Cytological method using the auto fluorescence of white corpuscles for the early diagnosis and the monitoring of infections
View Patent ↗The present invention relates to an in vitro method for diagnosing the infectious state of an individual on the basis of a sample of white corpuscles arising from a biological specimen taken from an organ potentially infected by a pathogenic microorganism of said individual, comprising at least the following two steps: i) measuring the mean cellular intensity of the autofluorescence of said sample, and ii) comparing the intensity measured in step i) with a control value, so as to determine the infectious state of said individual. The diagnostic method of the invention uses a routine optical material making it possible to work in wavelength regions which are compatible with the cellular autofluorescence, and thus constitutes a rapid, reliable and inexpensive aid for the diagnosis or monitoring of an infection in an individual.
1. An in vitro method for diagnosing the infectious state of an individual, comprising at least the following steps:
i) measuring the mean cellular NAD(P)H autofluorescence intensity of a sample of leukocytes of a fluid arising from an organ of said individual with a fluorescence microscope that excites the cells with a wavelength ranging from 300 nm to 600 nm; and
ii) comparing the intensity measured in step i) with a control value, so as to determine the infectious state of said individual.
2. The method according to claim 1 , characterized in that the leukocytes of said sample are selected from monocytes and/or polymorphonuclear neutrophils.
3. The method according to claim 1 , characterized in that, when the state of said individual is infectious, said individual is suffering from a bacterial, viral or fungal infection.
4. The method according to claim 1 , characterized in that said fluid is a pulmonary fluid, ascites fluid, cerebrospinal fluid or fluid of any other potentially infected organ.
5. The method according to claim 1 , characterized in that the autofluorescence intensity of the cells of said sample is measured on cells in suspension.
6. The method according to claim 1 , characterized in that the autofluorescence intensity of the cells of said sample is measured on cells that are chemically fixed and placed on a slide compatible with the observation of fluorescence.
7. The method according to claim 1 , wherein said sample of cells is prepared in a monolayer on a slide of transparent material prior to step i), according to a method comprising at least the following steps:
a) depositing cells of said sample in at least one cytocentrifugation system;
b) centrifuging so as to project the cells on said slide;
c) chemically fixing the cells thus projected on the slide to form a cell spot, with at least one drop of about 4% paraformaldehyde (PFA);
d) rinsing the previously fixed cell spot;
e) allowing the previously rinsed cell spot to dry;
f) adding to the previously dried cell spot at least one drop of a mounting medium compatible with the observation of fluorescence;
g) affixing a transparent cover slip on the cell spot arising from step f).
8. The method of claim 7 , wherein the at least one centrifugation system is centrifuged at about 600 rpm for about 5 minutes.
9. The method of claim 7 , wherein chemically fixing the cells thus projected on the slide to form a cell spot are comprises chemically fixing with at least one drop of about 4% paraformaldehyde (PFA) for about 10 minutes.
10. The method of claim 7 , wherein the previously fixed cell spot is rinsed with phosphate buffered saline (PBS) buffer.
11. The method of claim 7 , wherein the transparent cover slip is a transparent glass cover slip.
12. The method according to claim 1 , characterized in that, when the leukocytes of said sample are mostly polymorphonuclear neutrophils, the state of said individual is infectious if the autofluorescence intensity measured in step i) is significantly lower than the control value.
13. The method of claim 1 characterized in that, when the state of said individual is infectious, said individual is suffering from a bacterial infection.
14. The method of claim 1 characterized in that the autofluorescence intensity of the cells of said sample is measured on cells that are chemically fixed and placed on a transparent glass slide.