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

Abstract

ObjectivesSurvival 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.MethodsSeveral 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.ResultsDelaying 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.ConclusionsThe 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.

A Comparative Study of SMN Protein and mRNA in Blood and Fibroblasts in Patients with Spinal Muscular Atrophy and Healthy Controls

“…In humans, similar findings have been reported in a smaller number of tissues that include brain [43], muscle [43], whole blood [32, 36], PBMCs [12, 15, 18, 22, 29, 33, 34], fibroblasts [20, 26, 29] and buccal cells [36, 37]. SMN protein levels have also been investigated in body fluids, most notably in cerebrospinal fluid as an exploratory biomarker in a phase 1 study of intrathecal administration of antisense oligonucleotides [44], but also in urine, plasma and saliva [33, 36, 37].…”

“…Although SMN protein levels have been studied in many (experimental) cell types [12, 15, 18, 20, 22, 26, 29, 32–34, 36–43], there are no comparative studies of SMN expression in tissues that can be easily obtained. Significant differences in SMN protein levels have recently been found in platelets, red blood cells and PBMCs, which underlines the importance to investigate tissue-specific SMN expression [32, 36].…”

“…SMN protein levels in PBMCs and fibroblasts were quantified using the standardized SMN ELISA (2012, #ADI-900-209, Enzo Life Sciences, Farmingdale, NY) [33, 34] and expressed as nanogram per 1 gram of total protein.…”

Baseline results of the NeuroNEXT spinal muscular atrophy infant biomarker study

“…We found variability in PBMC yield from patient samples and were not able to process some samples because of insufficient material. Since the start of this project, it is now clear that measurement of SMN protein levels from PBMC samples collected using the CPT tubes is not optimal and a whole blood methodology is now available 36, 37. The protocol was modified for subsequent longitudinal visits to include collection of whole blood so that future analysis of SMN protein in this study may be improved.…”

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

“…Lines of evidence using quantitative assays suggest that SMN expression across species and tissue type usually decline with age. 154,161,168 Whether this decline is potentiated by disease stage and severity in patients remains unclear, without larger quantitative studies. In summary, factors influencing the measurement of SMN protein in samples and specimens include tissue and cell type, buffer conditions, and age of the specimen or cell line donor should also be weighed when designing any new screening assays.…”