of Anaesthesia 1997 ; 78 : 227 – 231 Flow cytometry

Sir,—With regard to the article by Heine and colleagues,1 we believe that several points require clarification. The authors claim that the technique using 123-dihydrorhodamine is more specific than the more well known chemiluminescence technique for measuring neutrophil respiratory burst. Such claims remain unsubstantiated. Readers may infer from the article that the chemiluminescence method is of less value than the authors’ technique. The chemiluminescence technique is not non-specific. It is based on the production of light by activated cells in the presence of enhancers such as luminol or lucigenin. The production of light is dependent on the presence of superoxide anion and hypochlorous acid (produced from superoxide). Singlet oxygen does not produce light in this system.2 The only apparent benefit of the dihydrorhodamine technique is its flow cytometry base, allowing the study of a single cell population within a mixed cell environment. However, in the study by Heine and colleagues1 such benefit is negated by separation of individual cell populations. The cell separation technique as presented is unclear. The layering of whole blood onto FicollHypaque density medium would result in a mononuclear cell layer with all granulocytes sedimenting with erythrocytes. However, the light scatter diagrams show mixed cell populations comprising monocytes, lymphocytes and granulocytes. It is difficult to see how the data relate to the methodology given or the need for cell separation in the first place. The propofol carrier, 10% Intralipid, inhibited respiratory burst of granulocytes in an identical manner to that of propofol.1 However, any effect of Intralipid on either light scatter or light quenching in the flow cytometry system is not discussed. In addition, cited literature has been misquoted. Intralipid was not shown to suppress T cell-mediated immunity as stated.3 Other studies have confirmed this finding.4 The effects of lipid emulsions on cell function are dependent on cell type and whether or not the study involves isolated cells in vitro or cells after in vivo lipid exposure.3–6 In addition, the antioxidant effects of propofol have not been discussed. Finally, the use of isotonic saline to dissolve thiopentone and methohexitone preparations inevitably resulted in hypertonic solutions. This may be inappropriate because hypertonic solutions induce a stress response, affect cell function and may also affect respiratory burst activity.7 8 Pre-exposure of cells to anaesthetic agents before stimulation with PMA1 implies that the results may represent the effect of the drugs on protein kinase C rather than on NADPH oxidase directly. This factor is not discussed. Protein kinase C is also affected by hypertonic solutions.9 H. F. GALLEY N. R. WEBSTER Anaesthesia and Intensive Care Department of Medicine and Therapeutics University of Aberdeen Aberdeen 1. Heine J, Leuwer M, Scheinichen D, Arsniev L, Jaeger K, Piepenbrock S. Flow cytometry evaluation of the in vitro influence of four i.v. anaesthetics on respiratory burst of neutrophils. British Journal of Anaesthesia 1996; 77: 387–392. 2. Halliwell B, Gutteridge JMC. Role of free radicals and catalytic metal ions in human disease. Methods in Enzymology 1990; 186: 1–85. 3. Monson JR, Ramsden CW, MacFie J, Brennan TG, Guillou PJ. Immunorestorative effect of lipid emulsions during total parenteral nutrition. British Journal of Surgery 1986; 73: 843–846. 4. Monson JRT, Sedman PC, Ramsden CW, Brennan TG, Guillou PJ. Total parenteral nutrition adversely influences tumour directed cellular cytotoxic responses in patients with gastrointestinal cancer. European Journal of Surgical Oncology 1988; 14: 435–443. 5. Fischer GW, Hunter KW, Wilson SR, Mease AD. Diminished bacterial defences with Intralipid. Lancet 1980; ii: 819–820. 6. O’Donnell NG, McSharry CP, Wilkinson PC, Asbury AJ. Comparison of the inhibitory effect of propofol, thiopentone and midazolam on neutrophil polarization in vitro in the presence or absence of human serum albumin. British Journal of Anaesthesia 1992; 69: 70–74. 7. Junger WG, Liu FC, Loomis WH, Hoyt DB. Hypertonic saline enhances cellular immune function. Circulatory Shock 1994; 42: 190–196. 8. Sato N, Kashima K, Shimizu H, Uehara Y, Shimomura Y, Mori M. Hypertonic glucose inhibits the production of oxygen derived free radicals by rat neutrophils. Life Sciences 1993; 52: 1481–1486. 9. Larsen AK, Jensen ES, Hoffmann EK. Activation of protein kinase C during cell volume regulation in Ehrlich mouse ascites tumour cells. Biochimica et Biophysica Acta 1994; 1222: 477–482.