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Cellular Immunophenotyping of Cerebrospinal Fluid From Patients with Neurological Disorders


Anup Regunathan Photo

Johns Hopkins University (Maryland)

Abstract

Immunophenotyping of the cerebrospinal fluid (CSF) cells by flow cytometry has proven to be a powerful method that has already provided an important insight into the MS disease process. Previous experiments have optimized combinations of commercially-available flourochrome-conjugated antibodies to distinguish and quantify 14 major subpopulations of immune cells in the blood and CSF to assess the extent of their in vivo activation. The aim of this study is to develop a new analysis template that distinguishes the size and granularity of identical cell types in the blood and CSF. If our hypothesis, that analysis of size and granularity may provide additional information about activation status of different immune subtypes was correct, then one could obtain functional information from immunophenotyping panels that do not contain activation markers, such as CD25 or CD80.

Cells that are under scrutiny are stained with a set of developed antibodies and processed by a flow cytometer. Using the data, we use FACSDiva, accompanying software, to gate the cell population. “Gating” is the process by which we subjectively separate specific cell populations from the total events. Through this method, we are able to identify the levels of cell types in patients with various diseases and observe patterns to draw correlations between diagnoses.

Overall data comparisons between RRMS patients treated with daclizumab and those without treatment reveal differences in the absolute numbers of immune cell subtypes. Patients that received treatment have decidedly lower absolute number counts for all cells of the adaptive immune system. In treated patients, the CD4+ small and CD8+ large T cell population proportion are decreased. In our hypothesis, a larger size, along with higher granularity, represents activated cells. Daclizumab HYP (DAC-HYP) is a CD25 blocking MS treatment undergoing a clinical trial. As CD25 is an active marker for leucocytes, DAC-HYP is expected to inhibit their activation. This expectation was fulfilled in the observed gating as it is shown that the sizes of T and B cells are decreased along with granularity.

The results show the “normalization” of intrathecal immune abnormalities in daclizumab-cohort as compared to RR-MS patients. Specifically, decreasing numbers of activated (large) plasma-blasts and B cells and increasing numbers and normalization of the monocyte phenotype in daclizumab-treated patients are observed. Overall, this type of analysis has a potential to identify those aspects of the intrathecal immune responses that are specifically pathogenic in untreated MS patients.

Last updated November 27, 2013