Measurement of Oxyradicals from Leukocytes in Whole Blood Lodged in a Microchannel Array

The oxyradical production by leukocytes is crucial for killing invading bacteria, while it has been widely discussed as a tissue injuring factor. However, it is still unclear, probably because of lack of simple and reliable measurement methods, to what extent it varies among different subjects and either to what extent it is affected by environmental factors including diet. Meanwhile, our previous studies have shown usefulness of parallel arrays of many identical capillary-size channels for the measurement of rheological or mechanical properties of blood and other cells, Our flow measurement system is now commercially available as MC-FAN (Micro Channel array Flow ANalyzer). The aim of the present study is to describe an application of MC-FAN to the measurement of the oxyradical production using chemiluminescence for leukocytes in whole blood. Fresh heparinized whole blood was caused to flow through a microchannel array (width 7μm, length 30μm, depth 4.5μm, and number 8736) under a 20cmH2O suction with the addition of Salmonella typhimurium cells (2×107CFU/ml) or S. typhimurium lipopolysaccharides (100ng/ml) and luminol (200μM). When 100μl passed, the flow was ceased and leukocytes lodged in the microchannel array were subjected to the photoemission measurement by a VIM camera system or a photomultiplier-DC amplifier system. The passage depth (4.5μm) under the glass plate covering the microchannel array allows only single cells in the depth direction; this explains why photons emitted in the observation direction are not absorbed by red cells. In addition, the microchannel array concentrated leukocytes there and hence gave a sufficient number of leukocytes in the measurable area, i.e., a sufficient intensity of photoemission thatt allowed a real time measurement, Thus, a simpler and more reliable method has been established. An unexpectedly large variation in the oxyradical production by leukocytes was observed among healthy subjects.