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.

Hypertrophic protein-losing gastropathy. A retrospective analysis of 40 cases in The Netherlands. The Dutch Ménétrier Study Group.

Hypertrophic protein-losing gastropathy is a rare clinical entity of unknown etiology. Seventeen of 50 GI Units in The Netherlands, surveying their patient material, documented at least 1 positive case. Altogether, 40 patients (25 male and 15 female; mean age, 44.3 years) fulfilled the usually accepted criteria. Main complaints were epigastric pain (65%), asthenia (60%), anorexia (45%), weight loss 45%, edema (37.5%), and vomiting (37.5%). Hypoalbuminemia of < 35 g/l was found in 81%, and an abnormal enteric protein loss (51CrCl3) in 22 of 26 tested patients (85%). The mean basal acid output was 0.99 mmolH+/h. Stomach radiology in 35 patients showed giant folds mainly of the corpus mucosa; endoscopy confirmed the hypertrophy of the folds in all cases (in four confirmed by endosonography) and the presence of adherent mucus. Occasionally a concomitant gastric ulcer was found. Endoscopic biopsies were usually of limited value for the histologic diagnosis, mainly suggesting the possibility of hypertrophic gastropathy, excluding gastric cancer or lymphoma. In the follow-up 80% was treated with antacids, H2-receptor antagonists, mucosa-protectives, omeprazole, or combinations. No single agent appeared of major value. Eradication of Helicobacter pylori occasionally reduced symptoms. Twenty-two patients (55%) improved with or without medical therapy, during a mean follow-up of 7.6 years. Five patients (12.5%) underwent gastric surgery; four improved. In total, 26 patients (65%) improved, with partial or total regression of hypoalbuminemia. Eight patients died, three of gastric cancer, and five of cancers localized elsewhere.(ABSTRACT TRUNCATED AT 250 WORDS)