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Open Access Technical advance

LeukoCatch, a quick and efficient tool for the preparation of leukocyte extracts from blood

Daisuke Okuzaki12, Shoichi Kimura3, Norikazu Yabuta1, Toshinari Ohmine4 and Hiroshi Nojima12*

Author affiliations

1 Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan

2 DNA-chip Development Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan

3 Fukae Kasei Co., Ltd., 3-2-44 Takatsuka-dai, Nishi-ku, Kobe, Hyogo 651-2271, Japan

4 Laboratory for Clinical Investigation, Osaka University Hospital, Osaka University, 2-15 Yamadaoka, Suita, Osaka 565-0871, Japan

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Citation and License

BMC Clinical Pathology 2011, 11:9  doi:10.1186/1472-6890-11-9

Published: 17 August 2011

Abstract

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.

Keywords:
cell extract; leukocyte; diagnosis; PBMC; proteomics