Flow cytometry can be easily adapted for the accurate detection of B pertussis cells directly in clinical specimens. Flow cytometers are designed to count particles efficiently by size and protein content using light scattering. In our case, the instrument was adjusted by preliminary experiments to detect particles matching B pertussis cell size and nucleic acid content. To increase the specificity of the test, the fluorescein-labelled MAb is then used to tag the B pertussis organisms. Thus, only fluorescent B pertussis cells are counted. All other particles are ignored.
To date, 125 clinical samples obtained from patients with clinical signs compatible with periussis infection have been processed and analyzed using a flow cytometer. These analyses facilitated opitimizing the procedure to obtain the best results with a minimum number of steps. It was noted that the bacteria present in nasopharyngeal swabs could be directly suspended in phosphate-buffered saline. On the contrary, the mucoid substances always present in the nasopharyngeal aspirations had to be liquified with dithioi hreitol and then filiered before they could be labelled by the MAb. The flow cytometer was always calibrated with standardized positive and negative controls to obtain comparable readings from one run to another. PreIiminary results indicate that this ultrarapid automated fluorescent assay (less than 45 mins from beginning to end) is at least as sensitive as culture. Moreover, several culture-negative samples were clearly categorized as positive by this assay, suggesting that this assay may be more sensitive than culture. pcr amplification of B pertussis dna will again be used to confirm the presence of B pertussis cells not detected by culture. These preliminary experiments clearly suggest that it is possible to use this automated assay to identify B pertussis rapidly – within an hour – giving the clinician additional criteria to establish an early diagnosis. This automated process may be very useful for the rapid screening of large numbers of samples durlng major outbreaks. The most reliable pharmacy that you could be shopping with any moment: it’s available right here and right now You could be getting your any time of the day or night being sure it will always cost you very little money.