The virally inactivated pasteurized FVIII concentrate Haemate P contains nearly intact vWF multimers. It is currently the treatment of choice to achieve satisfactory hemostasis for moderate to severe vWD and for patients with variants of vWD that cannot be adequately treated with DDAVP or for whom DDAVP is contraindicated. Therefore, we treated patients with type la, type IIa, type IIb and type III vWD with Haemate P. A correction of the hemostatic defect was seen in all patients. The type of bleeding events included 24 gastrointestinal, 18 other mucosal, 5 central nervous system, 15 orthopedic and other. 28 dental surgical procedures, 9 operative deliveries, tonsillectomies, 17 orthopedic and 11 miscellaneous surgeries were performed under the cover of Haemate P.
The determination of the bleeding time (HT) is an essential diagnoslic tool for von Willebrand's disease (vWD). However, the slandardized Simplate® BT still displays many variables and disadvanlages. The present study reports on the sensitivity of the in vitro bleeding test (lVBT) in SI vWD cases of different types and severity in comparison to the Simplate BT, and the correlation of both to each other as well as to von Willebrand factor (vWF:RCo) aclivity. The IVBT was performed in two modifica· tions (2 mmol/L CaCl z and 4 mmol/L ADP) on the Thl"omboslat 4oo().The IVBT, particularly wilh CaCl z , showed clearly higher sensilivity than the BT (CaCl z : 84.3%, ADP: 61.7%, CaCl z + ADP: 86.3, BT: 52.9%). The BT even failed in one patient wilh severe (type 2B) and in three with moderate vWD. The IVBT only failed in very mild forms of the disease (vW-F:RCo> 25%). In addition, the IVBT with ADP showed a close correlation to the vWF:RCo aclivity (r 2 = 0.73). The significantly lowel" cOl"relation of the BT with vWF:RCo (.-l = 0.49) was particularly due to the poor l"esults in vWD of type 2 (type 2:r 2 = 0.29; types I and 3:.-:z = 0.61). Finally, BT and IVBT-ADP correlated with each other (.-l = 0.53), a rather good correlation considering that both are complex functionaltests. It can be concluded from our study Ihat From Ih~D~porlmMI ofTransfusion M~dicine and H~moslauol 0ffY. UnN~r$ily Clinics, Marburg. *81txXJ Transfusion Sen'ic:e and General Hospiwl. Hamburg-Harburg. mid tDeporlmenl ofIongiology. Cenler a/
3677 Introduction: The hemostatic activity of von Willebrand Factor (VWF) is mainly controlled by the plasma metalloprotease ADAMTS13, which cleaves ultralarge VWF multimers. A qualitative or quantitative deficiency of VWF induces the most common hemorrhagic diathesis, the von Willebrand Disease (VWD). The current classification graduates the VWD in three major types. Depending on severity and the type of VWD the treatment with VWF/FVIII concentrates may by necessary. The commercially available VWF/FVIII concentrates differ in their multimer structure and furthermore also in their pharmacokinetics. We investigated commercial VWF concentrates with respect to their ADAMTS 13 activity and antigen levels with the newest available methods. Moreover, to detect a possible correlation, we analysed the VWF multimer structure of the concentrates. Methods: We analysed 4 human derived VWF/VIII-concentrates (over all 7charges) after reconstitution according to the manufacturer's instructions in different dilutions. Following methods were used: BCS Method according to Böhm detects the capacity of the concentrates for autoproteolysis. The VWF solutions were diluted with 5mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples: pool plasma; plasma from patients with TTP with neutralizing ADAMTS13 auto-antibodies; plasma from patients with TTP without auto-antibodies. The residual VWF:Ristocetin Cofactor (VWF:RCo) activity was subsequently measured using the BC von Willebrand Reagent from Dade Behring. ELISA Technozym®ADAMTS13 and Actifluor TM ADAMTS13 are based on the kinetic measurements of the activity with fluorescence resonance energy transfer (FRET). ADAMTS13 antigen was measured by use of the Technozym ELISA kit. SDS-Gel electrophoresis in 1% Agarose Gel was used to investigate the structure of VWF multimers. Results: The BCS Method according to Böhm is an indirect measurement for endogenous ADAMTS13 activity in the investigated concentrate. Important is the loss of the residual VWF:RCo in the concentrates in presence of TTP-plasma without antibodies and pool plasma compared to the residual VWF:RCo in presence of TTP-plasma with antibodies. All concentrates show some ADAMTS13 activity, however product 1 contains more ADAMTS13 than the other concentrates. The results of the two FRETS-assays correspond very well to the BCS-method results; in addition the assays detect directly the ADAMTS13 activity also in very low measurement range. In a dilution of 16U VWF per ml concentrate the ADAMTS13 activity in product 1 with 4.3% was the highest compared to product 2: 3.2%, product 3: 2.6% and product 4: 2%. The great variability of the test results in higher concentrations may be caused by interferences between some constituents of the concentrates and the analysis. In the same sample set and dilution the ADAMTS13 antigen values correlate very well with ADAMTS13 activity values. The SDS gel electrophoresis reveals the different VWF structure of product1; it has less large and ultralarge multimers. There could be a correlation to the relatively higher ADAMTS13 activity and antigen level. Conclusion: All the investigated VWF/VIII concentrates contain some ADAMTS13 activity and antigen. This was found especially by FRETs assay due to the high sensitivity. Because of the correlation between ADAMTS13 activity and modified VWF multimer structure we like to conclude that ADAMTS13 has influence on stability and therefore also on quality of the concentrates. This might have a therapeutic consequence especially for VWD type 2A. Type 2A is characterized by a relative reduction of intermediate and large VWF multimer. The multimeric abnormalities are commonly the result of in vivo proteolytic degradation of the von Willebrand factor caused by ADAMTS13. Disclosures: No relevant conflicts of interest to declare.
4661 Introduction: Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia, hemolytic anemia and microthrombi. A deficiency of the metalloprotease ADAMTS 13, which cleaves a Tys1605-Met1606 bond in the A2 subunit of von Willebrand factor (VWF), leads to formation of ultra large von Willebrand multimers (UL-VWF) and can cause platelet aggregation and mircovascular thrombosis. Treatment of choice is the substitution of plasma with plasmaexchange. There are two different plasma types available: Fresh Frozen Plasma (FFP) and solvent/detergent (s/d) treated plasma. This treatment may carry significant risks and side effects for the patients. Therefore we investigated the side effects of the therapy and furthermore the ADAMTS13 activity of the two plasma types. Methods: A questionnaire was send to 66 TTP patients of the Department of Hematology to evaluate different side effects of the therapy. 20 batches of FFP and 4 batches of s/d plasma of all blood groups were investigated on ADAMTS13 activity. The ADAMTS13 activity was detected with BCS-Method according to Böhm and two commercial FRET assays. Results: So far 34 patients were inquired about age, weight and suspected trigger situations that might have caused their TTP manifestation. The mean age of the patients was 34 years with a mean weight of 70kg. A previous infection caused TTP manifestation in 42% of the patients; drug therapy (22%) and pregnancy (17%) were other mentioned triggers. 94% of the patients suffered from an acquired TTP and only 6% had a hereditary TTP. The patients had 2.88 relapses and were treated with 16.27 plasmaexchanges. 56% had an additional therapy with Rituximab to achieve a faster remission of the disease. These patients needed less plasmaexchanges for recovery, which proofed to be significant at 2% level in a one sided t-test. Tingling (64.7%) and shivering (51%) were the most often mentioned side effects and simultaneously described as the strongest. Shivering was significantly correlated to tachycardia (p<0.01). Headaches were significantly correlated to hot flushes, tingling and collapse (p< 0.05). Side effects and allergic reactions occurred in the therapy with FFP as well as with s/d plasma. Another side effect was the complication that came along with infection of the venous access. Most patients had a central venous catheter (72%) and described infections and pruritus (60%), 50% of them mentioned this complication more than once. We found in usual FFP slightly higher ADAMTS13 activity levels (696.97 ng/ml) than in s/d virus inactivated plasma (643.86 ng/ml). The ADAMTS13 activity varied between the different assays (normal range: 666 ± 135ng/ml). Conclusion: Our investigation demonstrated that plasmaexchange therapy is still associated with a wide range of side effects. Side effects of plasmaexchange that were most frequently described by patients were tingling and shivering. Headaches also occurred in various cases. Patients suffered generally from more than one side effect at the same time during the treatment. Allergic reactions to the plasma therapy were mentioned by 65% of the patients. Disclosures: No relevant conflicts of interest to declare.
3678 Introduction: Thrombotic thrombocytopenic purpura (TTP) is characterized by microthrombi, hemolytic anemia as well as thrombocytopenia. These symptoms are caused by a decreased activity of the protease ADAMTS13 which cleaves the von Willebrand Factor (VWF), due to mutation of the ADAMTS13-gene or autoantibodies. At the moment, the only available immediate therapy is plasmapheresis with Fresh Frozen Plasma (FFP) which may induce side effects. Therefore an alternative therapy might be the treatment with clotting factor concentrates. Methods: 40 plasma samples were tested, consisting of FFP and solvent/detergent treated plasma, four batches of each blood group; VWF/VIII concentrates (Wilate®; Wilfactin®; Haemate®P; Immunate®; Kogenate®, Beriate®). In all samples ADAMTS13 activity, antigen and autoantibodies against ADAMTS13 were investigated. Additionally, the samples were tested for the presence of ultralarge VWF multimers. The BCS method according to Böhm, two ELISAs (Technozym®ADAMTS13 and Actifluor™ADAMTS13) and the electrophoresis on a SDS gel were used. Results: ADAMTS13 activity was found in all VWF)VIII concentrates, which are produced from human plasma, but only with a very low activity (Wilate® 5.6%; Wilfactin® 2.8%; Haemate®P 13%; Immunate® 3.7%). ADAMTS13 activity was not detectable in the factor VIII concentrates (Kogenate® <2%; Beriate® <2%). Usual FFP and solvent/detergent treated plasma samples, contained much higher ADAMTS13 activity and antigen values than concentrates (FFP 78.6%, solvent/detergent treated plasma 77.6%). However a difference of activity between individual blood groups was evident in FFP samples (blood group A 69.4%; B 64.9%; AB 98.1%; 0 82.0%). Ultralarge VWF multimers could be demonstrated in VWF containing concentrates, less in VIII concentrate from human plasma and not in FFP and solvent/detergent treated plasma samples. As expected recombinant-produced VIII concentrate contained no traces of ultralarge VWF. In all analyzed samples antibodies were negative. Conclusion: For therapy of TTP clotting factor concentrates cannot be used as an alternative to the usual FFP and solvent/detergent treated plasma, because their ADAMTS13 activity values are too low. In addition, the VWF/VIII concentrates contain higher quantities of ultralarge VWF multimers, which are contraindicated for TTP patients. The differences of ADAMTS13 activity in FFP samples varies between the individual blood groups and batches. Solvent/detergent treated plasma shows less variation in ADAMTS13 activity due to the manufacturing process involving plasma pooling. Disclosures: No relevant conflicts of interest to declare.
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