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Biomedical subjects

U Cassens

Publications and source records attributed to U Cassens.

33 records · Page 2Linked to original sources
PubMed1997

Efficacy and kinetics of bone marrow processing and enrichment of haematopoietic progenitor cells (HPC) by a large-volume apheresis procedure.

We investigated the efficacy of bone marrow (BM) processing by an automated large-volume apheresis procedure (6 x original BM volume) in 10 paediatric and adult patients undergoing BM harvesting before myeloablative therapy. Volume-dependent kinetics during apheresis were analyzed by sequential collection of processed cells into a six-fold collection bag system with consecutive analysis of the single bags. BM processing resulted in an 83.3% (+/- 21) recovery of mononuclear cells (MNC), a 97.9% (+/- 1.1) reduction of erythrocytes (RBC) and a 87.7% (+/- 2.9) volume reduction. To determine volume-dependent kinetics of haematopoietic progenitor cell (HPC) enrichment during apheresis, leukocytes (WBC), mononuclear cells (MNC), CD34 cells and colony-forming cells (CFU-GM) were serially quantitated in subsequent collection bags. Large-volume BM processing significantly enhanced absolute yields of CD34+ cells (mean: 4.01 (+/- 2.81) x 10(6)/kg bw) and CFU-GM (mean: 1.92 (+/- 1.47) x 10(4)/kg bw) compared with the standard procedure (3 x BM volume) by 26.9% (+/- 10.9) and 27.2% (+/- 11.6), respectively. We concluded that large-volume apheresis for BM processing is an efficient technique significantly improving the yields of haematopoietic progenitor cells (HPC) without any relevant changes in the purity of the final product. Moreover, sequential collection and analysis of HPC represents a good model to investigate the volume-dependent kinetics and efficacy of BM processing.

Adolescent↗
PubMed1996

Rapid quantitative analysis of protein tyrosine residue phosphorylation in defined cell populations in whole blood and bone marrow aspirates.

We developed a rapid method for quantification of tyrosine phosphorylation in immunophenotypically defined cell populations in specimens of whole blood and unprocessed bone marrow. Samples were formaldehyde-fixed and cells were permeabilized. Phosphotyrosine residues and surface antigens were simultaneously stained by monoclonal antibodies and visualized by flow cytometry. The accuracy of the method was confirmed by demonstration of an increase of phosphotyrosine levels in pp60v-src transformed fibroblasts. In blood of healthy donors, monocytes and granulocytes showed higher levels of phosphotyrosine than lymphocytes. CD34+ peripheral blood stem cells showed slightly increased tyrosine phosphorylation compared to autologous lymphocytes. Significantly elevated levels of phosphotyrosine were demonstrated in leukaemic blasts compared to lymphocytes (P = 0.01).

Acute Disease↗
PubMed1996

[Functional evaluation of erythrocytic antibodies by photometric detection of erythrophagocytosis in the monocyte monolayer assay].

In haemolytic disease of newborn (erythroblastosis fetalis) the in vivo behaviour of erythrocytic IgG antibodies is of particular significance. The monocyte monolayer assay (MMA), which determines by microscopy the number of erythrocytes phagocytosed by monocytes, is an important functional test for the qualitative assessment of erythrocytic IgG antibodies. We set up a photometric MMA and compared it with the microscopic MMA. In both tests we employed commercially available rhesus antibody sera and examined 8 sera of pregnant women with mild and severe courses of haemolytic disease of newborn by the photometric MMA. Good agreement was found between the microscopic and photometric MMA (r = 0.93). Over and above this the photometric MMA correlated with the course of haemolytic disease of newborn in 7 out of 8 cases (with one false-positive finding). The photometric technique permits rapid, sensitive and reproducible determination of erythrophagocytosis in microtitre plates. This method is based on the photometric detection of haemoglobin of phagocytosed erythrocytes via a peroxidase reaction. Standardised photometric MMA could in future be more widely applied especially in haemolytic disease of newborn for the functional characterisation of erythrocytic antibodies, the incidence of intra-assay and inter-assay errors being low.

Coombs Test↗
PubMed1996

Effect of storage conditions on the CD34+ counts in flow cytometric analysis after anticoagulation of samples with EDTA.

The transplantation of peripheral blood precursor cells (PBPC) is becoming of interest for autografting patients with a wide variety of haematological and other malignancies. For rapid quality control of PBPC apheresis products, flow cytometry is applied to quantify the number of CD34+ events. We studied the effect of different storage conditions on the number of CD34+ counts in EDTA-anticoagulated aliquots of PBPC grafts. Within 24 h, CD34+ signals decreased when samples were stored at room temperature (RT, 20 +/- 2 degrees C) compared to the results obtained directly after cytapheresis. The signal rate equalled or exceeded the baseline values after 24 h when aliquots were deposited at room temperature and subjected to agitation. Storage at 4 degrees C revealed no significant changes. These data indicate that quality control of PBPC samples by flow cytometry significantly depends on storages time, temperature and other conditions like the agitation of the specimen.

Adolescent↗
PubMed1996

Storage of peripheral blood stem cell samples alters flow cytometric CD34+ results.

With the use of monoclonal antibodies against the CD34 antigen, flow cytometry (FC) permits rapid assessment of the quality of hematopoietic grafts. We examined PBSC samples (n = 40) to investigate possible influences of storage time and temperature on FC results. After cytapheresis, a sample of the PBSC product was collected and divided into 4 aliquots. Immediate analysis was performed on one aliquot. The other 3 specimen were stored for a) 24 h at room temperature (RT, 20 +/- 2 degrees C), at room temperature with agitation and c) at +4 degrees C. For flow cytometric analyses, samples were labeled with two CD34 markers (HPCA-2, Becton Dickinson Corp. [BD], USA; QBend-10, Immunotech Corp. [IT], Germany). After 24 h CD34+ signals had decreased by 25.4% (BD) and 27.0% (IT) in average, when samples were stored at room temperature in comparison to the results obtained directly after cytapheresis (p < 0.05). At RT in combination with agitation, there was an increase in signal rates compared to baseline values, probably due to binding of CD34 antibodies to myeloid or non-viable cells (+ 9.2% [BD] and 11.2% [IT]). At a storage temperature of +4 degrees C, CD34+ events did not decrease significantly (-0.7% [BD] and -0.2% [IT]). Our data demonstrate that FC results may be influenced by temperature, agitation and storage time.

Adolescent↗
PubMed1996

Improved automated bone marrow processing and enrichment of CD34+ cells by a large-volume apheresis procedure.

We investigated the efficacy of bone marrow (bm) processing by a large-volume apheresis procedure using a self constructed sixfold collection bag system for sequential cell collection and analysis for 5 pediatric patients. Quantitation of leukocytes (WBC), CD34+ cells and colony-forming cells (CFU-GM) within the single bags showed a relative time-dependent decrease of all cell fractions during leukapheresis, whereas the relative amount of mononuclear cells (MNC) droped only slightly. At the same time the large volume apheresis (6 x original bm-volume) clearly enhanced the absolute yield of CD34+ cells compared to the standard procedure (3 x bm-volume) for more than 20%. We conclude that large-volume apheresis for bm processing is an efficient technique to improve the yields of progenitor cells.

Adolescent↗
PubMed1994

Active uptake of serotonin as viability parameter of stored platelets.

The platelet-plasma serotonin (5-HT) exchange in different apheresis products and platelet concentrates (PCs) from buffy coats was studied for quality control over a period of 7 days. In PCs with steadily decreasing pH due to inappropriate gas exchange the platelet 5-HT concentration increases significantly (> 10% positive netto uptake) during the 1st day of storage. Thereafter platelet serotonin drops rapidly with t1/2 = 1 day at pH < 6.5, but platelet counts were constant and 5-HT uptake was inhibited concomitantly. Plasma 5-HT increases as pH decreases. This can be enforced by competitive inhibition of serotonin uptake due to imipramine which also moderates the total 5-HT loss. However, in PC with stable pH the operative 5-HT pump keeps steady-state conditions. The efficacy of the transmembrane uptake mechanism can be adequately monitored by the assessment of the 5-HT platelet/plasma ratio as a new platelet viability parameter.

Blood Platelets↗
PubMed1994

Detection of HLA antibodies by an enzyme-linked photometric immune phagocytosis inhibition test.

The immune phagocytosis inhibition test (IPI) has been described as a sensitive method for the detection of cytotoxic and non-cytotoxic HLA antibodies. We performed a photometric IPI and compared this technique with the conventional microscopic IPI. In further investigations we used the photometric IPI in comparison with the lymphocytotoxicity test (LCT) for the detection of HLA antibodies. The photometric IPI showed a high correlation to the microscopic IPI. In tests with different known HLA antibodies the photometric IPI reached a sensitivity of 85.1% versus 80.5% in the LCT, and a specificity of 92.3 versus 100% in the LCT. Diluted patient sera showed a higher sensitivity in the photometric IPI. We conclude that the photometric IPI can be used as a convenient and sensitive technique for the detection of HLA antibodies.

Autoantibodies↗
PubMed1994

[Measurement of alloreactive anti-recipient helper T-cell precursors after allogenic bone marrow transplantation].

Acute graft-versus-host disease (GvHD) is a severe complication after allogenous bone marrow transplantation (BMT). The compatibility of major histocompatibility complex antigens (MHC) is the strongest stimulus for GvHD, which also occurs in patients with a genetically MHC-identical sibling donor. In such cases it would be helpful to recognize anti-recipient (interleukin-2-producing) T-lymphocyte precursors to detect a minor histocompatibility antigen (mH), which escapes from typing methods. To quantitate the alloreactive immune response initiating acute GvHD, we established the helper T-lymphocyte precursor test (HTL-p) as described by Theobald et al. in 'GvHD direction', using a limiting dilution assay. Serial dilutions of the donor peripheral blood mononuclear cells (PBMCs) were cultured for 14 days with constant numbers of stimulator PBMCs from the recipient, followed by an unspecific restimulation step with phytohemagglutinin (PHA) or specific restimulation with EBV-transformed blast cells from the recipient. The Il-2 production of specific T cells was assessed by addition of CTLL-16 cells, whose proliferation was measured by an ELISA. We suppose that the HTL-p test is a good tool for measuring the number of anti-recipient T-lymphocyte precursors as a predictive value for the intensity of GvHD.

Bone Marrow Transplantation↗
PubMed1994

[Enzyme immunoassay for the determination of serotonin in stored platelet concentrates].

A new enzyme immunoassay (EIA) was applied on measurements of serotonin (5-HT) in stored platelet concentrates (PC). PC were prepared by apheresis of two separators (Cobe, Fresenius) or from buffy coats and stored for a period of 7 days at 22 degrees C and agitating. Measurements were done in pellets and suspension medium (plasma). Detection of 5-HT was specific and sensitive within the bounds of 2 and 1,000 ng/10(9) PLT.

Blood Component Removal↗
PubMed1990

[Photometric detection of erythrocyte phagocytosis in the monocyte-macrophage assay].

The relative rate of red blood cell (RBC) in vitro phagocytosis by monocytes was measured photometrically by the diaminobenzidinehemoglobin reaction. Our results demonstrate, that the in vitro monocyte-macrophage assay (MMA) allows a specific and sensitive estimation of low RBC-phagocytosis rates. Therefore, the MMA is a useful tool for further characterisation of antibody-monocyte interaction and is of clinical importance in blood group antibody analysis.

Erythrocytes↗
PubMedDate not supplied

Quantification of PCR products using microparticles and flow cytometry.

The analysis of genetic biomarkers has become an important tool in clinical diagnostics. This includes the identification of disease-related genetic alterations, the detection of pathogenic infective germs on DNA or RNA level and the quantification of the expression of marker genes indicating an altered physiological status. It has been previously described that the combination of polymerase chain reaction (PCR), microparticles and flow cytometry represents a universal platform technology for the routine analysis of such biomarkers. Here we demonstrate the applicability and flexibility of this technology by means of various applications. The quantification of interferon gamma (IFNG) mRNA in irradiated white blood cells is shown as well as the detection of latent infections with cytomegalovirus (CMV). Besides the quantification of single amplification products, the flow cytometric assay is also capable of analysing products of a multiplex PCR. As an example, we describe the identification of spontaneous deletions in the genome of a hybrid cell line using a co-amplified gene (RAB1) essential for the cell survival as an internal control. Furthermore, we show that the use of a green laser (532 nm, 50 mW) substantially increased the sensitivity of the assay compared to conventional flow cytometers using a 488 nm (25 mW) laser. We conclude that the analysis of PCR products using microparticles and flow cytometry fulfils the criteria of clinical routine diagnostics regarding (i) sensitivity, (ii) specificity, (iii) reproducibility and (iv) automatibility.

Animals↗