失血性休克大鼠凝血因子的变化研究

本研究观察SD大鼠失血性休克过程中凝血因子的变化 ,探讨凝血连锁反应在休克发生、发展过程中的作用机制。制作大鼠失血性休克模型后 ,4 0只大鼠随机分为休克前组、休克 1、2、4、6、8、12和 2 4小时组。分别检测各组假血管性血友病因子 (vWF) ,凝血因子Ⅷ (FⅧ ) ,组织因子 (TF) ,D 二聚体 (D D) ,纤维蛋白原 (FIB)的血浆浓度及观察凝血酶原时间 (PT)、活化部分凝血酶原时间 (APTT)变化。结果表明 :与休克前比较 ,各休克组PT、APTT逐渐延长 ,于休克后 4 - 6小时显著延长 (P <0 .0 0 1) ,APTT平均 5 9.7秒 ,PT平均 30 .2秒。休克后 ,血浆D 二聚体显著增加 (P <0 .0 0 1) ,于休克 8小时达到峰值。血浆vWF、FⅧa、TF、FIB于休克早期增加 ,随着休克的发展 ,纤维蛋白原于休克 2小时开始明显下降 (P <0 .0 0 1) ,休克 12小时降至最低值。TF、vWF、FⅧa分别于休克 6小时、8小时开始明显下降 (P <0 .0 0 1或P <0 .0 5 )。凝血因子消耗比率 :FⅧ为 (86 .1± 1.8% ) ,纤维蛋白原为 (89.6± 0 .6 % ) ,假血管性血友病因子为 (5 5± 1.4 % ) ,组织因子为 (6 2± 2 .5 % ) ,其中 ,FⅧ、纤维蛋白原消耗比例较大。失血性休克时以外源性凝血途径激活为主 ,内源性凝血途径作用较小。纤维蛋白原和D 二聚体、PT、APTT可用     

Effects of replacement fluids on coagulation system used for therapeutic plasma exchange.

Therapeutic apheresis is a widely used treatment alternative for several diseases. In 29 patients with different diseases, we have monitored the PT, aPTT, thrombin time (TT), fibrinogen, D-dimer, factor VIII, IX, X, XI, XII, VWF, Protein C, S, Active Protein C Resistance (APCR) and Antithrombin-III during TPE. Patients were divided into four groups based on the replacement fluids used: 3% VARIHES or ISOHES + 4% albumin (1:1) (group 1), fresh frozen plasma (FFP) (group 2), 3% VARIHES or ISOHES (group 3) and 4% albumin (group 4). In our study, the fibrinogen level decreased to 83% of the base line level after the end of 48 h therapy. The APTT, PT, and TT increased during TPE. However no statistical difference was observed between the groups. We found a significant change in factor levels with time, only the difference in factors IX and XI between the groups was significant. In addition, factor levels measured at 48 h were close to the levels measured before aphereses. In our study, time the related change in AT-3 values was significant. Time-related changes of the Protein S and APCR were not statistical significant significant but on the other hand, we found a significant difference in AT-III and Protein C values between groups. The side effects of HES on coagulation factors and tests were comparable to those of other replacement fluids. Its low cost makes it favourable.     

Non-frozen transports of whole blood samples do not cause relevant bias for global coagulation tests in clinical trials evaluating the drug safety

Sample shipments with dry ice have a large economic impact on clinical research. Therefore, the bias caused for global coagulation tests by non-frozen transports of whole blood instead of frozen plasma was investigated experimentally and by a meta-analysis of 6-year central laboratory data. In the experiment, aliquots from 14 healthy volunteers were kept as whole blood at 20+/-2 degrees C and as frozen plasma until an analysis of prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and antithrombin III (ATIII) at day 0, 1, 2, and 3 from collection. Within these 3 days only PT and aPTT demonstrated any changes: in blood samples kept at 20+/-2 degrees C these amounted about 10% for both. In frozen plasma, aPTT did not change whereas PT increased by 14%. In a meta-analysis of central laboratory data, PT and aPTT results were grouped across various phase II-IV trials by the type of sample transfer, either as frozen plasma on dry ice or non-frozen as whole blood. For the latter the mean difference to a reference group of phase I trials with same-day analysis was in line with the amount of bias found in the experiment (aPTT, 34.6+/-6.0 vs. 31.6+/-3.5 s; PT, 87.7+/-13.3 vs. 97.3+/-7.9%). The consistent bias resulted in shifted, but still normal distribution curves with a total rate of clinically relevant outliers of about 1.9% for aPTT and 2.4% for PT. Biases thus appear irrelevant for a common safety evaluation within clinical trials. Non-frozen whole blood transports for the measurement of global coagulation tests appear justified for this purpose, if protocols do not require frozen shipments for other reasons. However, transit time must not exceed 2 days and pre-analytical conditions should be consistent within the same trial.     

Potentiation of the function of Hageman factor fragments by high molecular weight kininogen.

Patients lacking high molecular weight (HMW) kininogen have profound abnormalities of the Hageman factor-dependent pathways of coagulation, kinin formation, and fibrinolysis. The ability of HMW kininogen to potentiate the Hageman factor fragments (HFf) activation of prekallikrein and Factor XI in plasma was studied. HFf only partially converted Factor XI to XIa and prekallikrein to kallikrein in plasma deficient in HMW kininogen (Williams trait), while enhanced activation of Factor XI and prekallikrein by HFf resulted after reconstitution with HMW kininogen. In a system using highly purified components, HMW kininogen increased the initial rate of prekallikrein activation whether the kallikrein formed was assayed by arginine esterase activity or kininforming ability. The potentiation of prekallikrein activation occurred over a 12-fold range of enzyme (HFf) concentration and was nonhyperbolic with respect to substrate (prekallikrein). HMW kininogen exerted its effect even in the absence of prekallikrein since the hydrolysis of acetylglycyl-lysine methyl ester by HFf was increased by HMW kininogen. These results suggest that one of the functions of HMW kininogen is to augment the catalytic action of HFf.     

Refreezing previously thawed fresh-frozen plasma. Stability of coagulation factors V and VIII:C

With the growth in autologous blood programs and the increased scrutiny of the indications for transfusion of fresh-frozen plasma (FFP), an increase has been seen in the number of occasions on which FFP was requested and thawed but then not transfused. The coagulation properties of FFP units that were refrozen and then rethawed were therefore studied. Fifty-eight units of plasma were studied, with each experimental unit of FFP paired with an identical control unit. Experimental units were frozen, stored at -65 degrees C, thawed, stored at 1 to 6 degrees C for various periods of time up to 24 hours, and then refrozen, stored at -65 degrees C, rethawed, and stored again in the refrigerator for up to 24 hours. Control units were frozen once at the time the experimental units were first frozen and thawed once at the time of the second thaw of the experimental units. Aliquots of plasma were sampled periodically and were later batch-tested for prothrombin time (PT), activated partial thromboplastin time (aPTT), and factor V and VIII:C activity. The results of coagulation testing of the twice-frozen plasmas were always within the normal range. There was a slight but statistically valid prolongation of the PT and aPTT and a decrease in the factor V and VIII:C levels for twice-frozen plasma compared with control plasma. The greatest decline occurred in the level of factor VIII:C. The measured deterioration in coagulation of twice-frozen FFP is unlikely to be of clinical importance. Refreezing FFP may eventually prove useful for rare donor, autologous, and massive transfusion programs.     

Coagulation parameters of CPD fresh-frozen plasma and CPD cryoprecipitate-poor plasma after storage at 4 degrees C for 28 days

A pilot study was performed on the storage of plasma and cryosupernatant plasma at 4 degrees C for up to 28 days. Eight bags, four of CPD fresh-frozen plasma (FFP) and four of CPD cryosupernatant plasma (CSP, plasma without cryoprecipitate), were sampled during storage for assays of pH; factors V, VIII, IX, and XI; fibrinogen; prothrombin time; activated partial thromboplastin time (APTT); plasma protein electrophoresis; viscosity; and C1q binding. No changes were found in viscosity or the plasma protein electrophoretic pattern, and there was no detectable immune complex formation. The fibrinogen concentration remained constant, and the prothrombin time showed a gradual increase of 2.5 seconds for both groups of plasma. The labile coagulation factor V decreased gradually for FFP and CSP to 58 and 64 percent of its initial value, respectively (51 +/− 8% and 54 +/− 6% of the value of fresh pooled plasma). Factor VIII decreased to 36 percent of its initial value in FFP (48 +/− 14% of fresh pooled plasma). In CSP, factor VIII decreased after 28 days to 7 percent of its initial value (7 +/− 1% of fresh pooled plasma). The APTT increased for FFP from 28 to 35.8 +/− 1.1 seconds and for CSP from 36 to 49.5 +/− 4.9 seconds. The only chemical change observed for both plasmas was a rise in pH, from 7.27 to 7.56, after 28 days. The results of this pilot study indicate that FFP can be stored at 4 degrees C for 28 days with sufficient recovery of coagulation factors to maintain hemostasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gamma irradiation with 30 Gy on the coagulation system in leukoreduced fresh-frozen plasma

BACKGROUND: The aim of this study was to investigate the effect of gamma irradiation with 30 Gy on the coagulation system in leukoreduced fresh-frozen plasma (FFP). STUDY DESIGN AND METHODS: In 74 FFP units that had been stored for 352 +/- 103 days below -30 degrees C, the following variables were determined in parallel in an irradiated and not irradiated half: prothrombin time (PT); activated partial thromboplastin time (APTT); thrombin time; antithrombin III; protein C; protein S; von Willebrand factor antigen; ristocetin cofactor; plasminogen-alpha(2)-antiplasmin; the coagulation factors fibrinogen, factor (F)II, FV, FVII, VIII, F IX, FX, FXI, FXII, FXIII, and activated factor XII (FXIIa); D-dimer; fibrin monomer; thrombin-antithrombin complex; prothrombin fragment 1 + 2 (F1+2); plasmin-alpha(2)-antiplasmin complexes (PAPs); and platelet factor 4. The FVII activity ratio was assayed to quantify activation of FVII. RESULTS: Irradiation with 30 Gy resulted in a reduction of APTT (35.0 +/- 4.1 sec vs. 34.4 +/- 4.1 sec; p = 0.00000006) and PT (89.8 +/- 8.2% vs. 90.7 +/- 8.0%; p = 0.002) and a significant increase of the activities of the coagulation factors FII, FV, FVII, F IX, FX, and FXII. FVIII activity decreased from 118 +/- 31 to 116 +/- 32 percent (p = 0.02). Activation of the coagulation system was shown by an increase in the FVII activity ratio (1.19 +/- 0.29 vs. 1.31 +/- 0.34; p = 0.0000001), FXIIa (0.81 +/- 0.50 ng/mL vs. 0.90 +/- 0.51 ng/mL; p = 0.006), and F1+2 (1.19 +/- 0.20 nmol/L vs. 1.24 +/- 0.20 nmol/L; p = 0.000005) after irradiation with 30 Gy, whereas an increase of PAP (16.2 +/- 11.5 ng/mL vs. 20.2 +/- 12.0 ng/mL; p = 0.0004) demonstrated activation of the fibrinolytic system. No negative influence of irradiation with 30 Gy on inhibitors of coagulation was observed. CONCLUSION: Gamma irradiation of leukoreduced FFPs with 30 Gy results in a significant but very weak activation of the coagulation and fibrinolytic system in FFPs.     

Radioimmunoassay of human high molecular weight kininogen in normal and deficient plasmas

An RIA for human HMW kininogen, capable of detecting 150 pg of antigen, has been developed. Antibody to HMW kininogen was purified by immunoaffinity chromatography, and double-antibody precipitation was used to separate free and bound antigen. Of the LMW kininogens only one of the forms tested (B3.2) showed significant cross-reaction (2%). Bradykinin and human plasma kallikrein both showed no cross-reaction, and monkey HMW kininogen showed identity to the human antigen. Intraassay and interassay coefficients of variation were 2% and 1.5%, respectively. Recovery of HMW kininogen added to 6 plasmas was 97.7% +/- 1.8%. Assay of 17 normal plasmas gave a level of 90.8 +/- 2.5 micrograms/ml HMW kininogen (mean +/- S.E.M.). A bioassay of the samples, based on specific release of kinin by purified plasma kallikrein, yielded a level of 90.2 +/- 2.8 micrograms/ml HMW kininogen (r = 0.83, p less than 0.001). In neither assay was any significant sex difference observed. No evidence of any antigenic fragments was seen upon gel filtration of normal plasmas. RIA measurements were also performed on seven plasmas reportedly deficient in HMW kininogen. Williams, Dayton, San Francisco, and Flaujeac plasmas all showed no significant cross-reaction, whereas Fitzgerald, Reid and Detroit plasmas showed 1.0%, 2.5%, and 3.5% of normal antigenic levels, respectively. This sensitive, convenient method should facilitate studies on the role of the kallikrein-kinin system in health and disease.     

Stability of prothrombin time and activated partial thromboplastin time tests under different storage conditions

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are common laboratory tests that are useful in the diagnosis of coagulation disorders and monitoring anticoagulant therapy. Recent expansions in the outreach laboratory services at our institution prompted us to investigate the shipping limitations for some tests, including PT and aPTT. Although we followed NCCLS guidelines for the collection of blood specimens, we observed falsely elevated PT and aPTT values due to the different storage conditions. The objective of this study is to determine the effect of conditions and duration of storage on PT and aPTT tests using plasma and whole blood samples, respectively. For this study, 36 plasma samples with normal and prolonged PT and aPTT were exposed to different storage conditions. Blood was centrifuged immediately and plasma was stored at room temperature (RT), refrigerated at 4°C, or frozen at −20°C. The samples were analyzed at 0 h and repeated at 6, 12 and 24 h under various conditions. Although statistically significant differences were observed for plasma samples for normal PT tests after 12 h at refrigerated and frozen storage conditions, the differences would not change the clinical interpretation of the results. On the other hand, samples stored refrigerated or at RT showed significant differences for aPTT at 24 h. These differences would change clinical interpretation, especially for samples with normal or near normal aPTT times. Interestingly, aPTT was significantly higher for samples stored frozen when compared to refrigerated and RT conditions at 6 h. Similar patterns were also observed on ten whole blood samples with normal PT and aPTT values. In conclusion, either plasma or whole blood samples can be accepted for PT testing up to 24 h and for aPTT testing up to 12 h only, when transported either at RT or at 4°C.     

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).     

Characterization of human high molecular weight kininogen. Procoagulant activity associated with the light chain of kinin-free high molecular weight kininogen

Human high molecular weight (HMW) kininogen has been isolated and was found to be a single chain protein of approximately equal to 120,000 daltons. Upon digestion with plasma kallikrein bradykinin is generated, and SDS gel electrophoresis of the kinin-free protein reveals an apparent loss in size of 15,000 daltons. The kinin-free kininogen retains full activity as a coagulation factor and consists of two chains: a heavy chain of approximately equal to 66,000 daltons disulfide-linked to a light chain of 37,000 daltons. The heavy chain of HMW kininogen shares antigenic determinants with LMW kininogen and possesses no detectable coagulant activity. The isolated light chain is shown to be responsible for the coagulant activity of HMW kininogen and contains a unique antigenic determinant that distinguishes HMW kininogen from low molecular weight kininogen.     

Protein C concentrate to restore physiological values in adult septic patients

OBJECTIVE: To describe the efficacy and safety of protein C (PC) concentrate to restore physiological values in adult septic patients having clinical contraindications to activated PC. DESIGN: Case series (pilot study). SETTING: Three adult ICUs of a University Hospital. PATIENTS AND PARTICIPANTS: Twenty adult patients affected by severe sepsis or septic shock with plasma values of PC < 50%. INTERVENTIONS: Patients were treated with PC concentrate (Ceprotin ((R))--Baxter) with a starting bolus followed by a continuous infusion for 72 h [3 IU/(kg h)]. MEASUREMENTS AND RESULTS: PC activity, WBC, platelets, D: -Dimer, fibrinogen, PT, aPTT, AT III, lactate, Sepsis-related Organ Failure Assessment (SOFA), Disseminated Intravascular Coagulation (DIC) score, adverse events, and mortality were measured. Baseline plasma PC activity was 34.5 +/- 9.1%. PC concentrate normalized the PC activity in all patients within 48 h, and then remained stable for the following days. At baseline, several patients showed abnormal PT, aPTT, platelets values, and lactate levels. During the study period, there was a significant increase of platelets, fibrinogen, PT, AT III, and a significant decrease of D: -Dimer, aPTT, DIC score, and lactate. No adverse reactions (hemorrhage or thrombosis) were observed. Mortality at 28 days was 35%. CONCLUSIONS: Our pilot study shows that the administration of PC concentrate to patients having contraindications to the treatment with activated PC was safe and possibly useful to control the coagulopathy triggered and sustained by sepsis. A randomized, double blind study in patients with severe sepsis and contraindications to activated PC administration would be advisable to state the safety and the possible role of this product in the treatment of severe sepsis.     

Coagulation defects in trauma patients: etiology, recognition, and therapy

Trauma patients have many reasons to have defects in coagulation. These can be caused by the trauma or because of pre-existing disorders. Trauma patients who are at risk for coagulation defects should be screened with the basic tests (aPTT, INR/PT, platelet counts, hematocrit, and fibrinogen), with therapy based on the results. Attention also should be paid to any other correctable factors such as hypothermia. Finally, pre-existing disorders can influence the patient's hemostasis greatly and may require specific therapies.     

Effects of telavancin on coagulation test results

Background: The lipoglycopeptide antibiotic, telavancin, may interfere with some laboratory coagulation tests including prothrombin time (PT) and activated partial thromboplastin time (aPTT). Objective: To evaluate the effects of telavancin on PT and aPTT assays in common use. Methods: Pooled normal human plasma was spiked with telavancin 10, 20, 100 or 200 μg/ml (equivalent to trough, 2 × trough, peak and 2 × peak clinical plasma concentrations, respectively) or diluent control (0.9% sodium chloride). Samples were analysed using 16 PT reagents and seven aPTT reagents. Results: Telavancin 200 μg/ml (corresponding to 2 × peak clinical plasma concentration), produced significant PT prolongation (> 9% difference vs. diluent control) with all the 16 PT reagents (range 12% to > 600%). At lower telavancin concentrations, PT prolongation was dose‐dependent and varied among reagents, but appeared greatest with preparations containing recombinant tissue factor. With telavancin 10 μg/ml (equivalent to trough), PT prolongation was 10% with HemosIL^® PT‐Fibrinogen Recombinant, while ranging from 5% to –1% with all other reagents. Significant (> 34% difference vs. baseline) and dose‐dependent aPTT prolongation was observed with all the seven reagents in samples spiked with telavancin 100 or 200 μg/ml (range 65–142% at 200 μg/ml). aPTT reagents containing a silica activator appeared to be more sensitive to telavancin interference. Telavancin 10 μg/ml was not associated with increased aPTT with any of the reagents tested. Conclusions: Telavancin has the potential to prolong both PT and aPTT in vitro. It is recommended that samples for PT or aPTT be obtained just prior to a telavancin dose (trough).     

普通冰冻血浆与新鲜冰冻血浆部分血浆组分的比较

目的:比较普通冰冻血浆(FP)和新鲜冰冻血浆(FFP)中血浆组分的差异。方法:随机选择北京市红十字血液中心提供的FP和FFP各20份,血浆融化后即刻分别检测凝血因子、纤溶系统及抗凝蛋白指标等12种血浆组分,即活化的部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血因子Ⅷ(FⅧ)活性、凝血因子Ⅴ(FⅤ)活性、纤维蛋白原(FIB)水平、血管性血友病因子裂解蛋白酶(ADAMTS-13)活性、血管性血友病因子(v WF)活性、D-二聚体(D-dimer,DD)、纤维蛋白降解产物(fibrin degradation products,FDP)、抗凝血酶(antithrombin,AT)、蛋白C(protein C,PC)、蛋白S(protein S,PS),并进行比较分析。结果:与FFP相比,FP中APTT明显延长(t=3.428,P<0.01),PT延长(z=-2.140,P<0.05),FⅧ活性明显降低(t=-3.372,P<0.01),但均在参考区间内;PS活性降低(t=-2.458,P<0.05);两种血浆中其余组分的差异无统计学意义(P>0.05)。结论:与FFP相比,FP缺乏某些凝血因子和抗凝蛋白,但可替代FFP应用于部分疾病的治疗。     

Interferences in coagulation tests--evaluation of the 570-nm method on the Dade Behring BCS analyser.

The Dade Behring BCS is a coagulation analyser with optical reaction detection (standard 405 nm). The present study was conducted to evaluate measurement at 570 nm for analyses in interfering plasma samples. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer in normal (n=50), lipaemic (n=60), icteric (n=113), and haemolytic (n=58) samples were measured at 405 and 570 nm. As they are unaffected by the optical properties of the sample, the mechanical STAcompact analyser (Roche Diagnostics) and an ELISA technique were defined as the "comparison" methods. The percentage of valid PT results using the 570-nm method varied from 54% (lipaemic samples) to 97% (haemolytic samples). Valid aPTT measurements were found in 67% (lipaemic samples) up to 93% (icteric samples) of samples. Fibrinogen measurement revealed valid results in 58% (lipaemic samples) to 100% (haemolytic samples) of samples. The number of valid D-dimer results varied from 28% (lipaemic material) up to 100% (haemolytic material). Significant inter-method differences were found: aPTT in lipaemic (BCS 405 vs. 570 nm) and icteric samples (STAcompact vs. BCS 405 and 570 nm); fibrinogen in lipaemic (BCS 405 vs. 570 nm), icteric (BCS 405 vs. 570 nm; STAcompact vs. BCS 570 nm) and haemolytic samples (STAcompact vs. BCS 405 and 570 nm). Differences between the BCS 570-nm and the STAcompact methods were in most cases low and less pronounced than between the BCS 570- and 405-nm methods, making the BCS 570-nm method an alternative to measurement at 405 nm. Limitations have to be taken into account regarding lipaemic plasma.     

Filtration of methylene blue-photooxidized plasma: influence on coagulation and cellular contamination

BACKGROUND: Virus inactivation of plasma can be achieved by photodynamic methods in the presence of phenothiazine dyes such as methylene blue (MB). Subsequent filtration may increase the efficacy of virus inactivation and reduce adverse effects of WBC contamination and MB. STUDY DESIGN AND METHODS: This study examined the effect of filtration with three different filters (MBF1, MBF2, and MBF3) on MB concentration, residual cells, coagulation factors, and activation measures of coagulation, fibrinolysis, and complement in MB-treated (1 microM/L) plasma units. RESULTS: Filtration reduced the concentration of MB by > or = 89 percent. WBCs were depleted by 92 percent (MBF1) and >99.9 percent (MBF2 and MBF3). Treatment with MB significantly decreased the coagulation potency from levels in untreated plasma, as measured by thromboplastin time ratio (112 +/- 18% vs. 95 +/- 11%), activated partial thromboplastin time (40 +/- 3 sec vs. 44 +/- 3 sec), thrombin time (16.9 +/- 1.1 sec vs. 18.6 +/- 1.5 sec), factor VIII (1.09 +/- 0.21 U/mL vs. 0.85 +/- 0.13 U/mL), and vWF (0.94 +/- 0.65 U/mL vs. 0.65 +/- 0.24 U/mL). Filtration did not further decrease these values, while factor XI (0.75 +/- 0.22 U/mL vs. 0.37 +/- 0.20 U/mL) and prekallikrein values decreased in MB plasma units filtered with the MBF3. In addition, activated factor XII (0.7 +/- 0.5 microg/L vs. 4.5 +/- 1.0 microg/L) increased. CONCLUSION: WBCs and MB can be eliminated from MB-treated plasma units by filtration. Differences in biocompatibility of the different filters, especially the influence on the contact phase of coagulation, must be taken into consideration.     

Plasma prekallikrein: quantitative determination by direct activation with Hageman factor fragment (beta-XIIa)

This report describes a plasma prekallikrein assay which, unlike methods that employ contact activation, is not affected by the factor XII or HMW kininogen content of the plasma analyzed. In this assay beta-XIIa, a potent fluid-phase activator of prekallikrein, is added to diluted plasma in the presence of 20% acetone (to inactivate kallikrein inhibitors) at 30 degrees C and the kallikrein generated is measured with the chromogenic substrate S-2302. Prekallikrein is fully activated under these conditions and the activity remains stable for at least 30 hr. The mean prekallikrein concentration in plasma samples from 24 healthy individuals was 1.50 +/- 0.35 (S.D.) S-2302 U/ml, corresponding to 20.3 +/- 4.7 micrograms/ml prekallikrein (the specific activity of highly purified human prekallikrein was determined to be 74 S-2302 U/mg). In contrast, the mean concentration in five plasma samples from patients deficient in HMW kininogen was 0.38 +/- 0.02 S-2302 U/ml. No activity was generated in prekallikrein-deficient plasma, and essentially normal levels (1.35 +/- 0.18 S-2302 U/ml) were measured in plasmas from three patients with factor XII deficiency. Plasma prekallikrein was also quantitated by radial immunodiffusion, which gave results similar to those obtained by functional assay with beta-XIIa. The determination of plasma prekallikrein by direct activation with beta-XIIa in the presence of acetone offers several advantages over the use of contact activators such as dextran sulfate. These advantages include complete inactivation of kallikrein inhibitors and total activation of prekallikrein (even in plasmas deficient in other contact factors) without simultaneous generation of plasmin.     

Fibrinolysis, thrombocytopenia, and coagulation abnormalities complicating high-dose interleukin-2 immunotherapy

High-dose interleukin-2 (IL-2) immunotherapy can cause hypotension, respiratory distress, interstitial edema, and thrombocytopenia, similar to endotoxic shock. We have observed that IL-2 has no direct effect on coagulation factors in vitro, but it has been observed to alter the coagulant properties of vascular endothelium. Accordingly, we investigated the possibility that IL-2 infusions initiate plasma fibrinolysis and disseminated intravascular coagulation (DIC). We studied the clinical course, platelet count, and coagulation profile in response to IL-2 infusion in seven patients, two with metastatic melanoma and five with metastatic renal cell carcinoma. Every patient experienced hemodynamic instability and thrombocytopenia, and one patient suffered an unusual complication, mesenteric thrombosis. No patient had appreciable changes in the prothrombin time or the partial thromboplastin time, nor did factors V or VIII decline in the two patients observed. In four patients examined, we found decreased titers of Hageman factor (factor XII), high molecular weight kininogen, prekallikrein, and plasma thromboplastin antecedent, as if these had been consumed by reactions of the intrinsic pathway of thrombin formation. Circulating D-dimer fragments were found in the plasma of every patient at some point during each infusion cycle, and we observed decreased titers of plasminogen in the four patients just mentioned, suggesting that IL-2 infusions initiated fibrinolysis. Taken together, the clotting factor derangements and related toxicity phenomena cannot be ascribed firmly to DIC. Activation of the intrinsic (contact) system of coagulation, however, may provide one link between the vascular endothelial surface alterations caused by IL-2 infusions and the development of the systemic toxicity that resembles septic shock.     

Thawing of fresh-frozen plasma with a new microwave oven

In the Federal Republic of Germany fresh-frozen plasma (FFP) is still the most important therapeutic agent for acquired coagulation disorders. However, thawing by waterbath (WB) requires about 30 minutes, which is too slow in emergency situations and carries the risk of bacterial contamination of the FFP. There are conflicting data about the use of microwaves for thawing. Therefore, we examined a new microwave oven (MWO; 2450 +/- 50 MHz), which was developed with our cooperation and allows thawing of FFP in 5 minutes, heating FFP to a surface temperature of 21.5 degrees C. A shaking WB (30 min, 37 degrees C) was also used in parallel for comparison. We measured activated partial thromboplastin time (aPTT), nonactivated PTT (NaPTT), fibrinogen, factors VIII:C, X, and XI, fibrinopeptide A, beta-thromboglobulin (beta-TG), thrombin-AT III-complexes, factor VIII-related antigen, C3c, C4, and the plasticizer di(2-ethylhexyl)phthalate (DEHP) in 84 units of FFP as paired samples from 42 double aphereses. Immediately after thawing there was no significant difference in the coagulation test results of FFP with low-cell contamination, regardless of the thawing procedure. Two hours later, after storage at room temperature, FFP thawed by MWO showed even less change than that thawed by WB (NaPTT, p less than 0.01; FX, p less than 0.01). The differences became more evident in comparison with FFP with higher cell contamination and could be observed immediately after thawing (FVIII:C p less than 0.001; FXI, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)