去冷沉淀血浆与普通血浆临床应用价值的比较

目的探讨去冷沉淀血浆中部分有效成分的实际含量,为临床选择不同血浆输注提供实验室依据。方法采集23袋（200ml/袋）ACD-B血液保存液保存的血液,4h内分离制备新鲜冰冻血浆,各袋均留2份标本;1份同新鲜冰冻血浆一起置-35℃保存,于第3天速融后检测总蛋白、白蛋白、纤维蛋白原、凝血因子Ⅴ、Ⅷ、Ⅹ含量;1份置4℃保存21d后检测总蛋白、白蛋白、纤维蛋白原、凝血因子Ⅴ、Ⅷ、Ⅹ含量;新鲜冰冻血浆于第3天取出制备冷沉淀,留取去冷沉淀血浆标本1份检测总蛋白、白蛋白、纤维蛋白原、凝血因子Ⅴ、Ⅷ、Ⅹ含量。结果去冷沉淀血浆中的血浆蛋白和凝血因子均低于普通血浆（P〈0.001）。结论去冷沉淀血浆的使用价值有局限性,不能与普通血浆等同使用。     

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 vivo stability of cryoprecipitate fibrinogen

Because of its lower hepatitis risk, cryoprecipitate has been advocated as a substitute for commercial fibrinogen. Previous literature on cryofibrinogen has demonstrated a short blood t1/2, rendering it unsuitable for therapeutic use. The in vivo clearance of 131I- cryoprecipitate was compared with that of 125I-standard fibrinogen. A small amount of cryoprecipitate was rapidly cleared and apparently was cryofibrinogen. However, the bulk of the cryoprecipitate was cleared with a normal half life, as was cryoprecipitated that was in 10-bag pools. The data indicated cryoprecipitate was an effective in vivo form of fibrinogen and thus the preferred fibrinogen source because of its combining normal t1/2 with single donor procurement.     

Protein composition of clots detected in pooled cryoprecipitate units

BACKGROUND: On rare occasions, upon thawing of stored cryoprecipitate components, clots are observed on visual inspection. Although it has been assumed that the clot reflects fibrinogen to fibrin conversion, there are few published studies that document that this assumption is correct. Our studies were conducted to further identify the protein characteristics of the clotted material. STUDY DESIGN AND METHODS: Clotted material isolated from four thawed cryoprecipitate pools was examined by solubilization procedures and electrophoresis analysis. RESULTS: Solubilization of much of the clotted material in phosphate‐buffered saline warmed to 37°C suggested the presence of soluble fibrin. Gel electrophoresis under reducing conditions showed that the most prevalent bands exhibited molecular weights corresponding to the α, β, and γ subunits of fibrinogen with a much lighter band exhibiting the molecular weight of fibrinogen γ‐γ dimer, consistent with the presence of partially crosslinked fibrin. The presence of the dimer indicated that the clotted material was caused by the action of thrombin, but also reflected the action of Factor XIIIa. No ongoing clot formation was observed. CONCLUSION: Our studies indicate that, on rare occasions, fibrinogen conversion to fibrin is responsible for observable clots in thawed cryoprecipitate pools. These clots are structurally heterogeneous, including both noncrosslinked (soluble) and crosslinked (insoluble) fibrin. This diversity in the fibrin structure may account for some of the diversity in the limited literature regarding their presence in cryoprecipitate pools.     

Cryoprecipitate prepared from plasma treated with methylene blue plus light: increasing the fibrinogen concentration

When cryoprecipitate is prepared from plasma which has been treated with methylene blue plus light (MB) for the purpose of virus inactivation, clottable fibrinogen content is 40% lower compared with units prepared from untreated plasma. Initial studies showed that when frozen MB plasma units were removed to +2 to +6 degrees C for 4 h and then returned to -40 degrees C prior to cryoprecipitation, fibrinogen recoveries increased from 24 to 42%. Although fibrinogen yield improved when plasma units were stored at +2 to +6 degrees C for varying lengths of time, FVIII levels decreased with increasing time. Conditioning for 8 h was studied in more detail. Groups of two plasma units were mixed together, divided into two equal units, frozen/thawed and treated with MB. One of each pair was stored continually at -40 degrees C, whereas the other was removed to +2 to +6 degrees C for 8 h. Samples were assayed for fibrinogen, FVIII, VWF:Ristocetin cofactor activity (RCo), VWF:Ag and VWF:Collagen binding (CB). The cryoprecipitate fibrinogen content increased to a mean of 207 mg unit(-1). VWF:Ag, VWF:RCo and VWF:CB recoveries also increased. FVIII recovery decreased from 50 to 45% (mean 124 iu unit(-1)). Conditioning has been validated for routine production of cryoprecipitate from imported plasma.     

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.     

Cryoprecipitate transfusion: assessing appropriateness and dosing in trauma

Background: Originally developed for patients with congenital factor VIII deficiency, cryoprecipitate is currently largely used for acquired hypofibrinogenemia in the context of bleeding. However, scant evidence supports this indication and cryoprecipitate is commonly used outside guidelines. In trauma, the appropriate cryoprecipitate dose and its impact on plasma fibrinogen levels are unclear. Objectives: The aims were to evaluate (i) the appropriateness of cryoprecipitate transfusion in trauma and (ii) the plasma fibrinogen response to cryoprecipitate transfusion during massive transfusion in trauma. Methods: Retrospective review (January 1998–June 2008) of indications, dose and plasma fibrinogen response to cryoprecipitate transfusion at a large teaching hospital. A fibrinogen of <1·0 g L^?1 within 2 and 6 h of transfusion was used for evaluating appropriateness. Results: Ten thousand five hundred and forty cryoprecipitate units were transfused in 1004 patients. Thirty‐seven percent and 31% were used in cardiac surgery and trauma, respectively. In 394 events in trauma, 238 (60%) and 259 (66%) were considered appropriate using the 2‐ and 6‐h cut‐off criteria, respectively. In patients who did not receive plasma components 2 h prior to cryoprecipitate, a dose of 8·7 (±1·7) units caused a mean increase in fibrinogen levels of 0·55 (±0·24) g L^?1, or 0·06 g L^?1 per unit. Conclusions: In our hospital, where transfusion guidelines are overseen by transfusion medicine specialists and technologists, and policies for rapid blood component and laboratory turnaround times exist, it is possible to achieve high rates of appropriateness for cryoprecipitate transfusion in trauma. The current recommended dose causes a modest increase in fibrinogen levels (0·55 g L^?1).     

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.     

A novel, automated method of temperature cycling to produce cryoprecipitate

BACKGROUND: Cryoprecipitate continues to find wide application in transfusion practice. Current AABB standards call for a minimum of 80 units (U) of factor VIII and 150 mg of fibrinogen per bag of cryoprecipitate. However, individual cryoprecipitates can vary greatly in content, with as many as 20 different factors known to affect the yield. STUDY DESIGN AND METHODS: Plasma was processed in a new, rapid, automated device (CryoSeal, Thermogenesis) with computer-controlled temperature cycling to produce cryoprecipitate. RESULTS: In repeat runs (n = 20), the automated procedure yielded a product containing 184 mg of fibrinogen and 158 U of factor VIII in 55 minutes. Additional studies using plasma pools to compare the quality of the machine-generated products to those of traditionally prepared cryoprecipitate showed comparative recoveries of 182 and 187 mg of fibrinogen and 172.1 and 129.7 U of factor VIII and no significant difference in the levels of plasminogen, protein C, or protein S. CONCLUSION: The new system offers an automated method of cryoprecipitate production in which the steps involved in temperature cycling are initiated sequentially, producing within 1 hour a preparation that is equivalent to standard cryoprecipitate.     

Cryoprecipitate use in 25 Canadian hospitals: commonly used outside of the published guidelines

BACKGROUND: Canadian Blood Services' disposition reports suggested considerable variation in cryoprecipitate use and prompted this national audit. STUDY DESIGN AND METHODS: Thirty‐one institutions were invited to participate in a 2‐month audit. Patient information and relevant laboratory and transfusion data were collected. Cryoprecipitate transfusions were categorized as appropriate if a fibrinogen level (taken 6 hr before/after transfusion) was not more than 1.0 g per L and inappropriate if the pretransfusion fibrinogen level was more than 1.0 g per L and posttransfusion fibrinogen level was more than 1.0 g per L or not performed. Appropriateness was categorized as undetermined if the pretransfusion fibrinogen level was not performed and the posttransfusion fibrinogen level was more than 1.0 g per L or not performed. RESULTS: Overall, 25 of 31 invited hospitals agreed to participate. A total of 4370 units of cryoprecipitate were transfused in 603 events to 453 patients representing 62 percent of cryoprecipitate issued to hospitals during the time period. Comparison of the number of units of cryoprecipitate per 100 units of red blood cells (RBCs) transfused by each institution showed significant variation in practice (mean, 9 per 100 RBCs; range, 2 to 27 units). The single most common indication for cryoprecipitate was cardiac surgery (45.4% of events). Overall, 24 percent of cryoprecipitate transfusions were considered to be appropriate (pretransfusion fibrinogen level ≤1 g/L in 19% and posttransfusion fibrinogen level ≤1.0 g/L in another 5%), 34 percent were inappropriate, and in 42 percent appropriateness could not be determined. CONCLUSION: A 2‐month audit of cryoprecipitate use in Canada revealed that the majority of cryoprecipitate use in Canada is not in accordance with published guidelines.     

Paradoxical Bleeding in Intensively Transfused Hemophiliacs: Alteration of Platelet Function

Platelet function tests (bleeding time, platelet adhesiveness, platelet factor 3 release, and platelet aggregation to ADP) were done on a group of severe factor VIII deficient hemophiliacs before, during, or after transfusion therapy with cryoprecipitate or factor VIII concentrate. Mild decreased platelet function was seen in nonbleeding hemophiliacs after a single transfusion of cryoprecipitate. More severe abnormalities of platelet function were observed in a group of patients receiving factor VIII therapy for severe hemorrhages or in the postoperative period. Several of these intensively transfused patients bled when their circulating factor VIII level was normal or near normal. In one patient, who was studied repeatedly, prolongation of the thrombin time and partial thromboplastin time occurred in association with high levels of fibrinogen. Fibrin split products (fsp) were increased in most of the patients during bleeding episodes. Mixtures of cryoprecipitate or factor VIII‐concentrate with normal plasma contained increased protamine precipitable material and fibrin split products. Paradoxical bleeding can occur in intensively transfused hemophilic patients and may be related to either abnormal platelet function or increased levels of circulating fibrin monomer, fsp, or fibrinogen interfering with thrombin‐fibrinogen interaction.     

Preparation of pooled cryoprecipitate for treatment of hemophilia A in a home care program

Cryoprecipitate is used infrequently in home therapy for patients with hemophilia A since freezer storage is required and resuspension and pooling of thawed cryoprecipitate is cumbersome. We evaluated procedures for preparation of cryoprecipitate in an "open system" so that four to six bags of cryoprecipitate could be pooled after production and refrozen for home therapy. Factor VIII activity for pooled cryoprecipitate was 132 +/- 30 (mean +/- SD), 125 +/- 45, and 145 +/- 47 units per bag pooled in three separate studies. Cultures from cryoprecipitate pools and individual cryoprecipitate bags did not show contamination in the "open system" or with the water bath thawing procedure. The mean increment in factor VIII activity per unit per kg infused was 0.02 units per ml and the mean half-life was 10.5 hours in three patients with hemophilia A. Pooled cryoprecipitate was shown to be clinically efficacious and acceptable for use in home care programs for hemophilia A.     

Preparation of cryoprecipitate from riboflavin and UV light-treated plasma

Background and objectivesThe Mirasol® pathogen reduction technology system for plasma is based on a riboflavin and UV light treatment process resulting in pathogen inactivation due to irreversible, photochemically induced damage of nucleic acids. This study was undertaken to evaluate the possibility of making pathogen reduced cryoprecipitate from riboflavin and UV light- treated plasma that meets the quality requirements specified by UK and European guidelines for untreated cryoprecipitate.Materials and methodsCryoprecipitate was made from riboflavin and UV light-treated plasma. Plasma units were thawed over a 20 h period at 4 °C, and variable centrifugation settings (from 654 g for 2 min to 5316 g for 6 min) were applied to identify the optimal centrifugation condition. Plasma proteins in cryoprecipitate units were characterized on a STA Compact, Diagnostica STAGO and Siemens BCS analyzer.ResultsNeither the centrifugation speed or time appeared to have an effect on the quality of the final cryoprecipitate product; however the initial solubilization of the cryoprecipitate product was found to be easier at the lower centrifugation setting (654 g for 2 min). Cryoprecipitate units prepared from Mirasol-treated plasma demonstrated protein levels that were less than levels in untreated products, but were on average 93 IU/unit, 262 mg/unit and 250 IU/unit for FVIII, fibrinogen and von Willebrand ristocetin cofactor activity, respectively.ConclusionCryoprecipitate products prepared from Mirasol-treated plasma using a centrifugation method contain levels of fibrinogen, FVIII and von Willebrand ristocetin cofactor activity, that meet both the European and UK guidelines for untreated cryoprecipitate. Flexibility in centrifugation conditions should allow blood banks to use their established centrifugation settings to make cryoprecipitate from Mirasol-treated plasma.     

Cryoprecipitate: an outmoded treatment?

Cryoprecipitate is an allogeneic blood product prepared from human plasma. It contains factors VIII, von Willebrand factor (vWF), fibrinogen, fibronectin and factor XIII. Its use was first described in the 1960s for treatment of patients with factor VIII deficiency. It has also been used to treat patients with congenital hypofibrinogenaemia. Now, the most common use of cryoprecipitate is fibrinogen replacement in patients with acquired hypofibrinogenaemia and bleeding. Despite almost 50 years of use, evidence of efficacy is limited. This review provides an overview of the history of cryoprecipitate use, the current debates on the use of this product and future developments.     

Comparison and stability of ADAMTS13 activity in therapeutic plasma products

BACKGROUND: The von Willebrand factor (VWF)-cleaving protease, ADAMTS13, is often deficient in cases of thrombotic thrombocytopenic purpura (TTP). The primary treatment of TTP is therapeutic plasma exchange (TPE) utilizing a variety of plasma products that help restore ADAMTS13 activity. However, multiple replacement products are available to choose from. Thawed plasma products have a variable refrigerated shelf life depending on the product type; stability of ADAMTS13 in thawed products stored at 1 to 6 degrees C has not been determined. STUDY DESIGN AND METHODS: ADAMTS13 activity was measured in three types of plasma products and cryoprecipitate. Fresh-frozen plasma (FFP) aliquots and cryoprecipitate-poor plasma (CPP) products were produced from 10 whole-blood (WB) donations. Twenty-four-hour plasma products were manufactured from 10 additional WB donations. ADAMTS13 activity in these products at time of thaw and after 5 days of storage at 1 to 6 degrees C was measured with a modified version of the FRETS-VWF73 fluorogenic assay. ADAMTS13 activity at time of thaw was measured in 10 units of cryoprecipitate and five related CPP products. RESULTS: ADAMTS13 is present in similar amounts in FFP, CPP, and 24-hour plasma products. Storage at 1 to 6 degrees C for up to 5 days did not significantly diminish ADAMTS13 activity. The concentration of ADAMTS13 in cryoprecipitate was significantly higher than that observed in plasma products. CONCLUSION: FFP, CPP, and 24-hour plasma products should be equally effective for ADAMTS13 restoration through TPE and should remain so for the duration of the shelf life of the thawed products.     

Clotting factors in supernatant plasma following cryoprecipitation

The supernatant plasma which remains after cryoprecipitation is a useful component for transfusion therapy. However, from prior studies it was unclear how well plasma clotting factors were preserved following cryoprecipitation. Coagulation factor assays were performed on fresh plasma, supematant plasma after removal of cryoprecipitate, and the same supernatant plasma following refreezing and thawing. Factor VIII and fibrinogen levels fell considerably after removal of cryoprecipitate, but there was also a significant decline in factor V. Refreezing and thawing the supernatant plasma had little effect on clotting factors.     

Coagulation parameters of thawed fresh-frozen plasma during storage at different temperatures

Once thawed, fresh-frozen plasma (FFP) should be used, according to guidelines, within 24 h. In hospital practice, this may be associated with wastage. This study has been performed to investigate the coagulation levels of thawed quarantine FFP as used in the Netherlands. Five units of quarantine FFP, obtained by plasmapheresis, were thawed and by sterile docking divided into satellite bags (SB). SB 2-4 were stored at room temperature (RT) for, respectively, 1, 3 and 6 h and SB 5-9 at 4 degrees C for 6, 12 and 24 h and 1 and 2 weeks. At each time point, activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, factor V (FV), factor VIII (FVIII) and ADAMTS13 activity were measured. During storage at RT for up to 6 h, no major differences were found in the levels of FV, PT, fibrinogen and ADAMTS13 activity. FVIII activity showed a decrease of 16% and the APTT was prolonged by 6%. During storage at 4 degrees C for 2 weeks, FV and FVIII were reduced by 35 and 45%, respectively. The APTT and PT were prolonged by 17 and 15%, respectively. Fibrinogen was decreased by 8%. No change in ADAMTS13 activity was found. FFP stored at RT for 6 h or at 4 degrees C for 2 weeks can provide sufficient support for adequate haemostasis except for patients with a known deficiency for FVIII and can be used for plasmapheresis in patients with thrombotic thrombocytopenic purpura (TTP).     

A heat-treated factor VIII concentrate prepared by controlled-pore glass adsorption chromatography

Four years' experience with a method for preparing a high-purity, low-fibrinogen, heat-treated factor VIII concentrate is reported. The process, batch adsorption of a cryoprecipitate extract with controlled-pore glass granules, removes 77 percent of the cryoprecipitate fibrinogen, resulting in a final concentrate-specific activity of 0.74 IU factor VIII per mg of protein at a yield of 194 IU factor VIII per kg of starting plasma. Heat treatment of the lyophilized concentrate for 72 hours at 60 degrees C results in less than 10 percent loss of factor VIII activity. This process does not require expensive fractionation equipment, is suitable for small-to medium-scale batch concentrate production and could be adopted by moderately well-equipped regional blood processing laboratories for the decentralized production of a high-quality, heat-treated factor VIII concentrate.     

Rapid controlled thawing of fresh-frozen plasma in a modified microwave oven

A microwave oven has been specifically modified to permit rapid thawing of fresh-frozen plasma (FFP) by using a rotating disc with a temperature sensor to hold the plasma bag. This modification makes it possible to mix the FFP continuously during thawing, and automatically shuts the oven off when the plasma reaches 21 degrees C. Comparisons were made between FFP thawed in the modified microwave oven and FFP thawed conventionally in a 37 degrees C waterbath. The following tests were done: total protein, albumin, and immunoglobulin concentrations; plasma fibrinogen, factor VIII, and factor IX activities; protein electrophoresis, albumin aggregation, hemolytic complement activity, and plasma particle count and size. In no case was there a significant difference between plasma thawed in the microwave oven compared with that thawed in the waterbath. Further, microwave thawing was reliable and rapid; all units of FFP thawed in less than 6 minutes, and the thawed plasma did not vary by more than 6 degrees C from the preselected final temperature of 21 degrees C. Thus, it appears that controlled thawing of FFP in a microwave oven specifically designed for this purpose is an effective and reliable method and has many advantages over conventional thawing of FFP.     

Fibrinogen in cryoprecipitate and its relationship to factor VIII (AHF) levels

Very little has been published to indicate the quantity of fibrinogen in cryoprecipitates. We assayed 88 preparations from five blood banks for factor VIII(AHF) and fibrinogen to assess whether the AHF assay can predict the fibrinogen content. Cryoprecipitate was considered to be consistent with FDA standards with 80 units of factor VIII/bag (40% yield from 200 ml plasma). Fibrinogen was considered adequate if 200 mg were recovered (40% yield, 200 ml plasma, normal range 150–350 mg/dl). The mean AHF was 145 units/bag and fibrinogen. In 64/88 bags, the fibrinogen and AHF were concordant, but in 24/88 bags the results were discordant. Although it appears safe to conclude that a bag of cryoprecipitate will average 250 mg fibrinogen, adequate control may require separate assays for fibrinogen.