RESUMEN
BACKGROUND: Whole-protein extracts from peripheral blood leukocytes are ideal for basic and clinical research. However, lack of a simple preparation technique has limited the use of such extracts. The aim of this study is to develop a simple and easy system that can selectively obtain leukocyte extracts without hemoglobin. METHODS: A filter that captures the leukocytes but not RBCs was set at the bottom of a 10-mL medical syringe by sandwiching it between plastic stoppers. The capturing efficiency of leukocytes with this tool, called LeukoCatch, was examined using human macrophage cells (MONO-MAC-6). The abilities of LeukoCatch system to capture the leukocyte proteins and to remove the hemoglobin from RBCs were tested by western blot analysis using human blood samples. RESULTS: This study presents the development of LeukoCatch, a novel tool that allows the preparation of leukocyte extracts from blood samples within 3 min without centrifugation. Tissue-cultured human macrophage cells were tested to determine the optimal filter numbers and pass-through frequencies of LeukoCatch, which was then applied to 2-mL blood samples. Samples were passed 2~5 times through a LeukoCatch equipped with 5 filters, washed twice with phosphate-buffered saline for red cell removal, and leukocyte proteins were extracted with 0.5 mL of elution buffer. Western blot analysis of the purified extract indicated that more than 90% of hemoglobin was removed by the LeukoCatch and that the protein recovery rate of leukocytes was at least 4 times better than that of the conventional centrifugation method. CONCLUSION: We conclude that LeukoCatch is useful not only for diagnosis at the bedside but also for basic research using blood samples or tissue culture cells.
RESUMEN
OBJECTIVE: We compared leukocyte counts obtained by cytometric analysis and Fuchs-Rosenthal (FR) chamber counting in different proportions of lymphocytes (Lym%) suspensions and cerebrospinal fluid (CSF). DESIGN AND METHODS: UF-100 (UF) was evaluated. For preparation of cell suspensions, gradient density centrifugation method was used. RESULTS: The regression equation for UF and FR chamber counting of the cell suspensions was y=0.88x+18.8 WBC/microL (r=0.832, n=106). For a few high Lym% samples, markedly underestimated WBC counts were obtained by UF. CONCLUSIONS: Underestimated WBC count is due not to systematic error but to random error. Counts of the "other" population by UF may be useful for detection of underestimated samples.