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We have already reported a neutrophil separation method for the multiple simultaneous measurement of neutrophil chemiluminescence. However, when the reported separation method was used, at least 4 ml of venous blood was needed to collect enough neutrophils for chemiluminescent measurement. Because of this blood volume, there is a limitation on applications of the multiple simultaneous method for neonates, infants, and in some clinical situations. To expand the application of this neutrophil chemiluminescence measurement into clinical and health science areas, we have developed a new method for separation of neutrophils from a relatively small amount of blood (500 microliters). In addition, the influences of remaining red blood cells and hemoglobin levels in the neutrophil fraction on the chemiluminescence were examined to determine the necessity for elimination procedures. The new separation method used a capillary tube (length, 130 mm; outside diameter, 5 mm; thickness, 0.8 mm) with density gradient reagents (Histopaque 1077 and 1119). After centrifugal separation (500 g, 30 min), the neutrophil fraction was isolated with 93.1 +/- 4.7% purity and 60.6 +/- 11.1% yield. This purity and yield were comparable to or better than those with the previously reported method, while levels of remaining red blood cells and hemoglobin were about the same. Remaining red blood cells and hemoglobin in the neutrophil fraction acted on the chemiluminescence as a quencher. For the correct estimation of neutrophil chemiluminescence, elimination of remaining red blood cells and hemoglobin in the neutrophil fraction is necessary. This new neutrophil separation method is a very useful method, especially for cases in which available blood amounts are limited.