[Protein Z deficiency in unexplained affinity to thromboses, bleedings or abortions]. / Protein-Z-Mangel bei ungeklärter Neigung zu Thrombosen, Blutungen oder Aborten.

UNLABELLED: A protein Z deficiency is presumably related with a threefold risk of venous and arterial thrombosis. Mucosal bleedings and post-operative haematomas can occur more frequently. This is seen in an increased in vivo bleeding time without other plasmatic coagulation disorders or thrombopathies. Pregnancy complications, especially abortions before the 15th week of gestation, are described as well. PATIENTS, METHODS: Since May 2011 the plasmatic concentration of protein Z has been tested in 684 patients of the Hämostaseologicum. RESULTS: In 74 patients a protein Z deficiency has been found. In other 45 patients protein Z was reduced because of the intake of phenprocoumon or coumadin. Of the 74 patients with diminished protein Z concentration 39 were marginally decreased (protein Z 1000-1500 µg/l). Of the 35 patients with a protein Z concentration <1000 µg/l 12 had had a thrombosis before (6 strokes, 3 DVT or PE, 1 arterial thrombosis, 1 retinal branch vein occlusion, 1 acute hearing loss). 7 had arterial hypertension, 2 suffered from diabetes mellitus. Of the patients who had a thrombosis 6 had a heterozygous factor V Leiden mutation. 10 had a microcirculation disorder (Raynaud's phenomenon), 4 had had bleeding complications before, 3 had a von Willebrand disease type I, 6 patients had had abortions and 4 were healthy. Of the 39 patients with protein Z concentrations between 1000 and 1500 µg/l 18 had experienced a thrombosis before (9 DVT or PE, 3 myocardial infarctions, 1 CHD, 3 strokes, 1 retinal branch vein occlusion, 1 PAOD I, 1 tinnitus). 5 additionally had arterial hypertension. 13 suffered from Raynaud's phenomenon, of which 7 had a hypotension. Of the patients with thromboses 3 had a heterozygous factor V Leiden mutation and one a protein C deficiency. 7 patients had had an abortion before. Bleeding complications were seen in 4 patients, of which 3 suffered from von Willebrand disease type 1.

[Risk pregnancies and how to treat them]. / Risikoschwangerschaften und deren Therapie.

UNLABELLED: Recurrent abortions are a common problem. A therapy with low-molecular- weight heparin is usual in deep vein thrombosis with thrombophilia, in woman with recurrent abortions or other risks, like EPH-gestosis or HELLP-Syndrom. PATIENTS, METHOD: The efficacy of a mono-therapy with LMWH (3000-16000 daily) in women with risk pregnancies has been examined prospectively. The dates of 676 pregnant women have been analysed and compared to the current literature about live birth rates without therapy and tot he results of other, similar studies. The live birth rate has been the target variable. RESULTS: We obtained main a live birth rate of 98.6%. There has been no record of serious adverse effects. We obtained a live birth rate of 95.8% if NMH therapy starts early, and a live birth rate of 100% if NMH therapy starts between week 20 and 25. For the live birth rate the existence of thrombophilic gene polymorphisms is irrelevant. CONCLUSION: The high live birth weight is depended on early starting the therapy with NMH. For the late risk it is favourable to start the therapy with heparin between week 20 and 25 week of pregnancy.

Experiences with recombinant FVIIa in the emergency treatment of patients with autoimmune thrombocytopenia: a review of the literature.

Patients with severe and refractory autoimmune thrombocytopenia (ITP) have significant morbidity and mortality rates. Currently, high-dose methylprednisolone and/or high-dose IVIgG are recommended for the emergency treatment of such patients with uncontrolled bleeding. However, some patients do not immediately respond to these therapeutic regimes and may require additional treatment. Recombinant activated FVIIa (rFVIIa) is a prothrombotic agent that appears to be useful in the treatment of patients with life-threatening bleeding. It has also been used in the treatment of several patients with thrombocytopenia. We administered rFVIIa into a patient with refractory ITP and performed a systemic review of all published reports to assess the available evidence on the efficacy and safety of this drug in patients with ITP. The results indicate that rFVIIa may help in the emergency treatment of patients with ITP who do not respond to other therapies.

Influence of peptidylarginine deiminase type 4 genotype and shared epitope on clinical characteristics and autoantibody profile of rheumatoid arthritis.

BACKGROUND: Recent evidence suggests that distinction of subsets of rheumatoid arthritis (RA) depending on anti-cyclic citrullinated peptide antibody (anti-CCP) status may be helpful in distinguishing distinct aetiopathologies and in predicting the course of disease. HLA-DRB1 shared epitope (SE) and peptidylarginine deiminase type 4 (PADI4) genotype, both of which have been implicated in anti-CCP generation, are assumed to be associated with RA. OBJECTIVES: To elucidate whether PADI4 affects the clinical characteristics of RA, and whether it would modulate the effect of anti-CCPs on clinical course. The combined effect of SE and PADI4 on autoantibody profile was also analysed. METHODS: 373 patients with RA were studied. SE, padi4_94C>T, rheumatoid factor, anti-CCPs and antinuclear antibodies (ANAs) were determined. Disease severity was characterised by cumulative therapy intensity classified into ordinal categories (CTI-1 to CTI-3) and by Steinbrocker score. RESULTS: CTI was significantly associated with disease duration, erosive disease, disease activity score (DAS) 28 and anti-CCPs. The association of anti-CCPs with CTI was considerably influenced by padi4_94C>T genotype (C/C: OR(adj) = 0.93, p(adj) = 0.92; C/T: OR(adj) = 2.92, p(adj) = 0.093; T/T: OR(adj) = 15.3, p(adj) = 0.002). Carriage of padi4_94T exhibited a significant trend towards higher Steinbrocker scores in univariate and multivariate analyses. An association of padi4_94C>T with ANAs was observed, with noteworthy differences depending on SE status (SE-: OR(adj) = 6.20, p(adj)<0.04; SE+: OR(adj) = 0.36, p(adj) = 0.02) and significant heterogeneity between the two SE strata (p = 0.006). CONCLUSIONS: PADI4 genotype in combination with anti-CCPs and SE modulates clinical and serological characteristics of RA.

Immune tolerance induction in patients with IgA anaphylactoid reactions following long-term intravenous IgG treatment.

To date, there is very little information regarding the pathomechanism of IgA anaphylactoid reactions and the management of affected patients. Five adult patients with common variable immunodeficiency (CVID) and a history of anaphylactic reactions due to the administration of immunoglobulin preparations were studied. The activity of anti-IgA was determined by the gel agglutination technique using IgA-coated beads. Antibodies to IgA were detected in the serum of all five patients. Initially, IgA 'depleted' intravenous (i.v.) IgG preparations were infused carefully into the patients until the activity of anti-IgA was decreased significantly or became undetectable. Subsequently, unselected i.v. IgG preparations were infused, and the activity of anti-IgA was abolished in all cases. Intravenous IgG long-term administration results in tolerance induction in patients with IgA anaphylactoid reactions. This tolerance appears to be related to antibody blockage in the circulation and an inhibition of antibody production. Most importantly, IgA appears to play an important role in the treatment of CVID. Patients with IgA anaphylactoid reactions can be treated safely with IgA containing i.v. IgG preparations following tolerance induction.

Application of the particle gel agglutination assay in the typing of single human leucocyte antigens.

We describe a simple and rapid particle gel agglutination assay (PaGIA) for typing of the human leucocyte antigens (HLA) HLA-A2, HLA-B7 and HLA-B27. Superparamagnetic streptavidin particles were coated with biotinylated monoclonal antibodies (MoAbs) to HLA-A2, HLA-B7 and HLA-B27. Anticoagulated whole blood samples from healthy blood donors (n = 118) with known HLA patterns were incubated with MoAb-coated particles, transferred into a standard ID-gel card, and subsequently centrifuged. Samples were evaluated macroscopically, with antigen-positive samples resulting in a visible agglutination reaction. A clear distinction could be made between all positive and negative samples tested. Fifty-seven samples were found to be positive for HLA-A2 (48%), 26 samples for HLA-B7 (22%) and 5 samples for HLA-B27 (4%).

Influence of blood donation on levels of water-soluble vitamins.

Iron depletion is a well-known side effect of blood donation. Research evidence also suggests an increasing prevalence of vitamin deficiency in apparently healthy subjects, but there is little information regarding the relationship between blood donation and vitamin status. A total of 217 volunteers (80 first-time and 137 repeat blood donors) were consecutively enrolled in the study. All subjects completed self-administered medical history and food intake forms, which included questions regarding alcohol consumption and smoking as well as on vitamin supplement, iron and contraceptive use (females). Vitamin B6, folic acid, vitamin B12 and biotin levels were measured using standard techniques. The mean vitamin levels of first-time and repeat blood donors did not significantly differ. Vitamin deficiencies occurred in both first-time and repeat blood donors but not on vitamin supplements. Vitamin status was affected by alcohol, nicotine and contraceptives. Blood donation does not decrease the level of water-soluble vitamins. Vitamin deficiencies occur in apparently healthy first-time as well as in repeat blood donors and can be prevented by vitamin supplementation.

Optimum storage conditions for cord blood-derived hematopoietic progenitor cells prior to isolation.

Optimum storage conditions of cord blood-derived hematopoietic progenitor cells before isolation remain unknown. We therefore evaluated CD34+ cells isolated from cord blood units (n=57) within 1 h after collection and following storage for 24, 48 and 72 h at either room temperature (RT) or 4 degrees C. Isolated CD34+ cells were analyzed for their cell count, immunophenotype, apoptosis rate, clonogenicity and transmigration capacity in response to stroma-derived factor 1alpha using direct-paired comparisons (n=27). CD34+, CD133+ and CD45+ positivity after isolation remained the same under all conditions. After 24 h, CD34+ cell counts and numbers of CFU-GM colonies dropped regardless of the storage temperature. After 48 h, the number of CD34+ cells increased compared to 24 h, if the cord blood had been stored at RT resulting in almost three times more CD34+ cells than at 4 degrees C. These cells had a lower early apoptosis rate and formed four times more BFU-E than those stored at 4 degrees C with equivalent plating efficiencies. CD34+ cells kept at RT for 48 h had the highest transmigration capacities, which paralleled an increased CXCR-4 expression. Cord blood should be stored at RT before CD34+ isolation and a storage time for 48 h should be preferred to 24 h.

[Preoperative identification of patients with impaired (primary) haemostasis. A practical concept]. / Präoperative Identifikation von Patienten mit (primären) Hämostasestörungen. Ein praktisches Konzept.

The findings of a large prospective study designed to identify primary and/or secondary haemostatic disorders before surgical interventions are presented. A total of 5649 unselected adult patients were enrolled to identify impaired haemostasis before surgical interventions. Each patient was asked to answer a standardized questionnaire concerning bleeding history. Activated partial thromboplastin time (aPTT), prothrombin time (PT), and platelet counts (PC) including PFA-100 (platelet function analyzer): collagen-epinephrine (C/E), and collagen-ADP (C/ADP) were routinely done in all patients. Additional tests, bleeding time (BT), von Willebrand factor (VWF:Ag, VWF:Rcof) and a further haemostaseological diagnostic was performed only in patients with a positive bleeding history and/or evidence of impaired haemostasis; e.g., drug ingestion. The bleeding history was negative in 5021 patients (88.8%) but positive in the remaining 628 (11.2%). Impaired haemostasis could be verified only in 256 (40.8%) of these patients. The vast majority was identified with PFA-100: C/E (n = 250; 97.7%). The sensitivity of the PFA-100: collagen-epinephrine was the highest (90.8%) in comparison to the other screening tests (BT, aPTT, PT, VWF : Ag). The positive predictive value (to detection of impaired haemostasis) of the PFA-100: collagen-epinephrine with the standardized questionnaire was high (82%), but the negative predictive value was higher (93%). The use of a standardized questionnaire and, if indicated, the PFA-100: C/E and/or other specific tests not only ensure the detection of impaired haemostasis in almost every case but also a significant reduction of the costs. Based on these data, national regards are formulated or under construction.

Ximelagatran for treatment and prophylaxis of recurrent events in deep vein thrombosis.

The treatment of acute venous thromboembolism and prophylaxis of recurrent events with heparin/low molecular weight heparin followed by vitamin K antagonists is limited by several factors. Oral direct thrombin inhibitors (ODTIs) showed a better pharmacological activity and might be an alternative in the treatment of venous thromboembolism. The Thrombin Inhibition in Venous Thromboembolism (THRIVE) program performed some studies developing the ODTI ximelagatran for this indication, and it is presented in the overview. The aim of the THRIVE I study was the dose finding, and that of the THRIVE IV study the applicability in hemodynamic stabile pulmonary embolism. A prospective, randomized, double blind trial was performed to compare oral ximelagatran with enoxaparin/warfarin for a 6-month treatment of acute venous thrombosis (THRIVE II and V). A second double blind study compared ximelagatran with placebo over 18 months after a 6-month anticoagulant therapy of acute deep vein thrombosis. The efficacy and safety of treatment of patients with acute deep venous thrombosis who received 2 infinity 36 mg ximelagatran was not inferior to that of patients who received a conventional anticoagulant for prophylaxis of recurrent events over 6 months. Ximelagatran 2 infinity 24 mg significantly reduced recurrent thromboembolic events compared to placebo without increasing the risk for hemorrhage. A reversible symptomless increase of alanine aminotransferase occurs in 6% to 9.6% of patients between months 2 and 4. The results of the follow-up studies suggest that thromboembolic events may recur in patients with acute venous thromboembolism after termination of treatment with both vitamin K antagonists and ximelagatran.

CD47 is expressed at normal levels in patients with autoimmune haemolytic anaemia and/or immune thrombocytopenia.

CD47 deficiency results in lethal autoimmune haemolytic anaemia (AIHA) and mild spontaneous thrombocytopenia in non-obese diabetic mice. It is unknown whether CD47 has an impact on AIHA of the warm type or autoimmune thrombocytopenia (ITP) in humans. Healthy blood donors (n= 11), patients with AIHA (n= 13), patients with ITP (n= 18) and one patient with Rh(null) phenotype were investigated. CD47 expression on red blood cells (RBC), platelets, granulocytes and lymphocytes and in plasma was determined by quantitative flow cytometry. All types of blood cells studied were found to carry CD47. Although CD47 expression on Rh(null) RBCs was decreased, there was no significant difference between CD47 expression on RBCs of healthy blood donors and on those of patients with AIHA or ITP. Similarly, CD47 was detectable in the plasma of the studied subjects. No evidence for a pathogenetic role of CD47 in autoimmune haemolysis or thrombocytopenia in humans could be demonstrated.

Observation of serum erythropoietin concentrations in female athletes for up to eight days after a marathon run.

Erythropoietin (EPO) was studied in 13 female marathon runners before and up to 8 days after a competition marathon run. The median baseline control value was 13.7 U/l. No change in EPO concentration was found immediately (15 min.) and one day after the run. However, a median increase in EPO concentration (18.1 U/l) was found on day three post-exercise (p< 0.05). On day 8 no change was found compared to pre-exercise values. This late increase in EPO concentration would seem to be responsible for the well known increase of red blood cell mass in long distance runners.

A novel antigen-specific capture assay for the detection of platelet antibodies and HPA-1a phenotyping.

BACKGROUND AND OBJECTIVES: The antigen-specific assays currently used for the laboratory investigation of platelet antibodies and antigens are technically complex and cannot be used in most routine laboratories. Here, we describe a simple antigen-specific capture assay (ASCA) for the detection of serum platelet antibodies and for human platelet antigen-1a (HPA-1a) phenotyping. MATERIALS AND METHODS: For the detection of platelet antibodies, platelets from healthy blood donors were incubated with biotinylated monoclonal antibodies to platelet glycoprotein complexes (GP), then solubilized and mixed with superparamagnetic streptavidin particles. Serum samples from patients with autoimmune thrombocytopenia (n = 39), from patients with platelet alloantibodies (6 HPA-1a, 1 HPA-2b, 1 HPA-3a, 6 HPA-5b), and from healthy blood donors (n = 70), were tested. All serum samples from the patients were investigated in parallel by the indirect monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). For HPA-1a phenotyping, superparamagnetic particles were coated with a monoclonal antibody to HPA-1a and mixed with diluted whole blood samples from healthy blood donors (n = 139), who had previously been genotyped for platelet alloantigens. Results The indirect MAIPA detected autoantibodies in 18%, and the direct MAIPA in 50% of patients tested. In contrast, the new ASCA demonstrated positive results in 77% of patients. All tested alloantibodies reacted positive by the ASCA, and all serum samples from healthy blood donors were negative. The results of HPA-1a phenotyping were in concordance with those of genotyping in all cases. CONCLUSION: In our opinion, the ASCA is easy to perform and much more sensitive than the currently available antigen-specific assays for the detection of platelet antibodies.

Report of a patient with heparin-induced thrombocytopenia type II associated with IgA antibodies only.

Heparin-induced thrombocytopenia type II (HIT II) is usually mediated by immunoglobulin G (IgG) antibodies that lead to platelet activation via the FcgammaIIA-receptor. Here we describe a patient who developed HIT II after aortocoronary bypass surgery. His serum contained an antibody that was detectable by the heparin-induced platelet activation assay (HIPA) and by the ID-HPF4 particle agglutination assay. The flow cytometric analysis showed that the antibody was of the IgA class.

Replacement of intravenous administration of anti-D by subcutaneous administration in patients with autoimmune thrombocytopenia.

Intravenous (IV) administration of anti-D in patients with autoimmune thrombocytopenia (AITP) may result in severe hemolysis and even death. Over a 3-year period, we gave anti-D only subcutaneously (SC), and none of our patients have developed any acute adverse reaction. Most importantly, SC delivery of anti-D produces largely the same beneficial effect as obtained by IV anti-D. We recommend replacement of IV administration of anti-D by SC administration in AITP.

A simple and practical agglutination assay for human leucocyte antigen-B27 typing.

BACKGROUND AND OBJECTIVES: The human leucocyte antigen (HLA) B27 is the most frequently typed single antigen that is associated with diseases. Here, we describe a simple and rapid particle agglutination assay (PaGIA) for HLA-B27 typing. MATERIALS AND METHODS: Superparamagnetic particles were coated with a monoclonal antibody to HLA-B27 and subsequently used for testing. Anticoagulated whole-blood samples were obtained from healthy blood donors (n = 194) with known HLA patterns and from patients (n = 51) who had been typed positive for HLA-B27 by flow cytometry. RESULTS: The particles agglutinated only after incubation with HLA-27-positive blood samples, using the ID-microtyping system. Positive reactions were clearly distinguishable from negative reactions in all samples tested. Flow cytometric HLA-B27 typing revealed an indeterminate result in one patient. CONCLUSIONS: The new HLA-B27 PaGIA is suitable for rapid typing of HLA-B27. The assay is simple and easy to perform, and can be implemented in any routine laboratory.

Detection of a new weak A blood-group allele (Aw11).

BACKGROUND AND OBJECTIVES: Weak ABO variants may escape tests using unlicensed sera. MATERIALS AND METHODS: Prior to transfusion, ABO grouping was performed using an automated system and in-house diluted sera, and manual and bedside test techniques. Genotyping and sequencing were performed using standard methods. RESULTS: Initially, the red blood cells (RBC) of the first-time blood donor were typed as B, but pretransfusion testing carried out using the bedside test indicated the presence of an additional A phenotype. Serological re-examination confirmed the bedside test results, and the allele in question was identified, by genotyping, as a new weak A variant (Aw11). CONCLUSIONS: The use of CE-marked and licensed antisera is recommended to avoid ABO mistyping.

Immunoadsorption patients with multiple sclerosis: an open-label pilot study.

BACKGROUND: Immunoadsorption (IA) is occasionally applied in patients with acute relapses of multiple sclerosis (MS). This pilot study was undertaken to determine whether IA might help in secondary progressive and relapsing-remitting multiple sclerosis. DESIGN: IA was performed at 1-week intervals in 12 patients with secondary progressive or relapsing-remitting MS. These patients had an extended disability status scale (EDSS) score of 4.5-7 and an EDSS increase of 0.5 within 6 months before inclusion in the study despite conventional drug therapy. The change in the EDSS and that in the MS functional composite (MSFC) score, which consisted of quantitative tests of arm function, ambulation, visual acuity and cognition, served as the primary outcome variables, which were measured at baseline and at 3, 6 and 12 months. Changes in quality of life and cerebral lesions by magnetic resonance imaging (MRI) were also assessed at baseline and after the last immunoadsorption (month 3). RESULTS: A significant reduction of the median EDSS change was observed after the treatment period, which reversed 3 months after the immunoadsorptions had been stopped. Ten of 12 patients remained stable during the first year of follow-up with no significant changes of the MSFC scores. No significant changes in magnetic resonance imaging T2-hyperintense brain lesions or in the number of gadolinium-positive lesions and in the patients' quality of life were observed. Western blot analyses demonstrated a reduction of serum myelin-specific antibodies, which were collected in the adsorber eluates. CONCLUSIONS: Removal of immunoglobulins, including myelin-specific antibodies by immunoadsorption, seems to delay disease progression as defined by EDSS, MSFC and MRI, while the patients' quality of life did not deteriorate.

Antibodies against protein Z and fetal loss: current perspectives.

Protein Z (PZ) is a vitamin K-dependent plasma protein that serves as a cofactor for the inactivation of factor Xa. A number of investigators found low PZ levels in patients with haemorrhagic as well as thromboembolic diseases, although there is no clear evidence of a pathogenic link between PZ deficiency and these clinical disorders. Nevertheless, low PZ levels have been found in association with early fetal losses, especially those occurring before the 15th week of gestation and in patients with detectable antiphospholipid and anti-PZ antibodies. The current diagnostic relevance and therapeutic consequences of these parameters will be discussed.