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E Seifried

Publications and source records attributed to E Seifried.

At least 37 records · Page 2Linked to original sources
PubMed2002

Paying for blood donations: still a risk?

It is presently disputed whether studies indicating a higher risk of infectious diseases among paid blood donors are lessons of the past, or still hold relevance. Comparative studies published between 1968 and 2001 were assessed for a possible trend of change in the relative risk for infectious disease markers between paid and unpaid blood or plasma donors. Studies reporting that paid donors had lower risk were found, but most studies, including recent ones, continued to report that paid donors have higher rates of infectious disease markers than unpaid donors. By log-linear regression analysis of the relative risk estimates for infectious disease markers among paid and unpaid donors from 28 published data sets, evidence was not found to indicate that the difference in risk for infectious disease markers between paid donors and unpaid donors had diminished over time (P = 0.128, not significant). Paid donors are still more likely than unpaid donors to donate blood in the period during which infectious donations escape detection by blood-screening tests (the "window-period"). Therefore, paid donations have a higher risk that labile blood components (such as red blood cells and platelets) are infected. Additional safety measures for handling plasma donations, and the preparation, purification and viral-inactivation steps employed for the production of plasma derivatives, may render the difference in infectious disease marker rates in donors irrelevant for plasma products. However, not all viruses are inactivated and paid donors were repeatedly found to have higher frequencies of markers for emerging agents. In a quality system, critical steps of the process should be addressed, and selection of the donor population is one of the first steps in this process. It is advised that blood establishments present yearly reports (with complete and raw data) to authorities on the incidence and prevalence of infectious disease markers among their donors as an ongoing surveillance on the "quality" of their donor populations. Paid blood or plasma donors still have higher rates for infectious disease markers than unpaid donors.

Blood Donors↗
PubMed2002

Generation and characterization of human hematopoietic cell lines expressing factor VIII.

Considering the plasticity of hematopoietic stem cells (HSC), they would be ideal targets for gene therapy of hemophilia A by virtue of their progeny providing immediate access to the bloodstream. However, several attempts to show expression of recombinant factor VIII (rFVIII) by primary hematopoietic cells and cell lines have failed; this failure was attributed to the inability of HSC to secrete rFVIII. Here we describe the generation of stable, FVIII-secreting hematopoietic cell lines representing different blood-cell types using a bicistronic lentiviral vector encoding for a B-domain-deleted FVIII (FVIII Delta B) and enhanced green fluorescence protein (EGFP). Transduced cell lines with erythroid and/or megakaryocytic background, (K562-F8 and TF-1-F8) secrete high levels of FVIII in the order of 76.4 and 41.6 ng FVIII:C/ml, whereas moderate and low levels are observed in B lymphoblastoid Raji-F8 cells and the T leukemia line Jurkat-F8 which secrete 6.73 and 1.83 ng FVIII:C/ml, respectively. The capacity to secrete rFVIII appeared to depend on factors related to the cell lineage rather than on the transduction efficacy. Stimulation of transduced cells with the protein kinase C (PKC)-activator phorbol myristate acetate (PMA) resulted in a marked augmentation of rFVIII secretion and enhanced green fluorescent protein (EGFP). Incubation with 0.1 and 1 ng/ml PMA resulted in up to 2.7-fold (K562-F8, Raji-F8) and 1.8-fold (293T-F8) increased rFVIII secretion. The established cell lines should be helpful in further elucidating mechanisms that are able to improve FVIII secretion in hematopoietic cells on a post-translational level and suggest reanalysis of hematopoietic cells as target for gene therapy of hemophilia.

Base Sequence↗
PubMed2001

Protection against severe disease is conferred by DERAA-bearing HLA-DRB1 alleles among HLA-DQ3 and HLA-DQ5 positive rheumatoid arthritis patients.

Experimental studies in transgenic mice have suggested that HLA-DQ predisposes to rheumatoid arthritis (RA), but could also modulate disease severity by presenting peptides derived from self-DR molecules. In particular, a short amino acid sequence, (70)DERAA(74), in the third hypervariable region of HLA-DRB1 confers protection for the disease, while particular HLA-DQ [DQB1*0501/DQA1*01 (DQ5) and DQB1*03/DQA1*03 (DQ3)] molecules predispose to the disease. We have therefore analyzed the allelic distribution of HLA-DRB1, DQA1, and DQB1 and the presence of rheumatoid factor and nodules among 199 German RA patients and 196 healthy controls. Our results show that HLA-DQB1*03/DQA1*03 (or DRB1*04) predisposes to RA more than HLA-DQB1*0501/DQA1*01 (i.e., DRB1*01 and DRB1*10). Homozygosity for DQ3 confers the strongest genetic risk for RA (OR = 19.79 compared to OR = 10.05 for two doses of shared epitope (SE) positive HLA-DRB1 alleles). Furthermore, patients carrying both predisposing DQ and (70)DERAA(74)-positive HLA-DRB1 alleles are more often rheumatoid factor (RF) negative than patients carrying predisposing DQ alleles alone. Only one out of 14 patients (7%) with a protective combination (DQ3/(70)DERAA(74) and DQ5/(70)DERAA(74)) had rheumatoid nodules compared to 67 out of 144 patients (46.5%) with predisposing DQ alleles alone (OR = 0.12, 95% CI: 0.02-0.72, p = 0.004). These results demonstrate a protective role of (70)DERAA(74)-positive DRB1 alleles against disease severity among RA patients.

Alleles↗
PubMed2001

Yield and future issues of nucleic acid testing.

Despite the much lower actual yield than that estimated for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) nucleic acid testing (NAT)-only positives in the USA and Germany, look-back procedures have revealed that no HCV transmission has occurred in Germany since the introduction of NAT. This indicates sufficient sensitivity of the pool-PCR approach. The slow ramp-up of hepatitis B virus (HBV) however, may require a different approach. It has been shown in Germany that the pooling of samples followed by virus enrichment results in a significant yield. Single donation testing for HBV would not increase the yield, because virus enrichment from mini-pool results in a similar sensitivity to that of single donation testing. Both strategies may be useful for extending future NAT to HBV screening. New candidate viruses for NAT are Parvo B19 and hepatitis A virus (HAV) because of their extreme resistance to inactivation procedures. Their low pathogenicity and epidemiologic characteristics, however, make them candidate viruses only for pooled source plasma. The main future issues of NAT will be related to the automation of pooling, extraction and amplification as a single homogeneous process. Depending on the throughput, automated single donation NAT as demonstrated by the 'Tigris' system may be an option, as far as all transfusion-relevant viruses will be included. In the near future high throughput systems will rely on pooled donor samples, most probably in conjunction with efficient enrichment procedures. For these systems, automation of the extraction and amplification process will be one of the first steps. These procedures will also limitthe costs of NAT and keep it available for use with future candidate viruses.

Biological Factors↗
PubMed2001

Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92.

For years activated natural killer (A-NK) cells have been explored with respect to their efficacy in anticancer therapy, but, except for some anectdotal reports, no clear clinical benefit has been shown. However, as the understanding about the interactions of NK cells and tumor cells advances, the use of A-NK cells might be revisited with more sophisticated approaches that pay tribute to mechanisms which allow tumor cells to escape immune surveillance. Here the highly cytotoxic NK cell line NK-92 seems to be an attractive alternative for use in adoptive immunotherapy, because it was shown to exhibit substantial antitumor activity against a wide range of malignancies in vitro as well as in xenografted SCID mice. NK-92 cells are characterized by an almost complete lack of killer cell immunglobulin-like receptors (KIRs) yet conserved ability to perforin and granzyme B-mediated cytolytic activity, which make them unique among the few established NK and T cell-like cell lines. NK-92 is the only natural killer cell line that has entered clinical trials. Here we discuss the current status of development of this cell line for adoptive immunotherapy (AIT) of malignancies and review our first clinical experience in patients with advanced cancer who have received repeated transfusions of irradiated NK-92 in a phase I/II trial. Also we discuss issues that address safety aspects of immunotherapy with clonal cell lines and describe further manipulations, which hold the potential of significantly improving the clinical outcome of AIT with NK-92.

Cell Culture Techniques↗
PubMed2001

TaqMan 5'-nuclease human immunodeficiency virus type 1 PCR assay with phage-packaged competitive internal control for high-throughput blood donor screening.

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5'-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5'-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5'-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a >or=95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1.

Blood Donors↗
PubMed2000

Platelet glycoprotein expression in patients with myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are characterized by a haematopoetic insufficiency that can lead to acute leukemia. A multistep pathogenesis caused by a clonal stem cell defect affecting several differentiation pathways has been proposed for MDS. Contrary to the better characterized alteration of lymphoid and myeloid differentiation, defects in thrombocytopoesis in MDS remain less clear. In the present study, we analyzed the expression of platelet glycoprotein (GP) Ia/IIa, IIb/IIIa, Ib/IX, and IV in 21 MDS patients (12 RA, 2 RARS, 4 RAEB, 1 RAEB-T, 2 CMML) and healthy controls by flowcytometric analysis and quantitation of platelet GP RNA using fluorescence-based PCR. We observed a reduced cell surface expression of GPIb (p<0.01) and GPIIb/IIIa (p<0.01), while GPIa/IIa and GPIV expression was only marginally different between patients and controls. In contrast, there was a two-fold increase of platelet GPIb and GPIIb RNA and a three-fold increase of GPIV RNA among MDS patients. Increased levels of platelet GPIb and GPIIb RNA were significantly more prominent among patients with RAEB(-T)/CMML (p<0. 05) in comparison to patients with RA/RARS. In conclusion, we demonstrate alterations in the cell surface expression and RNA content of platelet GPs in MDS patients. These data are consistent with dysmegakaryocytopoiesis and a defect in thrombocytopoiesis among MDS patients resulting from the clonal stem cell defect in MDS.

Adult↗
PubMed2000

Detection of nucleic acid sequences from bacterial species with molecular genetic methods.

While blood products become more safe in terms of viral contamination, the risk of transfusion-related bacterial infection has re-emerged to one of the major hazards in transfusion medicine. In recent prospective studies the rate of contaminated platelets ranged from 0.04 to 0.5%, and a rate of transfusion reactions between 0.007% and 0.046%. It is generally agreed that most of the organisms isolated from donated blood originate from the normal skin flora or from the environment. As it is unlikely that antiseptic methods can achieve absolute sterilization of the skin before venepuncture, blood banks have to rely on laboratory tests to detect contaminated blood products before release. But most of the currently available methods detecting bacterial contaminations do not have the potential to be sensitive and fast enough for a routine contamination screening in transfusion services. Here we present two alternative strategies based on molecular genetic techniques (Real-Time-PCR and Haystack processing) that detect or semi-quantify bacterial rRNA gene sequences for the majority of bacterial species. In addition we discuss some aspects on target selection, routine preparation and residual 16S-rDNA-contamination of enzymes.

Bacteria↗
PubMed2000

Iron-overload and genotypic expression of HFE mutations H63D/C282Y and transferrin receptor Hin6I and BanI polymorphism in german patients with hereditary haemochromatosis.

Gene variations of HFE, a HLA-class I like molecule, are highly associated with hereditary haemochromatosis (HH). Functional as well as molecular studies of the HFE protein have indicated that the molecule is involved in iron metabolism and that the HFE gene variations observed among HH patients affect its interaction with the transferrin receptor (TfR). In the present study, we have therefore analysed the relationship between the HFE gene variants, C282Y and H63D, and body iron status among 85 German HH patients. In addition, two TfR gene polymorphism, TfR-Hin6I and TfR-BanI, were typed that have been reported to define ethnically distinct haplotypes. As controls we used 251/159 healthy German blood donors. Seventy-eight (92%) patients were C292Y homozygous, the H63D mutation was present in five (6%) patients with none of the patients being H63D homozygous. Serum transferrin, transferrin saturation and liver iron content were determined prior to therapeutic intervention. Among C282Y homozygous patients serum ferritin levels (2294 +/- 3174 vs. 463 +/- 224 microg L-1, P < 0.0001) and transferrin saturation (86 +/- 18% vs. 62 +/- 25%, P = 0.048) were elevated significantly compared with C282Y and/or H63D heterozygous patients. In addition, the liver iron content (291 +/- 165 vs. 138 +/- 95 micromol g-1, P = 0.028) and liver iron index (6.4 +/- 2.8 vs. 3.2 +/- 2.3, P = 0.019) were increased among C282Y homozygotes compared with C282Y heterozygotes. In contrast, no difference was observed between patients and controls regarding the distribution of TfR-Hin6I and TfR-BanI alleles. These data indicate that the iron intake is higher among C282Y homozygous patients compared with C282Y heterozygous or C282Y/H63D compound heterozygous individuals and supports the functional role of the HFE protein in iron metabolism whereas the TfR gene variants seem to have no influence on iron uptake.

Blood Donors↗
PubMed2000

Evaluation of a new PCR assay with competitive internal control sequence for blood donor screening.

BACKGROUND: High-throughput nucleic acid testing for transfusion-relevant viruses by PCR requires contamination-proof methods with high sensitivity and validity. A new PCR reagent kit (TaqMan, PE BioSystems) reduces the risk of carry-over contamination by eliminating post-PCR processing. STUDY DESIGN AND METHODS: Oligonucleotide design was done with software specialized for designing the assays' (TaqMan) primers and probes. A template-derived competitive internal control sequence designed through site-directed mutagenesis was used to reveal failures in amplification. Assay sensitivity was determined for single-donor and single-patient testing and by spiking sample mini-pools. Three seroconversion panels were tested. RESULTS: Sensitivity is high, reaching 300 HBV genomes per mL of single-patient material on direct testing. A detection limit of 1000 HBV genome equivalents per mL of donor plasma is achieved for 96 pooled samples. The window period for HBV infection was reduced by 17, 10, and 63 days from that for HBsAg screening in three seroconverting donors. CONCLUSION: The assay provides sufficient sensitivity to be superior to HBsAg screening in transfusion medicine and will be useful in clinical laboratories because of its ease of handling.

Blood Donors↗
PubMed2000

Loco-regional thrombolysis for deep vein thrombosis: fact or fiction? A study of hemostatic parameters.

Loco-regional thrombolysis for deep-vein thrombosis (DVT) has been claimed to be equally effective and safe compared with systemic thrombolysis. It is not known whether a loco-regional thrombolytic effect exists and of what it might consist. To investigate this issue, we studied eight patients with DVT undergoing loco-regional thrombolysis with 20 mg alteplase infused over 4 h in a dorsal foot-vein of the affected leg, while the leg was kept tightly bandaged; alteplase infusions were repeated every 24 h, the number of therapy cycles (TC) was seven, and full-dose heparin was given. For coagulation analyses, 'loco-regional' blood samples were taken from a vein of the affected leg and 'systemic' samples were taken from an antecubital vein. After a median number of six TC, good partial reperfusion was achieved in 4/8 patients, moderate partial reperfusion in 2/8, major bleedings occurred in 2/8, and minor bleedings in 1/8 patients. During the first TC, recombinant tissue-type plasminogen activator (rtPA) activity and antigen, as well as FgDPs and d-dimers, were elevated significantly loco-regionally over systemic values, and a complete breakdown of plasmin-inhibitor activity occurred with only a slight systemic reduction; no other differences were found. During successive TC, differences in rtPA-activity and -antigen levels decreased, and no significant differences were found for all other parameters. Thus, a local fibrinolytic effect was demonstrable during loco-regional thrombolysis for DVT; the magnitude of this effect diminished during successive TC, giving rise to the hypothesis that the fibrinolytic efficacy may be decreased due to growing, antifibrinolytic activity. The preserved, loco-regional plasmin-inhibitor activities during the later TC, in contrast to the complete breakdown during the first TC, suggest that part of the enhanced antifibrinolytic activity is due to loco-regionally increased plasmin-inhibitor activity. The ultimate goal of loco-regional thrombolysis, the induction of local fibrinolysis without systemic effects, has not, however, been achieved.

Adult↗
PubMed2000

NAT and viral safety in blood transfusion.

BACKGROUND AND OBJECTIVES: Nucleic acid testing was introduced at our blood transfusion service in order to reduce the diagnostic preseroconversion window for transfusion relevant viruses. MATERIALS AND METHODS: Up to 96 donor samples were pooled overnight by two Tecan Genesis RSP 150 pipettors. Pools were centrifuged at 48,000 g for one hour to enrich viruses. Viral nucleic acids were extracted from centrifugation pellets using modified Qiagen viral RNA kit. HIV and HBV sequences were amplified by in house TaqMan PCR's with patented primers and probes for HBV. HCV PCR was performed by Roche Amplicor V2.0. "Nadis" software was developed for pooling, resolving positive pools and data communication with main frame computer. RESULTS: PCR testing was introduced in January 1997 for HCV HBV and HIV-1 for all plasma products and for labile components at the 21st of April 1997. Throughput increased from 2000 to 4000 samples per day. PCR testing is done in parallel to serological testing and products are released eight hours after pooling is completed. Until January 2000, 1,078,940 donations have been tested in 13,274 pools. A total of seven PCR only positives were identified (3 HCV, 3 HBV, 1 HIV). CONCLUSION: The yield of PCR testing for transfusion relevant viruses confirms theoretical estimates on the residual risk of antibody screening.

Blood Donors↗
PubMed1999

Feasibility and efficacy of routine PCR screening of blood donations for hepatitis C virus, hepatitis B virus, and HIV-1 in a blood-bank setting.

BACKGROUND: Despite sensitive antibody-based blood-donor screening, a residual risk of transfusion-transmitted viral infections exists. Only direct monitoring by sensitive nucleic-acid tests would provide data accurately to measure the risk and to assess risk-reduction procedures. We investigated the feasibility and efficacy of routine screening of donors for hepatitis C virus (HCV), hepatitis B virus (HBV), and HIV-1 by PCR. METHODS: For PCR testing, individual donor plasma samples were pooled (96x100 microL) overnight by two automatic pipetting machines. Viruses were concentrated by centrifugation and nucleic acids were extracted. HCV PCR was done on the Cobas Amplicor system (Hoffmann-La Roche, Mannheim, Germany). HBV and HIV-1 sequences were amplified by single (non-nested) in-house PCRs and detected by agarose-gel electrophoresis. Detection limits were 1000-5000 genome equivalents/mL in the donor blood. FINDINGS: PCR testing was done in parallel to antibody screening with a maximum throughput of 3000 samples in 7-8 h. Positive samples were identified 1-2 days later. 111 of 373,423 donations (107 of 4500 pools) were PCR and antibody/antigen-confirmed positive. We found one HCV PCR-positive antibody-negative donation with normal alanine aminotransferase and one HCV PCR-positive donation with an elevated alanine aminotransferase (100 IU), which was negative in the AxSYM 2.0 and Matrix 1.0, but positive after control in the Abbott Prism test (Abbott GmbH, Wiesbaden, Germany). INTERPRETATION: PCR is a suitable and fast blood-donor screening procedure and contributes to a reduction in viral transmission by transfusion of blood components. In our selected donor population, the yield of detected contaminated donations from donors in the time window in which they are highly infectious but do not have any symptoms or detectable antigen and antibody concentrations (diagnostic window), confirms theoretical estimates.

Blood Banks↗
PubMed1999

Sequence analysis and population data of short tandem repeat polymorphisms at loci D8S639 and D11S488.

Short tandem repeat loci are ideal markers for forensic and paternity case work. A high degree of polymorphism, as determined by gross length measurement, is very often due to complex underlying sequence variation. In the present study, we have studied the sequence structure and population genetics of two short tandem repeat polymorphisms at loci D8S639 and D11S488 in German Caucasians from the region of Hesse. Sequence data revealed a considerable polymorphism at both loci. Locus D8S639 is characterized by a tetranucleotide (AGAT)n repeat pattern with (GAT) and (AGGT) repeats dispersed throughout several alleles. These microvariations lead to alleles differing by one base pair or alleles of identical size. At locus D8S639 we observed 17 allelic lengths comprising 25 different alleles. Alleles at locus D11S488 possess a compound repeat region consisting of (AAAG)n and (GAAG)n repeats. At locus D11S488 we observed 15 allelic lengths with a total of 24 alleles. Allelic lengths increased in size by 4bp increments corresponding to the addition of one tetranucleotide repeat unit. Population data of loci D8S639 and D11S488 revealed a high polymorphism with heterozygosity rates of 0.85 (D8S639) and 0.91 (D11S488).

Alleles↗