P. Hellstern

Manufacture and in vitro characterization of a solvent/detergent-treated human plasma

We have developed a modified solvent/detergent (S/D) treatment to inactivate viruses in human plasma using 1% w/w final concentrations of tri(n‐butyl) phosphate (TNBP) and Triton X‐100 and an incubation period of 4 h at 30°C. The procedure inactivates ≥106 chimpanzee‐infectious doses (CID50) of HBV, ≥105 CID50 of HCV, and ≥106.2 tissue culture infectious doses (TCID50) of HIV. After virus inactivation, eleven plasma batches were lyophilized and 12 batches were deep‐frozen until further use. The batches were characterized by extensive laboratory tests including measurement of clotting factors I–XIII, von Willebrand factor, plasminogen, inhibitors of blood coagulation and fibrinolysis, and other clinically important plasma proteins. All parameters were determined before and after S/D treatment. Twelve conventional single donor plasma units served as control. There were no marked losses of activities of clotting factors, antithrombin III, protein C, plasminogen, and C1‐esterase inhibitor due to treatment. After the S/D step, the levels of these parameters were within the normal range in all batches. The same holds true for total protein, immunoglobulins, albumin, complement factors C3 and C4, haptoglobin, hemopexin, caeruloplasmin, α1‐antitrypsin, and pH. Protein S and α2‐antiplasmin activites decreased by about 50% and were frequently found to be slightly below the lower limit of the respective normal range after treatment. The interindividual variations of all proteins analysed were significantly lower than in the single donor plasma units. The S/D procedure did not lead to increases of markers indicating activation of hemostasis. We conclude that lipid‐enveloped viruses can be inactivated by the S/D procedure described in this study without critical reduction of recoveries of plasma proteins.

Gray platelet syndrome: selective α-granule deficiency and thrombocytopenia due to increased platelet turnover

SummaryClinical and laboratory studies of two siblings, both suffering from gray platelet syndrome (GPS) are described. The patients had a mild bleeding disorder, their platelets were blue-gray in panoptic stains, and α-granules were markedly reduced, as shown by electron microscopy. The platelet content of platelet factor 4 and that of β-thromboglobulin were significantly reduced (3%–7% of normal). Platelet count was decreased (33–150×109/l) and small platelets were increased in platelet volume distribution. Bleeding time was prolonged on most occasions. Bone marrow aspiration was performed in one patient and revealed increased reticulin fibers, however, megakaryocyte count was normal. The mean platelet survival was 4.8 days using 111indium-labelled platelets. In this patient, platelet-associated IgG was within the normal range. Prednisone therapy failed to increase platelet count. Dental surgery was performed under cover of desmopressin and no bleeding complication occurred; however, no improvement of bleeding time was observed. The patient delivered a healthy male infant without hemorrhaging while under concurrent platelet transfusion therapy.

Factor VII and Activated‐ Factor‐VII Content of Prothrombin Complex Concentrates

Background and objectives: The aim of this study was to determine the potencies of factor VII (FVII) and of activated FVII (FVIIa) in prothrombin complex concentrates (PCC). Materials and methods: We examined 56 lots of PCC from 5 manufacturers. Three brands were licensed preparations, and 1 product series had been involved in thromboembolic complications. FVII and FVIIa were measured using a two‐stage amidolytic assay and a specific clotting assay, respectively. We also quantified FVII clotting activity by a one‐stage assay reflecting a mixture of FVII zymogen and FVIIa. Results: All PCC contained substantial amounts of FVII, and FVIIa could be detected in all lots. There were marked differences between manufacturers and some significant variabilities between batches. The two lots involved in thromboembolic events contained considerably more FVIIa than the PCC still licensed. The lowest FVIIa potencies were observed in an experimental product series, indicating that PCC can be produced without activation of FVII during the manufacturing process. Conclusion: FVIIa is present in all PCC containing FVII. High FVIIa potencies may contribute to the thrombogenic potential of these preparations, and determination of FVIIa potencies should be included in the in vitro characterization of PCC.

Arterial and Venous Thrombosis and Normal Response to Streptokinase Treatment in a Young Patient with Severe Hageman Factor Deficiency

The case history of a patient with severe factor XII deficiency (factor XII activity and concentration below 1%) is described. The case reported gives further evidence that factor XII deficiency leads to a prolonged euglobulin lysis time. This might be a risk factor indicating a proneness to thromboembolic events. The young man suffered from arterial and venous thrombosis, and underwent lower extremity amputation without bleeding complications, as well as thrombolytic therapy to save the other leg. There was a normal response to streptokinase, heparin, and phenprocoumon treatment.

The use of heat-treated factor VIII-concentrates in von willebrand's disease

SummaryIn vitro investigations have demonstrated a high F VIII:Rcof potency and a high F VIII:Rcof/F VIII R:Ag ratio of two heat-treated F VIII concentrates. We therefore studied the in vivo effectiveness of these preparations (F VIII HSR, Behringwerke Marburg and F VIII HTR, Travenol) in five patients with von Willebrands disease (vWd). In the steady state in vivo recoveries of F VIII:Rcof ranged from 73–153% after transfusion of F VIII HSR and from 11.5–17% after F VIII HTR respectively. The gain of F VIII-complex after F VIII HS was comparable to cryopecipitate (KryobulinR SP, Immuno AG Wien). All three products shortened the bleeding-time. Three of our five patients underwent surgery (Billroth I, papillotomy, laparatomy, open heart surgery) under F VIII HS cover without bleeding complications. The dose applied ranged from 20 to 40 U/kg at 8 or 12 h intervals for a period of approx. 14 days. Serum-transaminase elevations were observed in two of four patients after F VIII HT treatment. Although the risk of hepatitis of heat-treated F VIII concentrates remains to be determined, these products proved to be effective in vWd. The major advantages of these preparations are stability, rapid solubility, a low content of contaminating proteins, and a rapid, general availability.

The subcutaneous administration of the vasopressin analogue 1-desamino-8-D-arginine vasopressin in patients with von Willebrand's disease and hemophilia

SummaryTwenty-one patients suffering from mild von Willebrands disease (vWd) and patients suffering from mild or moderate hemophilia A received 1-desamino-8-D-arginine vasopressin (DDAVP) (Minirin®, Ferring AG) s.c. at a dose of 0.4 µg/kg body weight. Additionally, two hemophiliacs and 22 patients with vWd received DDAVP i.v. Within the observation period of 3 h Factor (F) VIII:C levels increased 2.4 × baseline levels in hemophiliacs, and the maximal effect was observed 3 h post DDAVP s.c. In patients with vWd post DDAVP s.c. (i.v.) a 2.7 (3.4), 2.1 (1.9) and 2.2 (2.8) fold increase for F VIII: C, F VIIIR:Ag and F VIII:Rcof was observed. In eight patients suffering from vWd with additional F XII deficiency a small and transitory but significant increase of F XII levels was detected post DDAVP s.c. No local or systemic side effects were observed. In five patients with vWd tooth extractions were performed without bleeding complications under DDAVP s.c. treatment. Two patients practiced self-treatment by injecting the drug s.c. at home. We thus conclude that s.c. DDAVP is an effective, reliable, and cost-reducing form of treatment that does not bring with it the risk of transmitting infectious diseases in patients with vWd and hemophilia and that can be administered at home.

Factor VII Half‐life after Transfusion of a Steam‐treated Prothrombin Complex Concentrate in a Patient with Homozygous Factor VII Deficiency

Factor VII (FVII) deficiency is a rare bleeding disorder which can be treated effectively with prothrombin complex concentrate (PCC) [ 1, 21. However, these preparations may transmit viral diseases or induce accelerated intravascular coagulation. PCCs are frequently used for the treatment of acquired defects in hemostasis, such as result from excessive loss of blood, liver disease or coumarin treatment. Transfusions of PCCs in cases of inherited deficiencies of coagulation factors are well suited to assess the efficacy of these preparations. We report on a male patient with moderate, crossreacting material (CRM)-positive FVII deficiency who underwent extraction of 12 teeth with prophylactic transfusion of a steam-treated PCC. He was 32 years of age with no history of bleeding and had a FVI1:C (as determined with a one-stage assay using reagents and standards from Immuno, Vienna) of 0.03 U/ml and a FVI1:Ag of 26% (using antibodies from Behring, Marburg). Reduced FVII levels could also be detected in his mother (0.56 U/ml) and his son (0.34 U/ml). One day prior to surgery the patient received 2,000 U of a steam-treated PCC (Prothromblex TIM4, Immuno, Vienna, containing 2,220 U FVII, as determined using the above-mentioned one-stage assay) to assess the in vivo recovery and half-life as described recently [3]. After transfusion of 2,OOOU PCC (28.6U FVII:C/kg body weight), FVII reached a peak level of 0.88 U/ml. The decay of FVII activity is shown in figure 1. Using a monoexponential model, a half-disappearance time of 6.6 h was estimated. Using a biexponential model, the distribution and elimination half-lives were 3.8 and 15.4 h, respectively (fig. 1). The calculated distribution volume was 2,571 ml, the in vivo recovery was 133%. After infusion of PCC, no signs of Fig. 1. Factor VII activity (triangles) after transfusion of 2,000 U PCC. The broken line represents the calculated curve of decay of FVII activity (y=0.708* exp (-0.003*t) + 0.155* exp(-O.O0075*t) + 0.03).

High-dose systemic streptokinase and acylated streptokinase-plasminogen complex (BRL 26921) in acute myocardial infarction: alterations of the fibrinolytic system and clearance of fibrinolytic activity

We report the results of two consecutive studies using intravenous bolus injections of streptokinase (SK) or acylated plasminogen-SK complex (BRL 26921) in patients with acute myocardial infarction (AMI). In the first study, 20 patients received either 750,000 units (U) SK (group IA, n = 10) or 1,500,000 U SK (group IB, n = 10) within 5-10 min intravenously. In the second study 10 consecutive patients received 750,000 U SK within 15 min (group IIA) intravenously. The following 10 consecutive patients received 30 mg BRL 26921 within 2 min (group IIB). Early reperfusion was found in 16 patients in the first study (8 in each group) and in 18 patients in the second study (9 in each group). The decrease of fibrinolytic activity was biphasic with a half-disappearance time of 112.5 min for BRL 26921 and 31 (IA) and 18 (IB) min for SK. alpha 2-Antiplasmin depletion and a decrease of fibrinogen was observed with no differences after bolus injections of SK and of BRL 26921.

Die Bolusinjektion von anisoyliertem Plasminogen-Streptokinase-Aktivator-Komplex (BRL 26921) als alternatives Konzept der systemischen Lyse bei akutem Myokardinfarkt

SummaryThe thrombolytic properties of anisoylated plasminogen streptokinase activator complex (BRL 26921) and clinical results of the treatment were studied in 10 consecutive patients with acute myocardial infarction. Exclusion criteria were general contraindications against thrombolytic therapy and a time interval of more than 4 h between the onset of symptoms and admission to the hospital. All patients received a 250-mg bolus of prednisolone prior to intravenous injection of 30 mg BRL 26921 within 2 min. A continuous infusion of heparin at a dose of 1,000 USPU/h was started 2 h after the injection. Blood pressure was monitored via an arterial line. Arrhythmias and changes in the ST segments were documented by conventional ECG recording and computerbased ECG monitoring. Coronary arteriography and left ventriculography were carried out within 72 h. Besides routine laboratory tests, serial CK and CK-MB activity measurements were carried out. We determined the following hemostaseological parameters before and 15 min, 30 min, 1 h, 4 h, and 12 h after application of BRL 26921: prothrombin time, activated partial thrombosplastin time, thrombin time, thrombin coagulase time, fibrinogen, streptokinaseplasminogen activator activity, plasminogen and alpha-2-antiplasmin.Our results (reperfusion in all patients angiographically and in 7 to 8 of 10 patients from noninvasive criteria) show that BRL 26921 is a highly effective thrombolytic agent in patients with myocardial infarction, when compared with highdose systemic fibrinolysis. Applied in dosages required for early reperfusion, it does not appear to be selectively thrombolytic and is not free of hypotensive effects in man. The decrease of fibrinolytic activity is biphasic with a half-disappearance time of 112 min.

In vivo recovery and half-life time of a steam-treated factor IX concentrate in hemophilia B patients

SummaryFactor IX (FIX) recovery and half-life was measured in ten hemophilia B patients under standardized conditions. Each patient received a steam-treated high-purity factor IX concentrate at a dose of 19–39 U/kg body weight. FIX activity was determined using a one-stage assay, which was calibrated against the international concentrate standard (reagents from Immuno, Heidelberg). The in vivo recovery ranged from 24% to 53% (mean value 37.7%) and the half-disappearance time (HDT) from 8–30 h (mean 16.7 h). In four of the ten patients, the distribution and elimination half-lives were estimated and ranged from 0.3 h to 3.9 h (mean 1.4 h) and from 28.6 h to 39.7 h (mean 33.1 h), respectively. In six patients FIX was redetermined using a different FIX deficient plasma and a plasma standard (reagents from Merz & Dade, Munich, FRG). Recoveries and HDT based on the results obtained with this method were significantly higher (68.2% vs 39.7%; p<0.05), and longer (14.8 h vs 10.6 h; p<0.05), respectively. FIX activity was also measured by both assay systems in 100 healthy subjects (50 males, 50 females). The reagents from Immuno yielded a mean value of 0.77 U/ml, while the mean FIX activity utilizing standards and reagents from Merz & Dade was 1.11 U/ml (p<0.000001). The coefficient of correlation between the FIX activity measurements, as determined in 100 healthy subjects and 6 hemophilia B patients using the different test systems, was r=0.9 (N=159; y=0.08+1.3 * x; p<0.001). Our data suggest that recovery and HDT of factor IX concentrate strongly depend on the assay and calibration conditions and that an international FIX activity plasma standard is urgently required.