinogen concentrate were compared. The median increment following 10 donor pools (two bags) of cryoprecipitate was 0.26 g/l, compared to 0.44 g/l following 2 g of fibrinogen concentrate.ConclusionWith its superior safety profile from infectious diseases, this provides further evidence to support the use of fibrinogen concentrate.     

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.     

In vitro formation and i vivo clearance of fibrinogen: fibrin complexes.

Fbrinogen:fibrin complexes have been previously described in various thrombotic disorders. To evaluate further the properties of fibrinogen:fibrin complexes, and theirin vitro and in vivo behavior, soluable fibrinogen:fibrin complexes have been formed invitro using mixtures of '131l-fibrinogen ('131l-F) and '125l-fibrin ('125l-fb). By means of Sepharose 4B chromatography, a macromolecular complex (peak one) containing both moieties could be separated from a lower molecular weight peak two containg noncomplexed material. The latter eluted at the same position as did intact fibrogen.Both the '131l-F and '125l-fb components of peak one were rapidly catabolized when injected into rabbits with residual blood activity at 24 hours of 8 per cent and 4 per cent, respectively. Peak two behavedas a simple mixture with corresponding 24-hour levels at 31 per cent and 3 per cent. Gel filtration of postinjuection plasma samples demonstrated that peak one remained as macromolecular complex. Preinjection crosslinking of the F:fb complex with factor xiii did not substantially change the blood clearance. Prior blockage of the reticuloendotheial system with Thorotrast or carbon resulted in impaired clearance of peak one. The data provide evidence that fibrinogen and fibrin can form a macromolecular complex which is stable both in vitro and vivo. Further, the reticuloendotheialsystem was shown to mediate the the in vivo clearance of this complex. This latterfinding may be of pathophysiologic significance.     

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. In vivo stability of disulfide-linked immunotoxin conjugates.

The stability in vivo and circulatory clearance of immunotoxins were assessed in rhesus monkeys. The immunotoxins studied were T cell-specific monoclonal anti-T11 antibodies conjugated by disulfide linkage to ribosome-inactivating toxins. Intact immunotoxin was detectable in the circulation of the monkeys following a single intravenous infusion. This was demonstrated by quantitative flow-cytometric analysis, gel-filtration, and sodium dodecyl sulfate-gel electrophoresis. This intact conjugate was shown to be functional in the plasma of the infused animals in an in vitro cytotoxicity assay. However, a number of factors contributed to bring the level of circulating immunotoxin to a less than optimal level. When conjugated to a ribosome-inactivating toxin, the antibody was cleared more rapidly than was the native antibody. Furthermore, following infusion, some breakdown of the conjugate occurred, resulting in the generation of detectable levels of circulating free antibody. The present data indicate the feasibility of using immunotoxins as therapeutic tools in man.     

Which is the preferred blood product for fibrinogen replacement in the bleeding patient with acquired hypofibrinogenemia—cryoprecipitate or fibrinogen concentrate?

The importance of the targeted treatment of acquired hypofibrinogenemia during hemorrhage with a concentrated fibrinogen product (either cryoprecipitate or fibrinogen concentrate) cannot be underestimated. Fibrinogen concentrate is a pathogen inactivated, pooled product that offers a highly purified single factor concentrate. Cryoprecipitate is a pooled product that comes with a spectrum of other coagulation factors which may further enhance (additional procoagulant effect) or even disturb (prothrombotic risk) hemostasis. The pros and cons of each product are discussed.     

Postfiltration factor VIII and fibrinogen levels in cryoprecipitate stored at room temperature and at 1 to 6 degrees C

The 13th edition of the standards of the American Association of Blood Banks specified storage at 1 to 6 degrees C for cryoprecipitated anti-hemophilic factor (Cryo) administered up to 6 hours after thawing if the Cryo is used for factor VIII (FVIII) content (Standard J4.210). Previous editions specified room-temperature (RT) storage for up to 6 hours. Currently, the temperature specification has been deleted. There are few data addressing the optimal storage temperature and maximum storage time for FVIII and fibrinogen in thawed Cryo. Thirty bags of Cryo were assayed for FVIII and fibrinogen. Each bag was divided into two aliquots; one was stored at RT and the other at 1 to 6 degrees C. Assays were performed immediately after thawing (Base) and 6 and 24 hours after thawing, respectively. All samples were filtered through 200-mu blood component infusion sets before assay. Three hundred analyses were performed, 150 each for FVIII and fibrinogen by conventional clotting technique. Data were analyzed by using a paired t test. Cryo stored at 1 to 6 degrees C for 6 and 24 hours showed an FVIII loss of 35 percent (p less than 0.0001) and 63 percent (p less than 0.0001), respectively. Cryo stored at RT for 6 and 24 hours had an FVIII loss of 8 percent (p greater than 0.05) and 20 percent (p less than 0.0001). Cryo stored at 1 to 6 degrees C for 6 and 24 hours had a fibrinogen loss of 20 percent (p less than 0.0001) and 43 percent (p less than 0.0001). Cryo stored at RT for 6 hours had no fibrinogen loss and a 2 percent loss at 24 hours (p greater than 0.05). These preliminary data show a significant loss of FVIII and fibrinogen activity in Cryo stored at 1 to 6 degrees C and filtered before assay. The FVIII and fibrinogen activity at RT is clearly maintained up to 6 hours after thawing.     

Variations in cryoprecipitate production

Several variables influencing the recovery of Factor VIII in the cold- precipitated protein fraction were examined. The actual rate at which the physical-chemical conversion of plasma from a solid to a liquid state occurs does not seem to affect the yield. Rather, it is the amount of time immediately postthaw and prior to centrifugation that determines the Factor VIII activity in the cryoprecipitate. With prolonged periods at 4 C the Factor VIII activity leaves the cold precipitate and lifts into the supernatant. Even distribution of the plasma layer and close attention to the thawing procedure facilitate Factor VIII recovery.     

Overestimation of fibrinogen concentration in cryoprecipitate by repeated freeze–thawing with long thawing period as used in the Clauss method

Background

Cryoprecipitate (CRY) is widely used for treating acquired hypofibrinogenemia. During our study to determine an optimal preparation method, we noticed that the measurement of fibrinogen concentration in CRY had a risk of overestimation. We analyzed this condition and mechanism.

Study Design and Methods

CRY was prepared from fresh frozen plasma (FFP) under four conditions: A, 30 h thawing time, 2 cycles; B, 24 h thawing time, 2 cycles; C, 30 h thawing time, 1 cycle; and D, 24 h thawing time, 1 cycle. Then, fibrinogen concentrations in CRY and cryosupernatant (CS) were measured by the Clauss method.

Results

Purification (CRY/CRY+CS) and recovery (CRY/FFP) rates in CRY prepared under 2-cycle conditions were higher than those under 1 cycle. However, recovery rates often exceeded 100%, particularly in the case of CRY prepared under A condition, and fibrinogen concentrations calculated by direct measurement were higher than those indirectly calculated from FFP and CS, suggesting an overestimation of fibrinogen values. The level of soluble fibrin monomer complex was considerably higher in CRY prepared under A than under D condition, indicating that CRY adopted a hypercoagulated state. We further found that repeated thawing/freezing increased fibrinogen values as measured by the Clauss method while mechanical vortexing did not.

Discussion

Our findings suggest that direct assessment of fibrinogen contents in CRY prepared by repeated freeze–thawing with a longer thawing period presents a higher risk of overestimation. For the purpose of quality control, we propose an alternative method to indirectly estimate fibrinogen concentrations in CRY from those of CS and FFP.     

冷沉淀的临床应用分析

目的了解冷沉淀在临床的应用情况。方法收集我院2009年1月-2011年1月冷沉淀输注情况,将冷沉淀的使用情况按专业科室分别归类。结果冷沉淀的使用主要集中在胸外ICU、血液内科,消化内科及普外科。其中血液内科、胸外ICU的用量最多。结论冷沉淀的止血功效逐渐被临床认识并在各类痰病中得到大量应用。     

Fibrinogen content of low-volume cryoprecipitate

Single-donor cryoprecipitate is the most convenient and reliable source of fibrinogen. A change by the regional Red Cross Blood Service to the production of low-volume cryoprecipitate led the authors to reexamine the fibrinogen content of cryoprecipitate units. The average fibrinogen content of individual low-volume (4 ml) units (n = 23) was 101 +/− 48 mg; in the 10-unit pools (n = 9 pools), content was 89 +/− 13 mg. Both measurements were considerably lower than previously published. By contrast, the mean fibrinogen content of regular-volume (15 ml) cryoprecipitate units (n = 8) was 142 +/− 50 mg. The fibrinogen was stable for at least 4 hours after thawing, and it survived refreezing and thawing.     

表皮葡萄球菌体外与纤维蛋白原的结合力研究

目的 :比较纤维蛋白原结合基因 (fbe)阳性和纤维蛋白原结合基因阴性的表皮葡萄球菌 (表葡菌 )体外与纤维蛋白原结合力的差异 ,探讨 fbe基因在表葡菌致异物感染中的致病作用。 方法 :采用ATB鉴定临床分离菌 ,聚合酶链反应 (PCR)方法检测有无 fbe基因 ,采用酶联免疫吸附法 (ELISA)测定并比较 fbe基因阳性和 fbe基因阴性表葡菌体外与纤维蛋白原的结合力。结果 :6 2株临床分离菌中有 4 6株为表葡菌 ,表葡菌中 fbe基因阳性的菌株为 34株 ,阳性率为 73.9% ,其他葡萄球菌均不含该基因 ;fbe基因阳性菌株和fbe基因阴性菌株体外与纤维蛋白原的结合力差异有显著性 (P <0 .0 1 )。 结论 :fbe基因阳性菌株和fbe基因阴性菌株体外与纤维蛋白原的结合力存在差异 ,提示 fbe基因可能为表葡菌致异物感染的致病因素之一。