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H Eichler

Publications and source records attributed to H Eichler.

At least 19 recordsLinked to original sources
PubMed2006

Introduction of a validation concept for a PCR-based Mycoplasma detection assay.

BACKGROUND: Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. METHODS: The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. RESULTS: The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. DISCUSSION: The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.

DNA, Bacterial↗
PubMed2006

Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries.

BACKGROUND: Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). METHODS: Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The leukocyte-containing fraction with the stem/progenitor cells was recovered from the filters by reverse flushing. Utilizing the routine traditional processing and testing procedures of each laboratory, in vitro parameters were determined, with samples obtained after collection, after processing and after freezing/thawing. The results were expressed as the percentage recovery of viable cells in processed vs. collected samples (performance 1; PF1) and in thawed vs. processed samples (performance 2; PF2). The composite results obtained by the seven laboratories were summarized. RESULTS: The median PF1 percentage recovery of total nucleated cells (TNC) was comparable with both traditional methods (HES 79%, T&B 86%) and statistically reduced with both filtration procedures (filter A 58%, filter B 61%). Mononuclear cell (MNC) PF1 recovery was highest statistically with the T&B method (91%) and reduced on using filter A (77%) and filter B (70%) and the HES method (72%). CD34+ cell recovery was judged to be essentially comparable with the four methods, although the range of unit recoveries differed. The percentage recovery of TNC and MNC in PF1 was influenced by the volume of the collected cord blood, especially with use of the filtration procedures. This correlated with TNC content. A greater percentage of red cells and platelets was removed during processing with both filter methods. The time to process cord blood preparations with filter A was significantly shorter than the other methods. Processing with the HES method took the longest time. The recoveries for TNC, MNC and CD34+ cells in PF2 did not appear to be influenced by the specific processing procedure. DISCUSSION: These data indicate that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.

Antigens, CD34↗
PubMed2005

Adult vascular progenitor cells and tissue regeneration in metabolic syndrome.

Adult vascular progenitor cells (for example endothelial progenitor cells, EPC) have been studied for their contribution to vascular repair and angiogenesis. These cells can differentiate from bone marrow cells as well as circulating cells carrying hematopoetic stem cell markers. In vivo, they take part in vasculogenesis in different animal models of limb ischemia, myocardial infarction and wound healing. In metabolic disease, the outgrowth and function of EPC in vitro is defective and numbers of EPC correlate with classical risk factors of cardiovascular disease suggesting a role of EPC in the development of vascular complications. Pilot studies for the treatment of myocardial infarction and limb ischemia with autologous bone marrow showed a distinct therapeutic benefit that is presumably mediated by vasculogenesis in damaged tissues. However, little is known about the nature of EPC and their capability to differentiate into functional cells for tissue regeneration. In this article, we review and discuss the hitherto identified physiological function of EPC, the mechanisms leading to dysfunction of these cells and potential therapeutic applications in patients with metabolic syndrome or diabetes mellitus and vascular complications.

Adult↗
PubMed2004

Comparison of total nucleated cell measurements of UC blood samples using two hematology analyzers.

BACKGROUND: The total nucleated cell (TNC) content of umbilical cord blood (UCB) units currently serves as the most important measure for determining suitability for transplantation. Hence it is important that TNC measurements are performed in an accurate manner. TNC content is evaluated routinely by hematology analyzers (HA) as WBC counts. The objective of the study was to compare TNC content utilizing two different HA, one utilizing an impedance channel and optical channel, and the other using only an optical channel. METHODS: The HA utilized in this study used two different modes of operation for lysis, regular mode (RM) and extended lysis mode (ELM). Cell-Dyn 3200 (CD3.2) utilizes optical technology for WBC measurements, involving WBC optical count (WOC) and nuclear optical count (NOC), whereas the Cell-Dyn 3700 (CD3.7) utilizes both the impedance (WIC) and optical technology (WOC) for WBC measurements. TNC content was determined with 17 identical samples using CD3.2 in one laboratory and CD3.7 in the other laboratory. Cord blood samples processed to concentrate nucleated cells by either of the laboratories were sent by overnight courier and assays were performed on the same day by both laboratories. RESULTS: For CD3.7, the WOC values were consistently lower than the WIC using the regular mode, but showed no significant differences (P>0.05). The WIC and WOC values were comparable on using the ELM and RM. For CD3.2, WOC values using RM and NOC values using ELM showed no significant differences (P>0.05), even though the WOC measurement was lower than the NOC values for most samples. The best comparison of TNC measurement between the two HA could be achieved by comparing CD3.7-WIC with CD3.2-NOC values. The results were equivalent (P>0.05) and 12 of 17 samples had equal to or less than 10% difference (mean 9.5%). DISCUSSION: TNC measurements of UCB samples were essentially identical using the WIC channel of the Cell-Dyn 3700 and the NOC channel of the Cell-Dyn 3200.

Erythrocyte Count↗
PubMed2004

Implementation of the INTERCEPT Blood System for Platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets.

BACKGROUND AND OBJECTIVES: The INTERCEPT Blood System for Platelets utilizes amotosalen-HCl (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet concentrates (PCs). To facilitate implementation of this technique into routine blood bank manufacturing procedures, this study evaluated the impact of different time settings of photochemical treatment on in vitro platelet function. MATERIALS AND METHODS: Platelets derived from apheresis (6.5-7.0 x 10(11) platelets) were resuspended in 240 ml of autologous plasma and 360 ml of platelet additive solution (PAS III) and split into two equal-sized PC units. Whereas one unit was not treated, the other was treated with 150 microm amotosalen and 3 J/cm2 UVA light followed by a compound adsorption device (CAD) step for reduction of residual amotosalen and photoproducts. In a first series of experiments (arm A, n = 7), PC units were photochemically treated after an overnight storage period of 16-23 h followed by a CAD step of 4 h. In a second series (arm B, n = 8), photochemical treatment occurred after a short storage time of 4 h with a subsequent CAD step of 16 h. Platelet function was evaluated by assaying blood gas analysis, glucose and lactate concentration, lactate dehydrogenase (LDH), hypotonic shock response (HSR) and the expression of CD62p, over a period of 7 days. RESULTS: Neither of the photochemical treatment procedures showed differences for pH, pCO2, pO2, HCO3, glucose consumption or platelet activation until the end of day 7. Increased lactate values detected for the treated units of arm A at the end of the storage period were independent from the PCT time setting. CONCLUSIONS: Photochemical pathogen inactivation with different initial resting periods between 4 and 23 h, and different CAD steps of 4 and 16 h, had no influence on the platelet in vitro function during 7 days of storage.

Adsorption↗
PubMed2003

Interventions for normal tension glaucoma.

BACKGROUND: Normal tension glaucoma is a clinical condition in which the optic nerve is pathologically excavated and the visual field is disturbed. Nevertheless it has been assumed that intraocular pressure plays a role in the progression of visual field defects in this disease, but other, mainly vascular factors, have been discussed as well. OBJECTIVES: The objective of this review is to assess the effects of medical and surgical treatments for normal tension glaucoma. SEARCH STRATEGY: Trials were identified from the Cochrane Central Register of Controlled Trials (CENTRAL) (which contains the Cochrane Eyes and Vision Group trials register), MEDLINE, EMBASE and BIOSIS Previews. Bibliographies of identified trials were searched to find additional trials. In addition, investigators and pharmaceutical companies were contacted. Date of last search: January 2001. SELECTION CRITERIA: This review includes randomised controlled trials in which medical or surgical interventions were compared to no treatment, placebo or other treatment in people with normal tension glaucoma. Two reviewers independently assessed the full text copies of the possibly relevant trials. Trial quality was assessed according to the methods set out in Section 6 of the Cochrane Reviewers' Handbook (Clarke 2000). DATA COLLECTION AND ANALYSIS: Data were extracted by two reviewers and results were compared for differences. Discrepancies were resolved by discussion. The heterogeneity of interventions, follow-up periods and outcomes did not allow for statistical combinations of the study results. MAIN RESULTS: According to the selection criteria on visual field loss, eight studies were included in this review. Only three studies focussed on patient relevant outcomes. In one trial a beneficial effect of lowering intraocular pressure was found, but only if data were corrected for cataract development. In two small studies a beneficial effect on visual field loss of brovincamine, a calcium antagonist was reported. REVIEWER'S CONCLUSIONS: In one study the effect of intraocular pressure lowering on visual field outcome was only significant when data were corrected for cataract development. The results for calcium antagonists are promising, but larger trials have to be performed. Studies that focussed on reduction of intraocular pressure or haemodynamic variables are not necessarily relevant for the outcome in people with normal tension glaucoma.

Antihypertensive Agents↗
PubMed2003

Effect of recombinant human deoxyribonuclease on the expression of cell adhesion molecules of thawed and processed cord blood hematopoietic progenitors.

BACKGROUND AND OBJECTIVES: The integrity of granulocytic cells and platelets is compromised within cryopreserved stem cell transplants, and consequent DNA release during the thawing procedure can therefore lead to clotting phenomena or microaggregate formation and that in turn may cause loss of progenitor cells. To circumvent this problem a new processing protocol was introduced using recombinant human deoxyribonuclease (rhDNase) to prevent cell aggregate formation. In addition, the impact of this new processing protocol on CD34+ umbilical cord blood (UCB) cells was assessed. MATERIALS AND METHODS: Fifty samples derived from 7 buffy coat (BC) volume reduced UCB units were cryopreserved, thawed, and processed with washing solutions that were supplemented with rhDNase in various concentrations. Thereafter, clotting and microaggregate formation was scored microscopically. In addition, expression of the adhesion molecules leukocyte function-associated antigen 1 (LFA-1, n = 6), intercellular adhesion molecule-1 (ICAM-1, n = 11), and L-selectin (n = 11) on CD34+ UCB cells was analyzed by flow cytometry after incubating the samples with either dimethyl sulfoxide (DMSO) 5.5%, rhDNase 10 or 50 U/mL, or a combination of DMSO 5.5% and rhDNase 50 U/mL. RESULTS: At a minimal concentration of 10 U rhDNase/mL, clotting or microaggregate formation could be prevented for all tested samples, whereas cell clots could be observed for concentrations up to 8 U/mL. The expression of adhesion molecules on untreated CD34+ UCB cells (L-selectin: 64.6 +/- 18.8%; LFA-1: 62.6 +/- 7.5%; ICAM-1: 14.8 +/- 4.1%) did not show any significant difference compared with cells that were incubated with up to 50 U/mL rhDNase (L-selectin: 62.2 +/- 19.3%; LFA-1: 63.1 +/- 5.9%; ICAM-1: 17.5 +/- 6.7%). However, after a combined treatment with DMSO 5.5% and rhDNase 50 U/mL, a slight but significant decrease in L-selectin expression could be observed (P < 0.03). CONCLUSION: The supplementation of rhDNase to a final concentration of 10 U/mL cell suspension proved to be effective in preventing clot formation under the conditions examined and did not lead to decreased expression levels of adhesion molecules. We therefore recommend the use of rhDNase for the prevention of clot formation and cell loss during the processing of thawed UCB transplants.

Antigens, CD34↗
PubMed2003

Tolerance of granulocyte donors towards granulocyte colony-stimulating factor stimulation and of patients towards granulocyte transfusions: results of a multicentre study.

Data of 507 granulocyte donations from 183 donors were evaluated. No severe granulocyte colony-stimulating factor (G-CSF)-related side-effects were observed. Three donors complained of severe itching following infusion of hydroxyethyl starch (HES). A high proportion (85%) of the donors stated that they would donate granulocytes again. The mean granulocyte yield was 4.3 x 10(10). High-molecular-weight HES resulted in a significantly higher yield compared with low-molecular-weight HES. Mild, but no severe, adverse transfusion reactions were observed in 16% of the recipients. A leucocyte alloimmunization rate of 24% was found. G-CSF stimulation and transfusion of G-CSF-mobilized granulocytes were well tolerated by donors and recipients, respectively.

Adolescent↗
PubMed2003

Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms.

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.

Amniocentesis↗
PubMed2002

Is prenatal HLA typing of uncultured amniocytes before the collection of related allogenic cord blood helpful?

The collection of related allogenic cord blood is gaining increasing importance in families with one child affected by haematopoietic disease. Within a family, there is only a 25% chance of a full HLA match between siblings. 50% of all collected cord blood samples cannot be used because of poor quality. Because of this, the determination of HLA type is useful for planning the collection of related allogenic cord blood transplants. We studied whether HLA typing is possible during late pregnancy if amniocentesis has not been performed during the first trimester. HLA -A, -B and -DRB loci were detected in amniotic fluid, as well as in corresponding cord blood and maternal blood using PCR-SSP. For the first time, HLA typing was performed from uncultured amniocytes. Unambiguous results were obtained from all samples. Fetal HLA-genotype in amniotic fluid was confirmed by typing results from corresponding cord blood. HLA typing of uncultured amniocytes during late pregnancy is a reliable and fast method. For the first time, prenatal HLA typing by amniocentesis after week 38 of gestation is possible in less than 8 h and without fetal risk.

Adult↗
PubMed2002

Use of recombinant human deoxyribonuclease (DNase) for processing of a thawed umbilical cord blood transplant in a patient with relapsed acute lymphoblastic leukemia.

This case report describes for the first time the use of a recombinant human enzyme deoxyribonuclease (rhDNase) containing solution for the processing of a thawed umbilical cord blood (UCB) unit prior to successful transplantation to avoid cell losses by clotting phenomena. A 6-year-old boy received an unrelated 2/6 HLA antigen mismatched UCB transplant for high-risk Burkitt type acute lymphoblastic leukemia. The UCB unit was provided as a volume-reduced sample after buffy coat separation with a final volume of 36 ml. To avoid the loss of nucleated cells due to cell clumping during thawing procedure cells were washed with a solution containing the rhDNase. No visible clotting of the resuspended unit occurred, and the patient was transplanted with 2.9x10(7) nucleated cells/kg body weight without any acute or chronic side effects due to rhDNase. On day +35, PCR analysis of bone marrow aspirate showed complete chimerism, and the child engrafted with an absolute neutrophil count greater than 0.5x10(9)/l on day +47. Platelet transfusion independence was achieved on day +120. In conclusion, the supplementation of rhDNase to the washing and resuspension solutions of a thawed UCB unit is effective to prevent cell losses prior to transplantation. However, further investigations must be performed to confirm the safety of this procedure.

Blood Transfusion↗
PubMed2002

Autologous concentrated platelet-rich plasma (cPRP) for local application in bone regeneration.

Platelets are rich in growth factors that may contribute to an accelerated tissue regeneration process. The therapeutic osteogenic effect of local platelet administration probably depends on the amount of growth factors delivered within. To improve platelet-derived factor preparations, the platelets have to be concentrated without loss of the granular growth factor load. An autologous procedure according to the Good Manufacture Practice (GMP) guidelines to prepare a high concentrate from platelet-rich plasma (cPRP) for clinical application in bone regeneration is presented. Platelet-rich plasma (PRP) was generated from the centrifugation supernatant of 250 ml anticoagulated autologous blood. The PRP was then further centrifuged at the day of operation to generate concentrated PRP (cPRP) in 8 ml plasma. The data from 34 patients (9 m, 25 f), ageing from 21 to 71 years (median 50.4) were evaluated. Both, the platelet counts as well as the TGF-beta1 concentrations in the cPRP were found to be 12-fold increased compared to the PRP values. Overall, a 17-fold increase in platelet concentrations was achieved compared to the patients' whole bloods and platelet counts correlated with increased levels of TGF-beta1 within the cPRP. These results give rise to quality control specifications for a safe and cost effective preparation of cPRP. The method has proven to be suitable for blood services to support the needs for treatment in oral and maxillofacial surgery.

Adult↗
PubMed2001

Prenatal HLA typing of uncultured amniocytes prior to the collection of related allogeneic cord blood.

DNA samples isolated from corresponding uncultured amniotic fluid, cord blood and maternal blood (n=5) were subjected to low resolution typing of the HLA-A, -B and -DRB loci by the polymerase chain reaction using sequence-specific primers (PCR-SSP). Furthermore, the effect of ethylene diamine tetraacetate disodium salt (EDTA) on the quality of genomic DNA isolated from amniotic fluid samples after long-term storage was evaluated. Unambiguous results of HLA typing could be achieved from all amniotic fluid samples stabilized with EDTA. PCR-SSP typing failed in DNA samples from amniotic fluid without the addition of EDTA. In all cases the fetal HLA type could be confirmed by the result from the corresponding cord blood typing. Contamination with maternal DNA led to additional weak PCR-SSP bands in one case, but data interpretation was still unambiguous. Reliable fetal HLA typing can be achieved directly from amniotic fluid and culturing of amniocytes is not required.

Amniocentesis↗
PubMed2001

Impact of plasmapheresis on platelet hemostatic capacity in healthy voluntary blood donors detected by the platelet function analyzer PFA-100.

Previous flow cytometry studies showed that platelet activation may occur during cytapheresis, cardiopulmonary bypass and hemodialysis. The aim of this pilot study was to determine if the impact of plasmapheresis leads to alterations in platelet hemostatic capacity. Plasmapheresis was carried out in 30 volunteers using an Autopherese C (Baxter) and an MCS3p (Haemonetics). Blood samples were collected and analyzed immediately before and after plasmapheresis. As a result of the plasmapheresis on the Autopherese C, the mean closure time (CT) for collagen/epinephrine (Col/Epi) increased from 118.2 +/- 25.1 to 149.9 +/- 35.0 s and from 88.7 +/- 16.9 to 98.5 +/- 26.3 s for collagen/ADP (Col/ADP), respectively. Seven subjects showed impaired CTs after plasmapheresis for Col/Epi. For Col/ADP, five subjects showed impaired CTs after apheresis. Statistical analysis (McNemar test) showed a significant difference before and after apheresis for Col/Epi (P = 0.024) but not for Col/ADP (P = 0.088). Similar results were shown for plasmapheresis carried out with the MCS3p. These findings show that plasmapheresis can cause an impairment of platelet function in healthy volunteers as measured by the PFA-100, an effect of so far unknown clinical significance.

Adult↗
PubMed2000

Cord blood as a source of autologous RBCs for transfusion to preterm infants.

BACKGROUND: This prospective study was conducted to gain experience as to whether it is technically possible to produce autologous RBCs in additive solution from cord blood (CB), to optimize the blood supply for preterm infants. STUDY DESIGN AND METHODS: CB was collected from 47 infants with a mean (+/- SD) birth weight of 1717 (+/- 699) g. Whenever possible, RBC components were prepared by standard centrifugation using a six-bag system. All samples were put in sterility testing quarantine for 5 days, and a maximum storage of 14 days from collection to transfusion was specified. The babies were given either the autologous RBCs or standard allogeneic RBC concentrates, if autologous blood was not available. RESULTS: In 81 percent of the samples, autologous RBC components could be processed (vol, 7-87 mL; Hct, 31-82%). But within the group of extremely low birth weight infants (body weight <1000 g), a mean CB net volume of only 37 mL was collected, and the RBC preparation was successful only in exceptional cases. Three CB samples (8.6%) tested positive in sterility testing. Of the 47 infants, 21 were treated with a total of 62 allogeneic and 4 autologous RBC transfusions. Most infants with a body weight over 1400 g did not need any RBC transfusion. CONCLUSION: The preparation of autologous RBCs from the CB of preterm infants is technically possible in principle. However, major concerns must be raised as to whether such preparations are of benefit in ensuring safe care of neonates with blood components, with respect to the high rate of bacterial contamination and the limited availability in babies with low birth weight.

Costs and Cost Analysis↗
PubMed1999

Novel Ras antagonist blocks human melanoma growth.

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both in vitro and in vivo in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5-50 microM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated ras genes or rely on Ras signal transduction more heavily than nonmalignant cells.

Animals↗