Rapid one-step protocol for exploring immune cells in cerebrospinal fluid

Flow cytometry analysis of the cerebrospinal fluid's immune cell subset profiles can be used to help in the diagnosis and prognosis of nervous system pathologies. However, the cells need to be analyzed rapidly after collection. Furthermore, immune cells are quite rare in CSF, samples are limited in volume and immune staining protocols, sometime including washing steps, are not fully standardized among laboratories. It is therefore useful and necessary to improve the pre-analytical and analytical processes. We have thus developed the one-step protocol for the analysis of CSF cells, without washing step, using a panel of labeled antibodies for both enumeration of red and white blood cells and the analysis of main immune cell subsets. We first investigated repeatability of this protocol for the main immune cell subsets count and staining and its stability over a 3-day period using a mild fixation protocol. Then, using this protocol, we evaluated the impact of delays in treatment after lumbar puncture. We demonstrate that cell count and staining are stable over 3 days in both CSF and whole blood samples. Direct labeling with no washing step combined with the cytometer's analytical performance enable precise and reproducible cell counting. Using this simple and robust protocol, we showed that monocytes are lost if the biological samples are not processed rapidly following the lumbar puncture. This one-step method allows for rapid, standardized, and reproducible multiparametric CSF immune cell analysis.