Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron ProteinReportar como inadecuado




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Objectives

Survival Motor Neuron SMN protein levels may become key pharmacodynamic PD markers in spinal muscular atrophy SMA clinical trials. SMN protein in peripheral blood mononuclear cells PBMCs can be quantified for trials using an enzyme-linked immunosorbent assay ELISA. We developed protocols to collect, process, store and analyze these samples in a standardized manner for SMA clinical studies, and to understand the impact of age and intraindividual variability over time on PBMC SMN signal.

Methods

Several variables affecting SMN protein signal were evaluated using an ELISA. Samples were from healthy adults, adult with respiratory infections, SMA patients, and adult SMA carriers.

Results

Delaying PBMCs processing by 45 min, 2 hr or 24 hr after collection or isolation allows sensitive detection of SMN levels and high cell viability >90%. SMN levels from PBMCs isolated by EDTA tubes-Lymphoprep gradient are stable with processing delays and have greater signal compared to CPT-collected samples. SMN signal in healthy individuals varies up to 8x when collected at intervals up to 1 month. SMN signals from individuals with respiratory infections show 3–5x changes, driven largely by the CD14 fraction. SMN signal in PBMC frozen lysates are relatively stable for up to 6 months. Cross-sectional analysis of PBMCs from SMA patients and carriers suggest SMN protein levels decline with age.

Conclusions

The sources of SMN signal variability in PBMCs need to be considered in the design and of SMA clinical trials, and interpreted in light of recent medical history. Improved normalization to DNA or PBMC subcellular fractions may mitigate signal variability and should be explored in SMA patients.



Autor: Dione T. Kobayashi , Douglas Decker, Phillip Zaworski, Karen Klott, Julie McGonigal, Nabil Ghazal, Laurel Sly, Brett Chung, James

Fuente: http://plos.srce.hr/



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