Related Experiment Video
Updated: Sep 14, 2025
Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis
Published on: November 17, 2020
Rapid one-step protocol for exploring immune cells in cerebrospinal fluid
Ewa Wallisky Millet1, Fabrice Malergue2, Gregorio Petrirena Hernandez3
1Beckman Coulter Life Science, Immunotech, Research and development, Marseille, France; Aix Marseille Univ, INSERM, INS, UMR 1106, Inst Neurosci Syst, Marseille, France.
Insights
A new one-step flow cytometry protocol enables rapid, standardized analysis of cerebrospinal fluid (CSF) immune cells. This method ensures accurate cell counting and subset profiling, crucial for diagnosing nervous system disorders, even when samples are delayed.
Area of Science:
- Neuroimmunology
- Clinical Diagnostics
- Flow Cytometry
Background:
- Cerebrospinal fluid (CSF) immune cell profiling aids in diagnosing nervous system pathologies.
- Current methods face challenges due to rapid cell degradation, limited sample volume, and non-standardized staining protocols.
- There is a critical need for improved pre-analytical and analytical processes for CSF immune cell analysis.
Purpose of the Study:
- To develop and validate a one-step flow cytometry protocol for analyzing CSF immune cells.
- To assess the repeatability, stability, and impact of sample processing delays on CSF immune cell analysis.
- To establish a rapid, standardized, and reproducible method for multiparametric CSF immune cell analysis.
Main Methods:
- Developed a one-step antibody-based protocol for CSF cell analysis without a washing step.
- Validated the protocol's repeatability and stability over 3 days using mild fixation.
- Assessed the impact of post-collection delays on cell counts and immune staining.
Main Results:
- The one-step protocol demonstrated high repeatability and stability for immune cell subset counts and staining over 3 days.
- Direct labeling without washing, coupled with cytometer performance, ensures precise and reproducible cell counting.
- Significant loss of monocytes was observed in samples not processed rapidly after lumbar puncture.
Conclusions:
- The developed one-step protocol provides a rapid, standardized, and reproducible method for multiparametric CSF immune cell analysis.
- This protocol addresses limitations of existing methods, improving diagnostic and prognostic capabilities for neurological conditions.
- Timely processing of CSF samples is essential to prevent the loss of critical immune cell populations like monocytes.
Abstract:
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.
