Measurement of the Phagocytic Activity of Human Peripheral Blood Using a Highly Sensitive Fluorometric Detection Device Without Hemolysis.

BACKGROUND/AIM: Phagocytes recognize pathogens that enter the body as well as other abnormal and foreign materials that may exist within an organism (such as dead cells, oxidized lipids, and denatured proteins), and phagocytose and eliminate them to maintain a healthy state. In a previous study a simple prototype device was used, under development by Hamamatsu Photonics (Prototype), that detects fluorescence to determine the phagocytic activity of the murine macrophage cell line J774.1. The present study aimed to determine whether it was possible to detect phagocytic activity in a slight amount of human peripheral blood without using hemolysis. MATERIALS AND METHODS: Three microliters of human peripheral blood was drawn from the fingertip and mixed with 30 µg of pH-sensitive fluorescent particles. The fluorescence intensity of the human peripheral blood sample was then measured using the Prototype in development, cultured for 2 h at 37°C, and then re-measured. The phagocytes were observed under fluorescence microscopy and the phagocytosis rate of CD11b-positive cells was verified with a flow cytometer. RESULT: The phagocytic activity of non-hemolyzed human peripheral blood was measured using the Prototype under development; fluorescence after phagocytosis was detected. Furthermore, this was confirmed by both fluorescence microscopy and flow cytometry. The precision of the measurements of human peripheral blood phagocytic activity was verified with the Prototype using samples from three healthy individuals. The relationship between blood sugar levels and phagocytic activity before and after meal times was determined. Concerning exercise, phagocytic activity tended to decrease, although salivary amylase level increased in the healthy individual examined after exercise. CONCLUSION: The simple Prototype can measure phagocytic activity in a small amount of peripheral blood without hemolysis. The device allows for rapid and minimally-invasive detection of changes in phagocytic activity, which has conventionally been difficult. These findings provide promising evidence that assessment of individual phagocytic capacity can be made easier using this novel device.

Development of an Evaluation Device for Phagocytic Activity of New Phagocytes Using Simple and pH-sensitive Particles that Do Not Require Pre-treatment.

BACKGROUND/AIM: Phagocytic activity is affected by a number of different stress and age-dependent factors. An easy measurement of phagocytic activity is thought to allow an indicator of an individual's health. In this study, we investigated conditions of measurement to easily evaluate the activity of phagocytosis of phagocytic cells (macrophages and neutrophils) using an easy-to-use prototype, which was improved from the device by Hamamatsu Photonics K.K., to detect neutrophil activity using subtle fluorescence. MATERIALS AND METHODS: pH-sensitive fluorescent particles (pHrodo-Green E. coli Bio particles, GE particles) were added to mouse-derived macrophage cell lines (J774.1) and then incubated for 2 h at 37°C. For negative control, the phagocytosis inhibitor cytochalasin D (CyD), was added prior to culture. Next, fluorescence intensity was measured by the Prototype to evaluate the phagocytic activity of macrophages and neutrophils. Phagocytosis was also confirmed by flow cytometry. RESULTS: The Prototype detected a steady fluorescence increase in 5 sec in J774.1 after phagocytosis, using GE particles as a negative control in the presence of CyD. Furthermore, detection was possible at 10(4) cells/test, a concentration where the flow cytometer had difficulty for detection. CONCLUSION: The Prototype enables measurement of the phagocytic activity within a short period of time, even with a small sample amount, thus establishing the basic conditions of measurements of phagocytosis.