Introducing 8-color flow-cytometry to the routine workup of cerebrospinal fluid

Background and aim: Cerebrospinal fluid (CSF) analysis plays an important role in the diagnostic workup of many neurological diseases. CSF cells are usually assessed by their morphology. Due to the limited amount of CSF cells available for analysis, immunocytometric methods have received little attention so far. Based on the development of new cytometers, we aimed to assess, whether the main immune cell subsets in CSF can be determined in a single staining during routine CSF workup.

Methods: 8-color flow-cytometry was used to phenotype the CSF cells. A single staining with monoclonal antibodies against CD45 (all hematopoetic cells), CD14 (monocytes), CD3 (T cells), CD4 (T helper cells), CD8 (cytotoxic T cells), CD56 (NK and NK T cells), CD19 (B cells, plasmablasts), CD138 (plasmablasts and plasmacells) was established that allows to determine the distribution of the major immune cell subpopulations and to identify abnormal cells in the CSF. Dependent on the CSF cell count, 1 to 6ml CSF was used for analysis.

Results and conclusion: We examined the CSF of 325 patients with different non-inflammatory and inflammatory diseases of the nervous system. Based on this analysis we determined disease specific immune cell distributions. The presence of plasma blasts was indicative of an acute or chronic inflammatory disease of the CNS. Decreased numbers of CD4+ T cells (or the ratio of CD4+/CD8+ T cells) was frequently seen in immunosuppressed patients in particular with HIV infection. The presence of CD45 negative cells were suggestive of meningeal infiltration by carcinoma cells, which often expressed the CD138 marker. Similarely abnormally high numbers of B cells or T cells with monoclonal staining pattern of other markers were highly suggestive of mengeosis lymphomatosa.

In summary, 8-color-flow-cytometric analysis with a single staining allows detailed phenotyping of CSF cells, even when the CSF cell number is in the normal range. The method extents routine CSF analysis and provides information that can not be assed by CSF cell morphology analysis.