Autologous stem cell therapy in the treatment of limb ischaemia induced chronic tissue ulcers of diabetic foot patients.

BACKGROUND AND AIM: Despite improvements in surgical revascularisation, limitations like anatomical factors or atherosclerosis limit the success of revascularisation in diabetic patients with critical limb ischaemia. Stem cells were shown to improve microcirculation in published studies. The aim of this study was to evaluate safety, feasibility and efficacy of transplantation of bone marrow derived cellular products regarding improvement in microcirculation and lowering of amputation rate. METHODS: Bone marrow mononuclear cells (BMCs) in comparison with expanded bone marrow cells enriched in CD90+ cells ('tissue repair cells', TRCs) were used in the treatment of diabetic ulcers to induce revascularisation. Diabetic foot patients with critical limb ischaemia without option for surgical or interventional revascularisation were eligible. Parameters examined were ABI, TcPO(2) , reactive hyperaemia and angiographic imaging before and after therapy. RESULTS: Of 30 patients included in this trial, 24 were randomised to receive either BMCs or TRCs. The high number of drop-outs in the control group (4 of 6) led to exclusion from evaluation. A total of 22 patients entered treatment; one patient in the TRC group and two in the BMC group did not show wound healing during follow up, one patient in each treatment group died before reaching the end of the study; one after having achieved wound healing (BMC group), the other one without having achieved wound healing (TRC group). Thus, 18 patients showed wound healing after 45 weeks. The total number of applicated cells was 3.8 times lower in the TRC group, but TRC patients received significantly higher amounts of CD90+ cells. Improvement in microvascularisation was detected in some, but not all patients by angiography, TcPO(2) improved significantly compared with baseline in both therapy groups. CONCLUSION: The transplantation of BMCs as well as TRCs proved to be safe and feasible. Improvements of microcirculation and complete wound healing were observed in the transplant groups.

Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics.

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Preoperative haemostasis testing does not predict requirement of blood products in cardiac surgery.

OBJECTIVE: Standard haemostasis screening tests are performed to reveal unknown congenital or acquired disturbances of plasma and/or platelet haemostasis. Since their diagnostic efficacy is often low, routinely performed haemostasis testing has been questioned. We investigated whether preoperatively assessed haemostasis testing can be used to predict the requirement of blood products. METHODS: We retrospectively assessed haemostasis parameters including platelet function testing by PFA 100 as well as the numbers of red blood cell (RBC) concentrates, fresh frozen plasmas (FFPs), and platelet concentrates (PCs) that were given peri-operatively and during the first two postoperative days in 2,831 cardiac surgery patients. Logistic regression analyses were used to select those parameters, which could predict blood product requirement. RESULTS: Of our study cohort, 56.5% needed RBCs, 15% FFPs, and 5% PCs. The need for RBCs was associated with significantly altered pre-operative values of most haemostasis parameters. However, by the use of logistic regression analysis fibrinogen was the only haemostasis parameter that was independently associated with the use of RBCs (odds ratio 1.56; 95% CI: 1.27-1.91; P <0.001). The predictive value of other parameters such as age, body weight, haemoglobin, and haematocrit was however much higher in comparison to fibrinogen (odds ratios: 1.92-3.50; P <0.001). It was not possible to develop a score based on haemostasis parameters to accurately identify patients at risk for RBC use. Moreover, we were unable to estimate the need for FFPs and PCs using preoperative haemostasis testing. CONCLUSIONS: Our data demonstrate that preoperatively performed haemostasis testing is not predictive in estimating the need for blood products in cardiac surgery patients.

IgG classification of anti-PF4/heparin antibodies to identify patients with heparin-induced thrombocytopenia during mechanical circulatory support.

Commercial immunoassays frequently detect anti-PF4/heparin antibodies during mechanical circulatory support (MCS), but only a small minority of patients develops heparin-induced thrombocytopenia (HIT). Whereas platelet functional tests can distinguish between platelet-activating and non-platelet-activating antibodies, commercial PF4-dependent immunoassays do not. Between 2003 and 2004, 113 patients were placed on MCS. Blood samples were obtained on postimplant day 5-7 for analyses by antibody assays and the functional heparin-induced platelet activation (HIPA) assay. Three distinct groups of patient sera were identified: platelet-activating anti-PF4/heparin antibodies (n = 10), non-platelet-activating anti-PF4/heparin antibodies (n = 53), and anti-PF4/heparin antibody negative (n = 50). Patients with platelet-activating antibodies had the highest risk for thromboembolic events (P < 0.005), whereas those with non-platelet-activating antibodies did not differ from antibody negative patients (P = 0.369). The enzyme-immunoassay and column agglutination assays, which cover all immunoglobulin classes, demonstrated adequate sensitivity and negative predictive value; yet, both lacked specificity with respect to the platelet-activating antibodies. If all antibody positive patients were further classified by an IgG-specific anti-PF4/heparin enzyme-immuno assay, specificity for platelet-activating antibodies increased. Whereas IgG-specific optical density (OD) values below 1.0 were likely for non-platelet-activating anti-PF4/heparin antibodies, higher values were progressively predictive for pathogenic platelet activation. The probability of the development of clinical HIT also increased steeply. In conclusion, platelet-activating anti-PF4/heparin antibodies are relatively common (about 9%) in patients on MCS and are associated with significantly higher thrombotic event rates. Low IgG-specific OD values (< 1.0) in the enzyme-immunoassay indicate low likelihood for the presence of platelet-activating antibodies. These results justify further validation so that anticoagulation during MCS becomes safer and adequate.

No evidence for an improvement of long-term survival by HLA matching in heart transplant recipients.

It has been assumed that better HLA matching improves midterm survival in cardiac transplantation. However, statistically reliable data on long-term survival according to HLA matching are scanty. We performed a retrospective analysis of all patients who underwent orthotopic heart transplantation at our heart center between 1989 and 2005. HLA typing data (major histocompatability complex [MHC] class I and II) were available in 923 patients and their heart donors. Univariate and multivariate analyses were performed to assess the impact of HLA matching on long-term survival. The average follow-up period was 6.1 +/- 4.3 years (range, 0.0 to 15.0 years). In total, the 923 patients accrued 5625 patient-years of observation. Zero, one, and two mismatches occurred at each locus in between 0.3% (HLA-B) to 6.6% (HLA-C), 16.6% (HLA-B) to 39.4% (HLA-DQ), and 55.4% (HLA-DQ) to 83.3% (HLA-B), respectively. Two hundred eleven patients died during follow-up (22.9%). Survival at 1, 2, 5, and 10 years was 87.7%, 86.2%, 78.4%, and 63.9%, respectively. In the multivariate analysis, age, transplant era, presence of MHC class I and II antibodies, and high urgency status but not HLA mismatches were independent predictors of long-term survival. Moreover, diagnoses other than dilated cardiomyopathy increased long-term mortality risk. In summary, our data demonstrate that HLA matching is not an independent risk factor for longterm survival in heart transplant recipients. However, several pretransplant factors and transplant era were independently associated with mortality risk.

Evidence against heterozygous coagulation factor V 1691 G-->A mutation with resistance to activated protein C being a risk factor for coronary artery disease and myocardial infarction.

The aim of our study was to determine the prevalence of the factor V mutation (position 1691 G-->A) in patients with angiographically diagnosed coronary artery disease and myocardial infarction and, as a control, in blood donors. This mutation has already been proved to be the main genetic risk factor for venous thrombosis. In order to detect this mutation in exon 10 of the factor V gene we established a microtiter plate based hybridization assay for the specific detection of wild-type and mutant sequences in factor V gene segments, obtained after amplification by polymerase chain reaction. This test enables us to screen a large number of samples. The mutation was detected in 29 of 317 coronary artery disease (CAD) patients (9.1%) and 18 of 190 blood donors (9.5%) investigated. The mean activated protein C resistance ratios were 3.18 and 3.11, with nearly identical distribution. No increased prevalence of the factor V mutation was found in the CAD group. In 10 of 29 CAD patients (35%) with the factor V 1691 G-->A mutation and in 124 of 288 CAD patients without the mutation (43%) there was a history of myocardial infarction. From our data we conclude that there is no increased risk of developing coronary atheroma or consecutive myocardial infarction resulting from the factor V mutation with protein C resistance.

Frequent detection of hepatitis B core antibodies in heart transplant recipients without preceding hepatitis B infection.

It is unclear whether heart donors positive for hepatitis B core antibodies (anti-HBc) can transfer hepatitis B virus (HBV) infection to immunosuppressed heart recipients, or whether passive transfer of anti-HBc simulates a hepatitis B infection. Therefore, we performed a case-controlled study in 46 heart recipients who all tested negative for hepatitis B antigen (HbsAg), antiHBc, and hepatitis B surface antibodies before heart transplantation. Twenty-three patients (group 1) received hearts from anti-HBc-positive donors, while 23 other patients (group 2) received hearts from anti-HBc-negative donors. After heart transplantation, anti-HBc were present in 65.0% of blood samples among group 1 and 47.8% of the blood samples among group 2 (P > .05). HbsAg was undetectable in blood samples of all patients of both study groups. The immunoglobulin preparation that we regularly use for immune suppression immediately after heart transplantation contained a relatively high concentration of anti-Hbc antibodies. The nearly identical presence of anti-HBc in both study groups indicated that passive transfer via immunoglobulin preparations rather than HBV infection is the cause for the anti-HBc detected in heart recipients. Since only a small volume of blood is transferred with the donor heart, it seems to be rather unlikely that the donor heart might be the source of anti-HBc. In summary, we observed no evidence for HBV infection in those heart recipients who received organs from anti-HBc-positive donors. Moreover, our data demonstrated that the presence of anti-HBc in heart recipients frequently occurs but does not necessarily indicate a preceding HBV infection.

The 536C-->T transition in the human tissue factor pathway inhibitor (TFPI) gene is statistically associated with a higher risk for venous thrombosis.

Tissue factor pathway inhibitor (TFPI) is an important regulator in the extrinsic blood coagulation pathway. Although the regulatory biochemical role of TFPI is evident, the clinical significance of this proteinase inhibitor remains to be elucidated. The definition of a clinical TFPI deficiency seems to be more complex than that of other coagulation inhibitors because the activity and concentration of circulating TFPI can not be considered a true measure of in vivo levels. Its determination in plasma samples by immunological methods or functional assays has been shown to be inadequate in the detection of a clinical deficiency. Therefore, we screened genomic DNA samples of blood donors and thrombotic patients for alterations in the TFPI gene to assess the influence of a modified TFPI in venous thromboembolic diseases. We detected a single nucleotide substitution in exon 7 (536C-->T) leading to a proline to leucine exchange at amino acid position 151 of the protein ([P151L]TFPI) and found the prevalence of heterozygous carriers in German unrelated blood donors to be 0.2% (n = 5120). Four unrelated persons out of 14 probands carrying the genetic variation could be linked to venous thrombosis. For calculation of a potential risk for venous thrombosis for carriers of the mutation we investigated healthy blood donors about thrombotic events. 7 out of 308 blood donors were found to have a history of venous thrombosis, one of them carried the TFPI mutation. Statistical calculation showed a significant relative risk for venous thrombosis for individuals with the trait (odds ratio, 9.3; confidence interval, 1.8-48.6; p <0.01).

Evaluation of a new reagent for preserving fresh blood samples and its potential usefulness for internal quality controls of multichannel hematology analyzers.

We describe a new, easy-to-use reagent, Cyto-Chex (Streck Laboratories, Omaha, Neb), that preserves fresh whole blood in a non-cross-linking, nonformalin manner. Target values assigned to fresh blood were essentially met after preservation and storage of up to 31 days. Respective mean analytic inaccuracies and short-term intra-assay coefficients of variation (n = 30) were as follows: WBCs, 6.7% and 1.99%; RBCs, 0.7% and 0.76%; hemoglobin, -1.8% and 0.79%; hematocrit, -0.3% and 0.75%; mean corpuscular volume, -1.0% and 0.78%; and platelets, 6.9% and 3.12%. Linearity of dilution-sensitive analytes was satisfactory over a wide range of dilutions after preservation of blood samples. Ten independent laboratories using 10 different instruments determined day-to-day interassay imprecision during four 7-day periods after blood preservation. Mean interassay coefficients of variation for participating laboratories were WBC, 1.92%; RBC, 1.00%; hemoglobin, 1.29%; hematocrit, 2.00%; and platelets 3.29%. Cyto-Chex enables long-term monitoring of instrument accuracy and precision with retained blood specimens of healthy persons. Blood from patient cohorts with various hematologic disorders and with a wide range of numeric abnormalities and/or parameter aberrations can be preserved satisfactorily with this reagent. The reanalysis of preserved patient blood samples is an important adjunct to the use of commercial control material in quality control programs of multichannel hematology analyzers.

Wound therapy with autologous bone marrow stem cells in diabetic patients with ischaemia-induced tissue ulcers affecting the lower limbs.

Previous studies suggest that autologous transplantation of bone marrow mononuclear cells is safe and effective in inducing therapeutic angiogenesis in patients with peripheral arterial occlusive disease (PAOD). Here we discuss a multidisciplinary approach to treating PAOD with a focus on the use of angiological diagnostic tools. We conclude that our autologous stem cell therapy is working in this patient and it is a potential new therapeutic option for diabetic patients with chronic foot ulcers induced by critical limb ischaemia.

Simple method for preparation of cryoprecipitate (CP) and cryodepleted plasma (CDP).

Cryoprecipitates (CP) and cryodepleted plasma (CDP) were prepared from fresh frozen plasma (FFP). Plasma was easily and cleanly frozen at -50 degrees C using a methanol-bath Ultracryostat, which has been available commercially for the past few years. From a random sample (n = 6), factors VIII:C, IX:C, V:C, fibrinogen, antithrombin III and fibronectin were determined. Concerning the total contents and the in-vitro-recovery of factor VIII:C (x:104 IU/53.5%) and fibrinogen x:175 mg/36.9%), the preparation method was as efficient as other equally common methods. Apart from the well-known applications, CP may be used for the substitution of fibronectin (x:46.2 mg/73.0%). The supernatant plasma of cryoprecipitation (CDP) can be utilized for substitution of coagulation disorders especially deficiencies of the prothrombincomplex and antithrombin III (x:IX:C:183 IU/76.2%; V:C:140.5 U/73.8%; AT III:162 U/80.2%).

High rate of false positives in blood donor screening for antibodies to hepatitis C virus. Cause of underestimation of virus transmission rate?

Anti-hepatitis C virus antibody screening of blood donors in different countries revealed prevalences ranging from 0.4-1.4%. These results were obtained with an enzyme immunoassay based on a recombinant hepatitis C virus antigen. We applied a specific inhibition assay (neutralization assay) and a recombinant immunoblot assay to determine the specificity of positive reactions in the enzyme immunoassay. Of 2836 blood donor sera tested, 10 (0.35%) were reactive in the enzyme immunoassay, however, only 3 sera (0.1%) proved to be specifically anti-HCV positive in the inhibition assay. The recombinant immunoblot assay gave similar results. The prevalence of anti-hepatitis C virus antibodies among blood donors has been overestimated in recent publications. Furthermore the high rate of false positives in the enzyme immunoassay may explain reports claiming that only a minor part of EIA positive blood units transmitted the hepatitis C virus to recipients. The inhibition assay was also applied to sera of haemophiliacs and of patients with hepatopathy which had reacted positively in the anti-hepatitis C virus antibody enzyme immunoassay. The anti-hepatitis C virus specificity was confirmed for all sera from the haemophiliacs group (100%) and for 77% of the hepatopathy patients group. Thus, the anti-hepatitis C virus enzyme immunoassay has a high predictive value when it is used to screen groups with high risks of parenteral hepatitis C virus infection, however, its predictive value is very low when it is used for blood donor screening.

Enzyme immunoassays for anti-hepatitis C virus antibodies improved specificity and analytical sensitivity by combination of three different recombinant viral proteins in second generation tests.

The detection of hepatitis C virus infection currently relies on a "virology without a virus" approach. So far, only viral nucleic acid has been isolated and sequenced by the methods of genetic engineering. The resulting viral sequence was then used to "design" proteins for diagnostic use as antigens in enzyme immunoassays (EIA). A first-generation EIA (EIA I), which uses a non-structural hepatitis C virus protein as antigen, detected 26 (0.6%) reactive sera out of a total of 4350 blood donors. An inhibition test using recombinant hepatitis C virus antigen, and EIAs using other, both synthetic and recombinant hepatitis C virus peptides were used as a specificity enhancing measure and as confirmatory tests, respectively. Only 7 of these reactives (0.16%, inhibition test) and 5 (0.11%, peptide EIA) were confirmed positive. Of the 26 initially reactive donor sera, 5 sera (0.11%) reacted positive in a second-generation anti-hepatitis C virus antibody EIA (EIA II), which uses two different recombinant non-structural hepatitis C virus proteins and one recombinant core protein. Seventeen (77%) of 22 haemophiliacs reacted positive in EIA I, and 19 (86%) did so in EIA II. There were no false positives in this cohort. Twenty-eight (19%) out of 148 liver disease patients showed a positive reaction in EIA I, and 31 (21%) were reactive in EIA II. Based on the results of the peptide enzyme immunoassay, 1 serum of this group was false positive in EIA I, while none of the sera of this group were false positive in EIA II.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction.

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.

Effect of cyclosporine A on the release of tissue factor pathway inhibitor from endothelial cells in heart transplant patients and cell culture.

We investigated the influence of cyclosporine A on the concentration of tissue factor pathway inhibitor and von Willebrand factor antigen in plasma of heart transplant outpatients. Tissue factor pathway inhibitor was quantified in plasma of blood donors (n = 50) and heart transplant outpatients (n = 50) by a chromogenic substrate assay with a mean of 32.4 micrograms/l and 98.2 micrograms/l, respectively. Von Willebrand factor antigen was determined with an enzyme-linked immunoassay with a mean of 90.9% for blood donors and 184.5% in plasma of heart transplant recipients. In addition, we investigated the effect of cyclosporine A on endothelial cell cultures over an incubation period of four days. A dose-dependent effect of cyclosporine A on the release of endothelial tissue factor pathway inhibitor and von Willebrand factor antigen was determined in a concentration range from 100 to 200 micrograms/l cyclosporine A. The tissue factor pathway inhibitor and von Willebrand factor antigen concentrations in the cell culture supernatant increased during the incubation time according to the cyclosporine A concentration 2-3 fold and 2 fold, respectively. For a further elucidation of the cyclosporine A effect we investigated the influence of cremophor EL, the vehicle of cyclosporine A. Cremophor EL alone did not increase the tissue factor pathway inhibitor release. However, the release was enhanced 2-4 fold after co-stimulation with the calcium ionophore A 23187 (10(-4) mol/l) in a concentration-dependent mode. We conclude that a generalized endothelial damage or activation is most probably caused by cyclosporine A and its vehicle cremophor EL. This process probably depends upon the increase of cytosolic free calcium, as described for the liberation of von Willebrand factor by endothelial cells.

[Fibrinogen and leukocyte number in coronary heart disease. Correlation with angiography and clinical degree]. / Fibrinogen und Leukozytenzahl bei koronarer Herzkrankheit. Korrelationen zum angiographischen und klinischen Schweregrad.

In 302 consecutively patients fibrinogen (Clauss method) and leucocyte count were related to the angiographic and clinical degree of coronary atherosclerosis. Fibrinogen (mg/dl) was statistically higher compared to control (C, 267 +/- 55) in patients with one-vessel disease (1-vd (306 +/- 67), 2-vd (331 +/- 73), and 3-vd (328 +/- 62)). Patients with coronary sclerosis (Scl, luminal irregularities and/or stenosis under 50%) showed a tendency to higher fibrinogen levels (291 +/- 58) as compared to controls. Leucocyte count (10(9)/L) compared to C (6.7 +/- 1.8) was significantly higher in 1-vd (7.6 +/- 2.0) and 2-vd (7.6 +/- 1.9). A subgroup analysis was performed with 100 patients having severe forms of angina pectoris (AP III according to the CCS classification, unstable angina). Hundred-sixteen patients with unstable angina (390 +/- 79), particularly with angina at rest during the last 48 h were characterized by the highest fibrinogen values (423 +/- 89, class III B/C Braunwald). Leucocyte count in patients with stable angina (7.2 +/- 1.4) and angina at rest (9.3 +/- 2.7) was significantly higher as compared to control (6.7 +/- 1.8). Hyperfibrinogenemia and relative leucocytosis correlate with the angiographic and clinical extent of coronary artery disease and may offer evidence of a higher degree of thrombogenesis associated with components of inflammation.

Immunoglobulin preparations from hepatitis C antibody-positive plasma donors: influence on diagnosis and risk of infection in heart transplant recipients.

All heart transplant patients in our clinic received intravenous immunoglobulins as a prophylaxis against cytomegalovirus infections or reactivations. Serum was sampled from 160 heart transplant patients within 4 months after surgery. In 98 samples (61%) hepatitis C virus (HCV)-specific antibodies could be detected by a "second generation" enzyme immunoassay. Of these HCV antibody-positive patients 89 were tested for a second time. At this time, 5-11 months later, in 66 patients (74%) the HCV antibody had disappeared. In the 23 still positively reacting patients, immunoglobulins were given in the last 6 months before serum sampling. Nine commercial immunoglobulin preparations were tested for HCV-specific antibodies and the presence of HCV RNA. Seven preparations were anti-HCV positive with titres in the range of 64-256, whereas reverse transcription and polymerase chain reaction did not detect HCV RNA in any immunoglobulin preparation. Passive antibody transfer rather than a HCV infection is the cause of HCV antibody detection in our patients. The presence of HCV antibodies in high concentrations in commercial immunoglobulin preparations may only be explained by an extremely high proportion of anti-HCV-positive single donations in the plasma pools used for immunoglobulin production. The passive HCV antibody transmission prevents anti-HCV serological monitoring of patients treated with these preparations. Additionally, there are reports on the transmission of hepatitis non-A, non-B via immunoglobulin preparations. Therefore, we recommend an anti-HCV screening of plasma donors.

Synthesis of tissue factor pathway inhibitor in human synovial cells and chondrocytes makes joints the predilected site of bleeding in haemophiliacs.

The synthesis of tissue factor pathway inhibitor (TFPI) was investigated in cloned human synovial cells and human chondrocytes. TFPI-specific DNA transcription products of these cells were isolated, and a full-length cDNA of about 1000 base pairs was amplified by reverse transcription and polymerase chain reaction. The amplified DNA was cloned into the vector pUC 18. The TFPI coding sequence was confirmed by double stranded sequencing and was identical with that previously published for human TFPI coding nucleotide sequence from human placental cDNA (1). The inhibitory activity of TFPI in the cell medium of cultivated human chondrocytes and cloned human synovial cells was determined by a specific chromogenic substrate assay of factor Xa activity. The inhibitory activity of TFPI in the medium of human chondrocytes and cloned human synovial cells was 630-720 mU/10(8) cells and 1080-1665 mU/10(8) cells, respectively. In addition, TFPI activity in cell culture media of human chondrocytes and cloned human synovial cell was suppressed by a polyclonal goat anti-TFPI antibody directed against the inhibitory domain I and domain II. In the chromogenic substrate assay, the anti-TFPI antibody completely suppressed the inhibitory activity of TFPI in the samples.