Automated quantitation of peripheral blood neutrophil activation in patients with myocardial ischaemia.

BACKGROUND: Coronary ischaemic syndromes are associated with neutrophil activation. The Bayer automated haematology analysers can detect increased light scatter of neutrophil populations, which correlates with neutrophil activation. We aimed to assess the role of an automated analyser in detecting systemic neutrophil activation in peripheral blood samples of patients with coronary ischaemia. METHODS: A prospective cross-sectional study was undertaken in 18 patients with chronic stable angina, 9 with unstable angina and 26 normal control subjects. Whole blood samples were taken to assess neutrophil count and light scatter, and serum samples were taken from some patients for assessment of Troponin T, C-reactive protein (CRP) and myeloperoxidase (MPO). In addition, whole blood was stimulated in vitro with interleukin (IL)-8 and N-formyl-methionyl-leucyl-phenylalanine (fMLP) to assess changes in neutrophil light scatter detected by the analyser. RESULTS: Neutrophil light scatter was increased in patients with chronic stable and unstable angina compared to normal control subjects (normal subjects 74.1 (73.3, 75.0) (mean arbitrary units (95% confidence intervals, (CI)) vs. 78.6 (76.9, 80.3) in the chronic stable angina group P<0.001 and 77.1 (75.3, 79.0) in the unstable angina group P<0.007). In vitro stimulation of whole blood produced comparable increases in neutrophil light scatter when morphological changes in neutrophils were demonstrable under electron microscopy. CONCLUSIONS: Automated measurement of neutrophil activation by light scatter is possible using the Advia 120 analyser and is superior to a neutrophil count in discriminating groups with angina. This technique may be useful in monitoring disease activity and progression in coronary artery disease and in guiding the use of anti-inflammatory therapies.

Automated quantitation of circulating neutrophil and eosinophil activation in asthmatic patients.

BACKGROUND: Asthma has been associated with eosinophil activation, measured in serum, sputum, bronchoalveolar lavage (BAL) fluid, and urine. A whole blood automated method was developed to assess eosinophil and neutrophil activity in terms of peroxidase content and cell morphology using the Bayer haematology analyser. The method was applied to an in vitro stimulation model when fMLP was added to whole blood and the samples were then analysed for changes in granularity and shape. In addition, cells stimulated with interleukin (IL)-8 were examined by electron microscopy. METHODS: A cross sectional analysis was performed on venous blood from non-atopic, non-asthmatic normal subjects (n = 37), mild (n = 46) and symptomatic (n = 22) asthmatic patients on inhaled beta(2) agonist only, and more severe asthmatic patients (n = 17) on inhaled and oral corticosteroid therapy. Samples were analysed by the haematology analyser and peroxidase leucograms gated using the WinMDI software program. RESULTS: There were significant differences in the amount of light scatter by the neutrophil populations in the symptomatic (p = 0.007) and severe asthmatic (p = 0.0001) groups compared with the control group. However, abnormalities in eosinophil populations were not observed. In vitro activation of whole blood with fMLP caused similar changes in neutrophil light scatter, suggesting that neutrophil activation is present in peripheral blood of symptomatic asthmatic patients. IL-8 caused a change in shape of the neutrophils seen using transmission electron microscopy. CONCLUSIONS: Evidence of neutrophil activation can be seen in whole blood from patients with asthma using a novel automated method. This may potentially be applied to other inflammatory diseases.

Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system.

Platelet activation results in changes in a number of cell surface molecules including an increase in P-Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose-dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post-venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P < 0.001) increase was observed on platelets anti-coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r = -0.69, P < 0.001, n = 20) and the percentage (r = -0.72, P < 0.001, n = 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as measured by the ADVIA 120 may be used to detect anticoagulant induced, as well as thrombin stimulated, in vitro platelet activation in blood anticoagulated with EDTA. Further, we conclude that platelet activation is negligible for up to 3 h in sodium citrate anticoagulated whole blood.

Red blood cell depletion and enrichment of CD34+ hematopoietic progenitor cells from human umbilical cord blood using soybean agglutinin and CD34 immunoselection.

Realization of the full potential of human umbilical cord blood (HUCB) as a source of transplantable hematopoietic progenitor cells (HPC) will be contingent on the development of a reliable method for ex vivo cell processing and stem cell enrichment. A first step in stem cell enrichment protocols involves depletion of the red blood cells (RBC) in the sample. We have compared several RBC depletion methods on the basis of recovery of total nucleated cells. It was found that 3% gelatin was effective at depleting RBC with a nucleated cell recovery of 72.4 +/- 7.3% (mean +/- standard deviation [SD]) in the HUCB samples. Several lectins were also used for processing HUCB and compared for their ability to remove RBC. Of these, soybean agglutinin (SBA) was found to remove RBC without substantial loss of HPC. Moreover, sequential treatment of HUCB with 3% gelatin and the AIS MicroCELLector SBA resulted in a product with < 1% hematocrit, 57% reduction in T cells, 35% nucleated cell recovery, and relatively high yields of burst-forming units-erythroid (BFU-E) (61%) and colony-forming units-granulocyte/macrophage (CFU-GM) (58%) and -mixed (CFU-GEMM) (66%). In a separate series of studies, enrichment of the CD34+ subset in HUCB was accomplished by processing HUCB with Ficoll followed by sequential treatment with the AIS MicroCELLector SBA and AIS MicroCELLector CD34. The CD34+ fraction was enriched for BFU-E (14-fold), CFU-GM (nine-fold), and CFU-GEMM (five-fold) with an overall purity ¿ > or = 92% CD34+ by flow cytometry. This report demonstrates that 3% gelatin and the AIS MicroCELLector SBA can be combined as an ex vivo processing technique to reduce the volume of the HUCB product by depleting RBC and T cells while still maintaining a high recovery of HPC. Moreover, separation of highly enriched CD34+ cells from HUCB is achievable and opens up the possibility of using CD34+ cells from HUCB for ex vivo progenitor cell expansion for transplantation, transfusion, and gene therapy.

Separation of lectin-binding cells using polystyrene culture devices with covalently immobilized soybean agglutinin.

The plant lectin, soybean agglutinin (SBA), has been widely used to separate heterogeneous populations of cells. In the field of bone marrow transplantation, SBA has been used for partial depletion of T cells from bone marrow allografts to reduce graft-vs.-host disease. SBA's high affinity for many different tumor cells has also indicated its use as a tumor purging agent for autologous bone marrow transplants. We have compared two methods of cell separation using either soluble SBA agglutination, or SBA covalently attached to an activated polystyrene surface. The nonbinding SBA-cell populations generated by these two procedures were very similar in terms of cell recovery, light scatter properties, and phenotypic profile. Notably, both SBA- fractions were enriched in cells with the known progenitor markers, CD34, CD33, and HLA-DR, and were relatively depleted of SBA binding cells. In addition, the activity of each SBA- cell population was measured in vitro in short-term progenitor assays. Here, both SBA- populations were significantly enriched for CFU-GM. When device-separated SBA- cell populations were seeded into long-term bone marrow culture, they produced both increased progenitor activity and cell proliferation compared to unseparated BMMCs. The polystyrene technology described here could reduce or eliminate many of the drawbacks of soluble SBA agglutination, making SBA cell separation a viable and convenient technique for clinical application.

A novel method for rapid enrichment of lactotrophs from dispersed anterior pituitary cells of the rat.

A polyclonal antibody to the rat D2 dopamine (DA) receptor was rapidly and covalently attached to surface-activated polystyrene cultureware (MicroCEL-Lector plates). Addition of a suspension of dispersed rat anterior pituitary cells resulted in the rapid (within 1 h) selection of cells possessing D2 DA receptors (i.e. lactotrophs). Four-fold enrichment (from about 20% in the suspension to about 80%) was routinely obtained, as judged by prolactin (PRL) immunostaining. The enriched cells were virtually free of fibroblasts and were much more homogeneous in appearance than untreated cells after 3 days in culture. Lactotroph-enriched cell cultures displayed similar functional characteristics as untreated cells when assessed by determining dose-response curves for inhibition of PRL secretion by the DA agonist N-propylnorapomorphine. This method may be generally applicable for the selective enrichment and purification of desired cell types from heterogeneous mixtures in tissue dispersions.

Separation of hematopoietic progenitor cells from human umbilical cord blood.

Human umbilical cord blood (HUCB) is a rich source of hematopoietic progenitor cells (HPC). Nevertheless, it has been previously reported that attempts to enrich for HPC resulted in substantial loss of progenitor cells. We have developed a cell processing technique for HUCB using 3% gelatin sedimentation and the AIS MicroCELLectorSBA and CD34. By this technique, we were able to deplete red blood cells (RBC) and obtain a partial T cell depletion, while maintaining 70-90% of the HPC. The results indicate that it may be possible to manipulate cord blood without significant loss of HPC, thus enhancing its utility for transplantation.

Rapid isolation of human CD34 hematopoietic stem cells--purging of human tumor cells.

Human CD34+ hematopoietic stem cells were purified using a new technology in which monoclonal antibodies are covalently immobilized on polystyrene surfaces. The CD34+ cell isolation scheme involved three sequential processes: (1) purification of bone marrow mononuclear cells; (2) enrichment of CD34+ cells using covalently immobilized soybean agglutinin; and (3) positive selection of CD34+ cells using polystyrene surfaces coated with the anti-CD34 monoclonal antibody ICH3. CD34+ cells purified by this process have both low-to-medium forward light scatter and low 90 degrees light-scatter properties. Moreover, the purified CD34+ cells are greater than 85% viable, express appropriate characteristic surface antigens, and are 10-50-fold enriched in short- and long-term hematopoietic activity. CD34+ cells collected in this manner from bone marrow samples contaminated with radiolabeled breast carcinoma, neuroblastoma, acute myelogenous leukemia, or small cell lung carcinoma cells were 99.9% depleted of the tumor cells. The CD34+ cell selection devices are sterile and are easily scaled-up to process clinical scale bone marrow samples.

Separation and growth of human CD4+ and CD8+ tumor-infiltrating lymphocytes and peripheral blood mononuclear cells by direct positive panning on covalently attached monoclonal antibody-coated flasks.

A direct positive panning technique was developed in order to achieve highly purified CD4+ and CD8+ cells. Fresh peripheral blood mononuclear cells or tumor-infiltrating lymphocytes derived from bulk cultures were applied to modified polystyrene surfaces to which anti-CD4 or anti-CD8 monoclonal antibodies were covalently attached. Adherent cells were allowed to grow in the original flask and were then harvested and cultured in IL-2-containing medium. This positive immunoselection technique resulted in CD4+ and CD8+ cell subsets with high cell viability and a high degree of purity. In several samples, the isolated cell subsets were subsequently subjected to a negative immunomagnetic bead selection in order to remove reciprocal cell subset contamination or double-positive CD4+/CD8+ cells. The isolated cells maintained their ability to proliferate, kept their phenotypic profiles, and remained functionally intact after long-term growth in culture without the further addition of mitogenic or allogeneic cell stimulation. This approach is a simple, rapid, and reproducible technique that might be useful on a large scale to isolate and to grow T-cell subsets for research and for clinical use.