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Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein

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...

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Autores principales: Kobayashi, Dione T., Decker, Douglas, Zaworski, Phillip, Klott, Karen, McGonigal, Julie, Ghazal, Nabil, Sly, Laurel, Chung, Brett, Vanderlugt, James, Chen, Karen S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511312/
https://www.ncbi.nlm.nih.gov/pubmed/23226377
http://dx.doi.org/10.1371/journal.pone.0050763
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author Kobayashi, Dione T.
Decker, Douglas
Zaworski, Phillip
Klott, Karen
McGonigal, Julie
Ghazal, Nabil
Sly, Laurel
Chung, Brett
Vanderlugt, James
Chen, Karen S.
author_facet Kobayashi, Dione T.
Decker, Douglas
Zaworski, Phillip
Klott, Karen
McGonigal, Julie
Ghazal, Nabil
Sly, Laurel
Chung, Brett
Vanderlugt, James
Chen, Karen S.
author_sort Kobayashi, Dione T.
collection PubMed
description 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.
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spelling pubmed-35113122012-12-05 Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein Kobayashi, Dione T. Decker, Douglas Zaworski, Phillip Klott, Karen McGonigal, Julie Ghazal, Nabil Sly, Laurel Chung, Brett Vanderlugt, James Chen, Karen S. PLoS One Research Article 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. Public Library of Science 2012-11-30 /pmc/articles/PMC3511312/ /pubmed/23226377 http://dx.doi.org/10.1371/journal.pone.0050763 Text en © 2012 Kobayashi et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kobayashi, Dione T.
Decker, Douglas
Zaworski, Phillip
Klott, Karen
McGonigal, Julie
Ghazal, Nabil
Sly, Laurel
Chung, Brett
Vanderlugt, James
Chen, Karen S.
Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein
title Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein
title_full Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein
title_fullStr Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein
title_full_unstemmed Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein
title_short Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein
title_sort evaluation of peripheral blood mononuclear cell processing and analysis for survival motor neuron protein
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511312/
https://www.ncbi.nlm.nih.gov/pubmed/23226377
http://dx.doi.org/10.1371/journal.pone.0050763
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