R. Eisert

Diagnostic workup of patients with acquired von Willebrand syndrome: a retrospective single‐centre cohort study

Summary.u2002 Background:u2002Diagnosis of acquired von Willebrand syndrome (AVWS) remains challenging. Diagnostic algorithms suggest the use of factor VIII (FVIII:C), von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), and collagen‐binding capacity (VWF:CB), but the sensitivity of these and other laboratory tests for the diagnosis of AVWS is unknown. Objectives:u2002To analyze the capacity of laboratory tests, including point‐of‐care testing (POCT), for the identification of patients with AVWS. Patients/methods:u2002Thirty‐five consecutive patients were enrolled with AVWS diagnosed because of a history of recent onset of bleeding, a negative family history of von Willebrand disease, and abnormal plasma VWF multimers. Results:u2002According to our inclusion criteria, all patients had bleeding symptoms, and the VWF high molecular weight multimers were either decreased or absent. Regarding POCT, PFA‐100 was inconclusive, due to anemia or thrombocytopenia, in 29%; the sensitivity was 80% in the remaining patients. The sensitivity of VWF:Ag (23%), VWF:RCo/Ag ratio <u20030.7 (26%), VWF:CB/Ag ratio <u20030.7 (46%), anti‐VWF antibodies (15%) and VWF propeptide/Ag ratio (22%) was too low to rule out the disease. A combination of VWF:Ag <u200350u2003IUu2003dL−1, VWF:RCo/Ag ratio <u20030.7 and VWF:CB/Ag ratio <u20030.8 yielded a sensitivity of 86%. Patients diagnosed only because of abnormal VWF multimers showed similar clinical characteristics as other patients. Conclusions:u2002Early diagnosis of AVWS is difficult, due to lack of sensitivity of the tests used. A substantial number of patients present with normal or increased test results, emphasizing the importance of multimer analysis in all patients with suspected AVWS.

Recovery and composition of microparticles after snap-freezing depends on thawing temperature

Consent regarding the correct processing and storage of blood microparticles is lacking and different protocols for the freeze–thaw cycle exist. Therefore, three different thawing procedures were evaluated regarding their influence on recovery and composition of microparticles. Microparticles were prepared by TRAP-6 or A23187 stimulation of platelet-rich plasma from smokers and nonsmokers (n = 8), from an endothelial cell line or directly obtained from platelet-free plasma of septic patients (n = 5). After snap-freezing in liquid nitrogen platelet-free samples were thawed at 37°, at room temperature or on ice and staining of microparticles was carried out with Annexin V-Cy5 as well as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Microparticle concentrations were determined by means of Trucount tubes. Recovery of platelet microparticles was significantly reduced when samples were thawed on ice (P = 0.001 for all antigens) compared with the two other techniques (P = 0.6 for 37° and P = 0.7 for room temperature, respectively) whereas microparticles of endothelial origin appeared to be less influenced. There was a strong trend towards altered microparticle composition as microparticle counts detected by CD41 staining showed a stronger decrease on ice than Annexin V enumeration (P = 0.07). For microparticle detection thawing of snap-fozen, platelet-free plasma samples should be carried out at room temperature or at 37°C in a water bath but not on ice.

Isotype controls in phenotyping and quantification of microparticles: A major source of error and how to evade it

BACKGROUNDnThe characterisation and quantification of cell-derived microparticles (MPs) using flow cytometry are often complicated by a low staining intensity and a non-discrete signal pattern of many cell surface antigens. Fluorescence-labelled isotype controls (ICs) are commonly used to set limits for the discrimination of antigen positive vs. negative events.nnnOBJECTIVESnThe influence of different ICs on the characterisation and quantification of MPs was studied. Antigen negative MPs stained with an antibody of interest were evaluated as an alternative control.nnnMETHODSnMPs were prepared from platelets, endothelial cell lines and leucemic cell lines and stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Results are given as the mean fluorescence intensity (MFI) or percentage of false-positive events above a fluorescence intensity > 1.nnnRESULTSnUsing identical instrument settings, seven different ICs (FITC-conjugates N = 3, PE-conjugates N = 4) resulted in a wide range of MFI and percentage of positive events with a mean coefficient of variation (CV) of 0.77. Instead, NMPs showed less variability with a mean CV of 0.50 and allowed a reliable and reproducible quantification of MPs when set as controls with < 2% false-positive events above an FI > 1. As a result, the expression of certain antigens (e.g. CD62P) was lower compared to previous reports in the literature.nnnCONCLUSIONSnDiversity in the staining intensity of isotype controls is a potential source of error in the characterisation and quantification of MPs by flow cytometry. The use of antigen negative MPs to adjust instrument settings is suggested.

Acquired haemophilia caused by non-haemophilic factor VIII gene variants

The aetiology of anti-factor VIII (FVIII) autoantibody formation in acquired haemophilia remains unknown. We hypothesised that encounter of antigenically different, allogeneic FVIII may challenge inhibitor formation after presentation on MHC class II. Eighteen consecutive cases with acquired haemophilia were enrolled (nine females, nine males). A control group comprised 50 male and 50 female healthy blood donors. The coding region of the FVIII gene and the HLA-DRB1 genotype were studied. The presentation of foreign FVIII variants on the patient’s MHC class II alleles was predicted using SYFPEITHI algorithm. A rare FVIII variant (E2004K) was found in one patient with acquired haemophilia after massive transfusion; the 2004 K allele was predicted to be presented on the patient’s HLA-DRB1*0101. Moreover, distribution of a polymorphism (D1241E) was significantly skewed comparing patients and controls. Three of three patients with transfusion-associated disease carried 1241D in homozygous or hemizygous form and were predicted to present 1241E (foreign), but not 1241D (self), on their HLA-DRB1*0301. Therefore, encounter of 1241E may result in the presentation of a new T cell epitope in these patients. The same conditions were not found in any patient with acquired haemophilia of other causes. The expected frequency in the general Caucasoid population undergoing transfusion is 3% to 4%. In conclusion, encounter of variant allogeneic FVIII presented on a suitable MHC background could be a risk factor for inhibitor formation.

Inhibitor-immunology-study. Evaluation of inhibitor development in haemophilia B.

UNLABELLEDnThe development of inhibitors in haemophilia B is one of the most important complications of replacement therapy, affecting mortality and morbidity. Inhibitor development is based on complex immunological factors, and to date, only little is known about its underlying mechanisms. Here, we present first results of the haemophilia B group of our Inhibitor-Immunology study.nnnPATIENTS, METHODSnSo far we have analysed 15 patients with haemophilia B. Four of them developed a high titre inhibitor; the remaining 11 had no inhibitor. We evaluated 9 SNPs in 8 genes (CD40, CTLA-4 , IL-1β, IL-10, TLR2 , TLR4, TLR9, TNF-α). We compared the distribution of these alleles between inhibitor and non-inhibitor haemophilia B patients and between haemophilia B patients and a normal male control population. HLA typing was performed in all patients. Results, discussion: There appears to be a trend towards a skewed distribution of TLR 9, IL-10 and CTLA4 alleles in haemophilia B patients. Due to the limited number these differences are, however, not statistically significant. The t-test of all patients with inhibitor versus without inhibitor was significant for HLA-A*03 and DPB1*0401 and borderline for DRB1*0201.

Clinical consequences of compound heterozygosity for protein S mutation Heerlen and p.Cys252Gly protein S mutation

Fig. 1. Molecular analysis of a protein S (PS)-deficient family. Pedigrees are shownwith the PS percentages and genetic determination where known. Squares represent males and circles represent females. Thedottedpattern shows thepresenceofheterozygousCys252Gly, and thediagonal stripes showthepresenceof theheterozygousHeerlenmutation. 1*Venous thrombosis occurred spontaneously at the age of 60, 2* exogenously triggered thrombosis at 74, 3* spontaneous thrombosis at 17 and two incidences of thrombophlebitis. Dear Editors,

Hemophilia Summer Camp in Germany, 1996–2005

Successful home treatment in persons with hemophilia is not only depending on medical knowledge and technical skills, but also on the patients’ and parents’ willingness to share responsibility for treatment. The German Hemophilia Society (DHG) and the Hemophilia Care Center at Hanover Medical School are collaborating to enhance our patients’ capability and motivation for home treatment.

Acquired Glanzmann Thrombasthenia in a Patient with Myelodysplastic Syndrome

Acquired Glanzmann thrombasthenia is a rare bleeding disorder which is characterized by a blank bleeding history, rapid onset of bleeding tendency with a significantly prolonged bleeding time but normal platelet counts and normal expression of platelet membrane glycoproteins (GP). Etiologically, platelet antibodies bind on or close by the GP IIb and/or IIIa and inhibit binding of fibrinogen and von Willebrand factor (VWF) to platelets [1]. These antibodies mostly emerge in the scope of lymphoproliferative or autoimmune diseases. An association with a myelodysplastic syndrome (MDS) which we observed in our presented patient has not been described in the literature to our knowledge. This coincidence supports the thesis of loss of T cell regulation in MDS and establishes the basis for new immunosuppressive therapeutic approaches to acquired Glanzmann thrombasthenia.

Update of the Inhibitor-Immunology-Study

We are presenting an update of our study in which risk factors for the development of inhibitors in patients with hemophilia are to be explored. The ultimate goal is to find out why some children suffering from severe or moderate hemophilia develop inhibitory antibodies during replacement therapy and others do not, and to define genetic and immunological risk factors.

Recombinant Factor VIIa for Major Surgery in Severe Factor XI Deficiency: Pharmacodynamic Monitoring Using Thromboelastometry

Factor XI (FXI) deficiency is a rare autosomal recessive disorder characterized by abnormal bleeding after trauma or surgery. FXI replacement therapy using fresh frozen plasma (FFP) or FXI concentrate has been associated with important side effects including inhibitor formation, arterial and venous thromboembolism and volume overload [1]. The type of FXI gene mutation strongly influences the risk of inhibitor formation. In an Israeli cohort study of 64 unrelated subjects with severe deficiency (FXI 90% of cases, a nonsense mutation Glu117Stop (known as type II mutation) and a missense mutation Phe283Leu (type III). Homozygotes for type II and III have a residual FXI activity of 1 and 10 IU/dL, respectively, whereas compound heterozygotes (II/III) have a mean residual activity of 3 IU/dl. Inhibitors against FXI were observed only in patients homozygous for the type II mutation, who were exposed to plasma-derived FXI. The cumulative incidence was 7 out of 21 (33 %) [2]. Thus, patients with a FXI residual activity < 1 % seem to be at high risk for inhibitor formation. The successful use of recombinant activated factor VII (rFVIIa) for surgical prophylaxis has been reported in two cases with severe deficiency and inhibitor [3, 4]. Moreover, in a pilot study 14 consecutive patients (5 severely deficient, one with inhibitor) underwent 15 procedures (5 major, 4 minor, 6 dental) [5]. For the major procedures, patients received 90 μg/kg 2-hourly for 13 doses followed by 90 μg/kg 4-hourly for 6 doses. All procedures were successfully performed without any evidence of bleeding. The use of rFVIIa is potentially advantageous in FXI deficient patient by avoiding the risk of inhibitor formation and volume overload. In the above mentioned cases, dosing of rFVIIa was similar to the treatment of patients with inhibitors against factor VIII (FVIII). It is unclear whether this dose is actually required in FXI deficient patients who seem to have a milder bleeding phenotype compared to patients with hemophilia and FVIII inhibitors.Adequate monitoring assays to support dosing decisions in these patients are currently not available. Here, a severely FXI deficient patient was treated using rFVIIa and pharmacodynamic monitoring was performed using rotation thromboelastometry.