An in vitro test that includes the use of two-color flow cytometry has been developed for use in screening of neutrophils for their ability to fight infections. Neutrophils constitute the first line of defense of the human body against infections. Neutrophils engulf invading bacteria in a process called" phagocytosis." The primary means by which they destroy the bacteria is the production of hydrogen peroxide and toxic oxygen radicals; this production is called "oxidative burst." Acquired defects in phagocytosis or oxidative burst can allow localized or generalized infections to develop. Such defects can be caused by toxins, drugs (including adrenocorticosteroids), and/or radiation. A rapid test that yields data on such defects could be helpful in choosing appropriate therapeutic measures prior to onset of overt clinical disease.
Most of the older in vitro tests developed for the same purpose require microscopy, are time-consuming, and involve much subjective judgement by highly trained technicians. One of the older tests is less subjective in that it yields quantitative data via an absorbance-spectrophotometric measurement of concentration of dye extracted from stained cells, but the disadvantage of this older test is that it requires a large amount of blood (as much as 10 mL). In contrast, the present test can be performed relatively quickly, requires a small amount of blood, and yields quantitative data.
In the present test, one uses stained cells of two species; the opportunistic pathogen Candida albicans and the intracellular pathogen Listeria monocytogenes. Candida is a common commensal that can cause serious disease if immunological defects occur. Listeria is a catalase-producing organism that is not killed after phagocytosis if there is a defect in oxidative burst. (Listeria is used routinely in immunotoxicity testing.) Batches of Candida and Listeria organisms are stained with one of three fluorescent dyes (fluorescein isothiocyanate, CellTracker™, or Mitotracker™) for the purpose of testing for phagocytosis of those organisms. Neutrophils are stained with either of two other fluorescent dyes (dichlorofluorescein diacetate or hydroethidine) to detect oxidative burst with respect to those organisms.
In preparation for the test, the three types of cells are stained, then washed to remove excess dye. Neutrophils are mixed with cells of the other two types, and each mixture is incubated in a tube at an appropriate temperature. After an appropriate time (e.g., 1 hour), the incubation is stopped by putting the tubes on ice. A dilute suspension of cells in liquid is made from each mixture, and a jet of the liquid is made to cross a beam of light of wavelength 488 nm generated by an argon-ion laser in a flow cytometer. By use of photodetectors and band-pass filters in combination with a dichroic mirror, intensities of red and green fluorescence are measured, both in the forward- and perpendicular-scatter directions. These measurements are processed to determine the percentage of fluorescent neutrophils and the peak modal channel of fluorescence of the neutrophils. They are also used to generate a display of fluorescence intensities in a histogram or in a two-color contour plot.
This work was done by Duane L. Pierson of Johnson Space Center and Raymond P. Stowe and Saroj K. Mishra of KRUG Life Sciences. For further information, access the Technical Support Package (TSP) free on-line at www.techbriefs.com under the category.
This invention is owned by NASA, and a patent application has been filed. Inquiries concerning nonexclusive or exclusive license for its commercial development should be addressed to
the Patent Counsel
Johnson Space Center
Refer to MSC-22654.