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In this preclinical evaluation we have compared the efficacy of three clinical CD34+enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34+ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88–98%), 86% (62–97%), and 49% (18–85%), and median yield of 65% (54–100%), 40% (21–74%), and 30% (8–55%), respectively. Subset analysis recognised a selective loss of CD34+/61+ after enrichment, most likely due to class I–II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2–4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between... [ABSTRACT FROM AUTHOR]

Primitive haemopoietic cells are required for studies in both the clinical and research fields and a number of systems have been developed to facilitate isolation of these haemopoietic cell populations. We have analysed the results from several European centres using positive selection of CD34+ cells from haemopoietic tissues (n = 110). Four selection techniques including immunoaffinity columns (Ceprate LC), immunomagnetic beads (Dynabeads, Baxter Isolex 50) and submicroscopic magnetic beads (MACS) were used and the selected CD34+ cells were assessed for purity, yield and enrichment of colony-forming cells (CFC). The mean purities for all samples ranged from 68.4–78.4% for MACS, 33.9–69.9% for Dynabeads, 46.9–66.8% for Ceprate LC and 43.2–65% for Baxter Isolex 50. Yields were variable with all techniques. On average CFC enrichment using the immunoaffinity columns was greater than that observed for the other systems. Some techniques appear to be problematic and may require further expertise to improve the results. Nevertheless, the study demonstrates that highly purified CD34+ cells can be isolated from various haemopoietic sources, though yield and CFC enrichment varies significantly depending on the technique selected. This extends our previous report indicating that not all selection methods generate similar results and that there are differences in the purity, number and colony-forming ability of the cells recovered. [ABSTRACT FROM AUTHOR]

Stable and metastable phase equilibria in the binary system GeSe2-Se have been investigated by means of optical and electron microscopy, electron probe microanalysis (EPMA), x-ray diffraction (XRD), thermal analysis, and adiabatic calorimetry. A revised equilibrium phase diagram is presented. A not previously observed λ-type phase transition was observed near ∼420 K (147 °C) for GeSe2. The intermediate phase, φ, which earlier had been considered as metastable, is shown to be stable through extensive annealing experiments and by adiabatic calorimetry. The φ phase, with composition near Ge3Se7, melts incongruently at 658±5 K (385±5 °C) to GeSe2 and a liquid with 85±2 at.% Se. The liquidus of GeSe2 and φ was determined by EPMA of annealed-and-quenched samples. Both a stable (φ-Se-liquid) and a metastable (GeSe2-Se-liquid) eutectic point were observed. The stable one was observed at 480±2 K (207±2 °C) and 92.5±1.0 at.% Se, whereas the temperature of the metastable eutectic (GeSe2-Se-liquid) was observed to be ∼10 K lower. Metastable phase equilibria, obtained on thermal evolution of glasses with different compositions, are described and discussed. [ABSTRACT FROM AUTHOR]

The content of stem cells was analysed in bone marrow samples from 75 multiple myeloma patients. In unstimulated bone marrow the percentage of CD34+ cells was significantly reduced in 11 patients previously treated with melphalan-prednisolone (MP)(median= 0.15%) compared to median 0.87% in 31 untreated patients (P=0.0001). The bone marrow cellularity in the two groups did not differ. There was no correlation between the number of courses or total dose of melphalan and content of CD34+ cells in the bone marrow. The clonogenicity as well as the ability to expand the marrow stem cell pool during growth factor treatment were also reduced in MP treated patients compared to untreated patients. Analysis of different subsets of CD34+ cells revealed no influence on the pre B cell compartment in the bone marrow by MP treatment, but the committed stem cells (CD34+CD38+) were reduced more than the uncommitted stem cells (CD34+CD38-) in the MP treated group compared to the untreated patients. Mobilisation to and harvest of total number of CD34+ cells from peripheral blood was also reduced in the MP treated group. There was, however, no difference in the distribution between CD34+CD38+ and CD34+CD38- populations in the leukapheresis products in the untreated and the melphalan-treated group, suggesting selective mobilisation of CD34+CD38+ cells and/or differentiation of CD34+CD38-cells during growth factor stimulation. We conclude that melphalan decreased the number of stem cells in the bone marrow, the ability to expand the stem cell pool and mobilise stem cells to the pheripheral blood. [ABSTRACT FROM AUTHOR]

Low-frequency electrostatic turbulence generated by the ion–ion beam instability was investigated experimentally in a double-plasma device. Real time signals were recorded and examined by a conditional statistical analysis. Conditionally averaged potential distributions reveal the formation and propagation of structures with a relatively long lifetime. Various methods for making a conditional analysis are discussed and compared. The results are discussed with reference to ion phase space vortices and clump formation in collisionless plasmas. [ABSTRACT FROM AUTHOR]

Determinations of committed haemopoietic progenitor cells, namely CFU-GM (colony-forming unit–granulocyte/macrophage) and of CD34-expressing haemopoietic cells as assessed by multiparameter flow cytometry are routine diagnostic tools in haemopoietic cell therapy. Generally, the tests are used to optimise the timing and management of cytapheresis and to assess the engraftment potential of the harvested cells. Both measurements, however, are at best surrogate markers, as an adequate routine test which effectively assesses the short- and long-term repopulating haemopoietic cell is not available. Nonetheless, cell threshold doses have been established. Above these thresholds rapid engraftment is almost invariable but below these thresholds the outcome is variable. In this study we have focussed on the imprecision in counting haemopoietic cells, as assessed as CFU-GM and as CD34-expressing cells. The data on both tests have been analysed from six European institutions. The coefficient of variation in CFU-GM colony counting was about 30%, whereas the coefficient of variation in flow cytometric counting of CD34-expressing cells was about 10%. These data suggest that the technical imprecision in enumerating progenitor cells, particularly CFU-GM, at low levels, might make a major contribution to the clinical variability observed after transplantation of sub-threshold progenitor cell dose. [ABSTRACT FROM AUTHOR]

In an attempt to optimise stem cell graft evaluation we have developed a method of quantifying the number of cells in a phenotypically defined population of cells, expressing a gene of interest by combining an RT-PCR method working on whole single cells with flow cytometry. The clinical potential is illustrated by two examples. First, the phenotypes of clonal cells in the bone marrow (BM) of a patient with multiple myeloma (MM), were determined by sorting cells phenotypically defined by their expression of surface antigens and then performing RT-PCR on the individual sorted cells using the rearranged immunoglobulin heavy chain (IgH) gene as clonal marker. All plasma cells with the phenotype CD38++/CD45RA- expressed the clonal marker, whereas it could not be detected in plasma cells with the phenotype CD38++/CD45RA+. A minor population of clonal cells with the CD38+/CD45RA- phenotype was found. Second, the number of committed (CD34+/CD38+) and non-committed (CD34+/CD38-) stem cells, expressing the chimeric fusion gene p210 BCR/ABL in the autograft from a patient with chronic myeloid leukemia (CML), was determined. The number of cells expressing BCR/ABL mRNA was nearly equal in the CD34+/CD38+ and CD34+/CD38- compartment (8.1 and 8.5%). The method presented can easily be applied to determine the phenotype of malignant cells, where a unique mRNA species exist. Furthermore, the method allows one to predict the outcome of antibody mediated purging experiment. [ABSTRACT FROM AUTHOR]

Interleukin 2 (IL-2) was shown to induce a small but significant increase in the level of cGMP after 20 min sumulation and a subsequent fall after 1 h in activated T lymphocytes. No change in the level of cAMP was observed. Addition of the cyclic nucleotide analogues dbcAMP or dbcGMP did not stimulate DNA synthesis. On the contrary, IL-2-induced [3H]thymidine incorporation was inhibited by these drugs. Further, the phosphodiesterase inhibitor theophyllin inhibited proliferation of activated T lymphocytes. Our results indicate that neither cAMP nor cGMP act as ‘second messengers’ for IL-2 but support the theory that cAMP is a negative regulator of cell proliferation. [ABSTRACT FROM AUTHOR]

In a study of the role of Ca-+ in the stimulation of activated T lymphocytes with interleukin 2 (IL-21) it was found that lL-2-indueed proliferation can occur independently of extracellular calcium. Further, there was no correlation between triggering of DNA synthesis and an increase in free cytoplasmic calcium. However, lL-2 induced an increased uptake of 45Ca-+ from the extracellular medium. Since there is no increase in free cytoplasmic ealcium, it must be assumed that this is caused by an increase in membrane-associated calcium. Further, the calcium channel-blocking agent, verapamil, and TMB-8, a putative inhibitor of mobilization of calcium from intracellular pools, both exerted a dose-dependent inhibition of IL-2-induced DNA synthesis in activated T lymphocytes. We conclude that calcium is not a second messenger in activated T lymphocytes stimulated by IL-2, but our results indicate that calcium may play a role at membrane level. [ABSTRACT FROM AUTHOR]

We have investigated the ability of allogeneic, irradiated T lymphocytes to induce proliferation and immunoglobulin (Ig) secretion in untreated peripheral blood B lymphocytes. Non-mitogen-activated co-cultures of isolated T and B lymphocytes from selected, full-house HLA-A,B and D/DR antigen-phenotyped donors were reconstituted in a ratio of 4:1. Proliferation was assessed on day 5-6 of culture by the ³H-thymidine incorporation technique, and the Ig secretion was monitored on day 6 with a protein A plaque-forming cell (PFC) assay. B lymphocytes were able to diffferentiate into PFC, and the number of plaques was significantly higher in cultures of cells with two HLA-D/DR antigen incompatibilities than in those sharing one antigen. In cultures of peripheral blood lymphocytes with no HLA-D/DR antigen difference, only a few PFC developed. HLA-A and B antigens had no influence on the response. Further, monocytes were not an absolute requirement for allogeneic activation of B cells. Sonicated T cells and culture supernatants from allogeneic T- and B-cells cultures were not able to induce PFC formation in B lymphocytes. Our results indicate that the PFC response obtained in non-mitogen-activated cultures of allogeneic T and B lymphocytes is dependent on HLA-D/DR disparity or on genes encoded in the HLA-D/DR region. [ABSTRACT FROM AUTHOR]

The aim of this flow cytometry study in acute megakaryoblastic leukaemia (AML-M7) was to describe the membrane phenotype of CD34+ progenitor subsets and compare these with the phenotypes expressed by other AML FAB types. Following conventional histopathological diagnosis mononuclear cells from bone marrow and blood were examined in seven patients with AML-M7 and compared with results from 26 sequential patients with AML-M0 to AML-M6. The CD34+ subsets in AML-M7 patients differed from that of patients with AML-M0 to AML-M6 as the CD34+ CD61+ and the CD34+ Glycophorin A+ subsets were median 31% and 20%, respectively, compared to 4% and 2% in the AML-M0 to AML-M6 (P = 0.0005). Only 1% of the CD34+ progenitors were CD34+ CD38+ in AML-M7 compared to 72% in other AML subtypes (P < 0.000). These findings suggest that the CD34+ cell compartment in AML-M7 consists of early lineage-specific progenitors. In conclusion, flow cytometry analysis of CD34+ subsets may improve the diagnostic safety in AML-M7 and consequently the prognostic significance of immunophenotyping in acute leukaemia. [ABSTRACT FROM AUTHOR]

The interrelationship between four subpopulations of human lymphocytes was studied in eight normal persons. Double marking and fractionation experiments established that lymphocytes with receptors for AET-treated sheep erythrocytes constitute a population complementary to cells that carry surface membrane immunoglobulins as they are identified by polyvalent rabbit anti-human immunoglobulin: the latter includes nearly all cells with complement receptors. Two functionally different receptors for antigen-bound IgG are identified by their binding avidity for rabbit-antibody-sensitized ox erythrocytes, defining populations of lymphocytes with high-avidity and low-avidity receptors, indicating two valuable differentiation markers. [ABSTRACT FROM AUTHOR]

Serological properties or ox erythrocytes (ORBC) make it possible to select cells which exhibit weak agglutinability despite strong antibody sensitization. This property and the non-binding of unsensitized ORBC to lymphocyte surface membranes make these cells excellently suited as indicators in techniques for the identification of erythrocyte-antibody (EA) and erythrocyte-antibody- complement (EAC) rosette-forming lymphocytes (RFC). This report describes the relevant serology for the selection of appropriate cells and antisera. Further, some of the technical aspects of these tests are discussed. A simple method for the sensitization of ORBC with complement is described. The basis for (his method is the naturally occurring complement-binding anti-ORBC antibodies of the IgM class in human sera. After zymosan treatment the sera are deficient in the fifth component of complement and hence non-haemolytic, which make these sufficient as sensitizing agents in the preparation of EAC indicator cells. The relations of the EA and EAC-REC to be established T and B lymphocyte subpopulations are revealed by the enrichment and depletion of lymphocytes rostting with 2-aminoethylisothiouronium bromide (AET)-treated sheep erythrocytes (SRBC). [ABSTRACT FROM AUTHOR]

Human lymphocytes isolated from defibrinated blood are characterized especially in relation to the total yield of lymphocytes and their subpopulations identified by membrane markers. The defibrination per se gives a significant loss of monocytes and granulocytes but no loss of lymphocytes, indicating that no selective loss of lymphoeytes occurs. In a comparison of heparinstabilized and defibrinated blood no difference in yield is found during every single step of the isolation procedure. Quantitation of E-RFC and SmIg-positive lymphocytes gives no differences in comparing the respective isolated suspensions of mononuclear cells. The observed difference in EA- and EAC-RFC can freely be ascribed to the difference in monocyte contamination. Further, no correlation is found between total lymphocyte yield and the relative number of the subpopulations identified, i.e. E-RFC, SmIg-positive, Fc-receptor- and complement-receptorbearing lymphocytes. Hence it is concluded that defibrinated blood is optimal as blood source for the isolation of mononuclear cells when lymphocyte subpopulations are studied and enumerated. [ABSTRACT FROM AUTHOR]

Sixty glaucomatous eyes of 60 patients treated with laser trabeculoplasty as primary therapy were reviewed retrospectively. There were 42 eyes with capsular glaucoma and 18 with simple glaucoma. The mean prelaser intraocular pressure (IOP) was 35.2 (SD = 6.5) mmHg. Success was defined as IOP less than or equal to 22 mmHg without medication. The probability of success was 0.73 at 1 year, 0.66 at 2 years, 0.57 at 3 years, and 0.50 at 4 years. Three eyes experienced progressive visual field loss or disc damage in spite of an intraocular pressure below 22 mmHg without medication. High prelaser pressure and the severity of the visual field defects were significant predictors of treatment failure. [ABSTRACT FROM PUBLISHER]

Presents a letter to the editor about chronic myeloid leukemia.