A new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells.

BACKGROUND: Human peripheral blood lymphocytes kept in culture after isolation die by an apoptotic process. Detection of apoptosis with labeled Annexin V to demonstrate loss of plasma membrane asymmetry is sensitive, specific, and easy using flow cytometry. This is true in lymphoblastic cell lines when combining Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). However, measurement of apoptosis by flow cytometry in isolated human lymphocytes using Annexin V-FITC/PI is disturbed by the presence of a variable percentage of erythrocytes in the isolated lymphocyte population. To overcome this problem, we have developed and tested a new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells. METHODS: Peripheral blood lymphocytes are isolated by density gradient centrifugation. Nucleus-containing cells are selected using CD45-phycoerythrin (PE). The lymphocyte subset of interest is selected using CD4, CD8, or CD19 energy-coupled dye (ECD) labeling. Apoptosis is detected using Annexin V-FITC with 7-amino-Actinomycin-D (7-AAD) to distinguish early apoptotic from late apoptotic lymphocytes. RESULTS: We have developed a new technique to detect apoptosis in isolated human peripheral blood lymphocyte subsets with good reproducibility, coefficient of variation < 17%. CONCLUSIONS: We now have a validated tool to study apoptosis in subsets of isolated human lymphocytes to increase our knowledge of pathogenesis and therapies in lymphoreticular malignancies.

Determination of the mean normal prothrombin time for assessment of international normalized ratios. Usefulness of lyophilized plasma.

To report a Prothrombin Time (PT) as International Normalized Ratio in controlling oral anticoagulant therapy, the Mean Normal PT (MNPT) is required. To correct for methodological differences in performing the PT test, each laboratory should determine its own MNPT for each batch of reagent using fresh blood samples from a large number of normal individuals. This would be a laborious procedure. Two models for simplified assessment of MNPT were investigated by two laboratories in a collaborative study. According to the models, the MNPT of a new batch of reagent is calculated, using the PT of a lyophilized control plasma measured with the new batch and a reference batch, as well as the MNPT of the reference batch obtained with fresh samples. Experimental results were obtained with 19 batches of bovine thromboplastin, 4 lyophilized normal control plasmas and fresh blood samples of 40 normal individuals. The PTs of the 4 lyophilized normal control plasmas were not identical to the MNPT of the fresh normal samples and also different from each other. Therefore, the uncorrected PTs of these control plasmas cannot be used as MNPT. In general, there was good agreement between measured and calculated MNPT, although some control plasmas gave better results than others. There were no significant differences between the results obtained by both calculation models.

A Multi-centre study to evaluate method dependency of the international sensitivity index of bovine thromboplastin.

In The Netherlands, a particular coagulometer method for prothrombin time (PT) determination with reduced sample and reagent volumes is used by 62% of the laboratories controlling oral anticoagulant therapy. This "micro-method" has been calibrated against the manual tilt-tube technique for PT determination by six Dutch laboratories. Each laboratory tested 20 fresh normal blood samples and 60 fresh patient blood samples using both methods with the same batch of bovine thromboplastin reagent, according to a detailed protocol. Both methods were comparable as to their precision, but PTs measured by the micro-method were significantly prolonged (p less than 0.001, Student's t-test) as compared to the manual method. This effect is stronger for samples of normal subjects than for patients' samples. It was assumed that the International Sensitivity Index (ISI) of the bovine thromboplastin for the manual method was 1.00 in each laboratory. The ISI-values of the bovine thromboplastin for the micro-method determined by the six laboratories ranged from 1.00 to 1.07 (mean 1.03, SD 0.03). Our results indicate that any other laboratory, using this thromboplastin and the micro-method, should obtain accurate assessment of the International Normalized Ratio from their own mean normal PT and an ISI which is 3% higher than the ISI supplied by the thromboplastin manufacturer for the manual tilt-tube method.

The adsorption behaviour of two commercial IgG-preparations onto a polystyrene latex surface.

The adsorption behaviour of two commercial preparations of human IgG onto a polystyrene latex surface was studied. The adsorption isotherms obtained differed markedly; one preparation showed a plateau value of 0.4 microgram cm-2 which was reached at 0.1 g l-1, whereas the other preparation showed no plateau value within the concentration range studied (0.1-7.0 g l-1). Characterization by means of iso-electric focusing and HPLC also showed differences between the two preparations. No differences were observed when immuno-electrophoresis was carried out. These results stress the necessity for proper characterization of proteins used in adsorption studies.

In vitro adsorption of oxalic acid and glyoxylic acid onto activated charcoal, resins and hydrous zirconium oxide.

Patients suffering from primary hyperoxaluria show elevated plasma concentrations of oxalic acid and glyoxylic acid. The in vitro adsorption of these compounds into activated charcoal, a series of neutral and ion exchange resins and onto hydrous zirconium oxide has been investigated. Hydrous zirconium oxide was the most effective sorbent studied for the removal of both oxalic acid and glyoxylic acid. In batch experiments, the zirconium oxide was capable of binding 5.5 mu mol oxalic acid and 8 mu mol glyoxylic acid per gram sorbent using 0.5 gram sorbent and 50 ml of solutions with initial concentrations of 100 mu mol . L-1 and an ionic composition resembling that of plasma. Recirculation of 2 L of the same solutions through 12 gram of a mixture of hydrous zirconium oxide and alumina for 6 hours at a flow rate of 12 ml . min-1, resulted in a final concentration of 70 mu mol . L-1 of oxalic acid and 50 mu mol . L-/ of glyoxylic acid.

The effect of radiolabeling of human fibrinogen on its adsorption behaviour on a polystyrene surface.

Human fibrinogen (HFB) was labeled with different radioactive labels (Technetium -99m and iodine -125) in various ways. Characterization by chromatographic and electrophoretic methods did not show differences between the labeled and the nonlabeled proteins. The effect of the label and the labeling method on the adsorption behaviour of 99mTc and 125I labeled HFB at a polystyrene surface was investigated. In all cases labeled HFB showed preferential adsorption as compared to nonlabeled HFB. The preferential adsorption was expressed in terms of a factor ø (van der Scheer et al. 1978a), which will be 1, when no preferential adsorption occurs. 99mTc - and 125I - HFB showed ø values from 1.48 - 1.88. It is concluded that only meaningful adsorption experiments with labeled proteins can be performed when the possible occurrence of preferential adsorption has been investigated by appropriate methods. The results of prior work on protein adsorption at biomaterials using radiolabeled proteins have to be reconsidered.

Urinary oxalate estimation.

A method of estimating urinary oxalate is described. Impurities which might otherwise interfere with the estimation are removed by means of an ion-exchange resin. The oxalate is then reduced to glycolic acid, which forms a coloured compound with chromotropic acid. It can then be estimated colorimetrically. No special equipment is required, oxalate recovery is good, and the method is reproducible and precise.

Oxalic acid determination in plasma.

A method is described for the estimation of oxalic acid in plasma. After removing interfering substances with an ion-exchange resin, oxalic acid is reduced to glycolic acid, which forms a coloured compound with chromotropic acid. No special equipment is required. A good recovery and precision are found.