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Comparing Flow Cytometry of Sputum, Bronchoalveolar Lavage, and Peripheral Blood Cells in Healthy Individuals
Clinical Immunology Vol. 99, No. 1, April, pp. 100 –101, 2001 All articles available online at http://www.idealibrary.com on
LETTERS TO THE EDITOR Comparing Flow Cytometry of Sputum, Bronchoalveolar Lavage, and Peripheral Blood Cells in Healthy Individuals To The Editor: We have read with interest the paper by Alexis et al. describing a comprehensive analysis of peripheral blood, BAL, and induced sputum phagocytes in healthy individuals (1). This adds to the understanding of the phenotype and normal function of airway cells. However, we have some reservations about the study. First, we note that although the inclusion criteria for the healthy individuals included an FEV 1 of ⬍90% predicted, subjects 4, 10, and 13 fall well below this. Also, the use of a total of 540 ml (2 ⫻ 270 ml) saline for BAL seems excessive for the study. We have been able to isolate adequate numbers of cells from BAL for flow cytometric analysis, in healthy volunteers and COPD patients, using 180 ml saline (2). In healthy volunteers, we have been able to achieve a mean return of 45% using 180 ml (3). BAL has been shown to induce an inflammatory response at 6 h postprocedure, with activation of macrophages, induction of pro-inflammatory cytokines, and influx of neutrophils (4). It is likely that this response will be greater with greater volumes of saline. The authors also comment that DTT has not been shown to affect cell surface markers, but a recent paper by Loppow et al. has demonstrated that some cell surface markers can be affected (5). Additionally, Qiu and Tan have shown a dose– response effect of DTT on cell surface antigen expression on lymphocytes, neutrophils, and eosinophils (6). This should be borne in mind when flow cytometric analysis is being performed on sputum processed using DTT.
REFERENCES 1. Alexis, N., Soukup, J., Ghio, A., and Becker, S., Sputum phagocytes from healthy individuals are functional and activated: A flow cytometric comparison with cells in bronchoalveolar lavage and peripheral blood. Clin. Immunol. 97, 21–32, 2000, doi: 10.1006/clim.2000.4911. 2. Kelly, M. G., Brown, V. G., Ennis, M., and Elborn, J. S., Rates of granulocyte apoptosis and necrosis in bronchoalveolar lavage in normal subjects and patients with chronic obstructive pulmonary disease. Eur. Respir. J. 16(Suppl 31), 252s, 2000. 3. Kelly, M. G., Brown, V. G., Ennis, M., and Elborn, J. S., Comparing bronchoalveolar lavage and induced sputum in normal controls and patients with chronic obstructive pulmonary disease. Eur. Respir. J. 16(Suppl 31), 252s, 2000. 4. Nelson, M. E., Wald, T. C., Bailey, K., and Wesselius, L. J., Intrapulmonary cytokine accumulation following BAL and the role of endotoxin contamination. Chest 115, 151–157, 1999. 5. Loppow, D., Bottcher, M., Gercken, G., Magnussen, H., and Jorres, R. A., Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers. Eur. Respir. J. 16, 324 –329, 2000. 6. Qiu, D., and Tan, W. C., Dithiothreitol has a dose–response effect on cell surface antigen expression. J. Allergy Clin. Immunol. 103, 873– 876, 1999.
M. G. Kelly* ,† V. Brown† M. Ennis† J. S. Elborn* *Department of Respiratory Medicine Belfast City Hospital †Department of Clinical Biochemistry Queens University Belfast, Northern Ireland This letter is doi:10.1006/clim.2001.5014
Reply To The Editor: On behalf of my co-authors, I thank Dr. Kelly and colleagues for their interest and comments regarding our article on a flow cytometric comparison of sputum phagocytes with BAL and blood phagocytes published in Clinical Immunology (97, 21–32, 2000). Dr. Kelly 1521-6616/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
raises some important questions that deserve in some cases clarification, and in others further explanation. First, Dr. Kelly notes that subjects 4, 10, and 13 fall below the “inclusion criteria” for healthy subjects for %FEV 1. In fact, the inclusion criteria were not included in the article and were actually ⬎80% for %FEV 1, not
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LETTERS TO THE EDITOR Comparing Flow Cytometry of Sputum, Bronchoalveolar Lavage, and Peripheral Blood Cells in Healthy Individuals To The Editor: We have read with interest the paper by Alexis et al. describing a comprehensive analysis of peripheral blood, BAL, and induced sputum phagocytes in healthy individuals (1). This adds to the understanding of the phenotype and normal function of airway cells. However, we have some reservations about the study. First, we note that although the inclusion criteria for the healthy individuals included an FEV 1 of ⬍90% predicted, subjects 4, 10, and 13 fall well below this. Also, the use of a total of 540 ml (2 ⫻ 270 ml) saline for BAL seems excessive for the study. We have been able to isolate adequate numbers of cells from BAL for flow cytometric analysis, in healthy volunteers and COPD patients, using 180 ml saline (2). In healthy volunteers, we have been able to achieve a mean return of 45% using 180 ml (3). BAL has been shown to induce an inflammatory response at 6 h postprocedure, with activation of macrophages, induction of pro-inflammatory cytokines, and influx of neutrophils (4). It is likely that this response will be greater with greater volumes of saline. The authors also comment that DTT has not been shown to affect cell surface markers, but a recent paper by Loppow et al. has demonstrated that some cell surface markers can be affected (5). Additionally, Qiu and Tan have shown a dose– response effect of DTT on cell surface antigen expression on lymphocytes, neutrophils, and eosinophils (6). This should be borne in mind when flow cytometric analysis is being performed on sputum processed using DTT.
REFERENCES 1. Alexis, N., Soukup, J., Ghio, A., and Becker, S., Sputum phagocytes from healthy individuals are functional and activated: A flow cytometric comparison with cells in bronchoalveolar lavage and peripheral blood. Clin. Immunol. 97, 21–32, 2000, doi: 10.1006/clim.2000.4911. 2. Kelly, M. G., Brown, V. G., Ennis, M., and Elborn, J. S., Rates of granulocyte apoptosis and necrosis in bronchoalveolar lavage in normal subjects and patients with chronic obstructive pulmonary disease. Eur. Respir. J. 16(Suppl 31), 252s, 2000. 3. Kelly, M. G., Brown, V. G., Ennis, M., and Elborn, J. S., Comparing bronchoalveolar lavage and induced sputum in normal controls and patients with chronic obstructive pulmonary disease. Eur. Respir. J. 16(Suppl 31), 252s, 2000. 4. Nelson, M. E., Wald, T. C., Bailey, K., and Wesselius, L. J., Intrapulmonary cytokine accumulation following BAL and the role of endotoxin contamination. Chest 115, 151–157, 1999. 5. Loppow, D., Bottcher, M., Gercken, G., Magnussen, H., and Jorres, R. A., Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers. Eur. Respir. J. 16, 324 –329, 2000. 6. Qiu, D., and Tan, W. C., Dithiothreitol has a dose–response effect on cell surface antigen expression. J. Allergy Clin. Immunol. 103, 873– 876, 1999.
M. G. Kelly* ,† V. Brown† M. Ennis† J. S. Elborn* *Department of Respiratory Medicine Belfast City Hospital †Department of Clinical Biochemistry Queens University Belfast, Northern Ireland This letter is doi:10.1006/clim.2001.5014
Reply To The Editor: On behalf of my co-authors, I thank Dr. Kelly and colleagues for their interest and comments regarding our article on a flow cytometric comparison of sputum phagocytes with BAL and blood phagocytes published in Clinical Immunology (97, 21–32, 2000). Dr. Kelly 1521-6616/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
raises some important questions that deserve in some cases clarification, and in others further explanation. First, Dr. Kelly notes that subjects 4, 10, and 13 fall below the “inclusion criteria” for healthy subjects for %FEV 1. In fact, the inclusion criteria were not included in the article and were actually ⬎80% for %FEV 1, not
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