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

<div><h3>Objectives</h3><p>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.</p> <h3>Methods</h3><p>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.</p> <h3>Results</h3><p>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.</p> <h3>Conclusions</h3><p>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.</p> </div

Study 1: Impact of short-term PBMC processing delays.

<p>PBMCs were collected via CPT tubes from 3 individuals and processed with delays of 0, 45 minutes, 2 h and 24 h prior PBMC isolation by centrifugation. <b>A:</b> Cell viability was similar at all timepoints for all subjects, ranging from 94–99%. <b>B:</b> Cell counts were consistent through 45 minutes, but were significantly reduced by 30–40% with delays of 2 h and 24 h compared to the 0 h timepoint. <b>C:</b> Total soluble protein was consistent with up to 2 h processing delays, however at 24 h there was a trend towards increased protein concentrations by up to 40%. <b>D</b>: SMN levels with 24 h processing delays were accordingly reduced when normalized by total protein. <b>E</b>: SMN levels by cell counts were similar with all delays examined, albeit with trends for higher variability than the SMN signal generated by protein normalization. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g001" target="_blank">Figure 1</a> error bars indicate the minimum and maximum values while the horizontal bar indicates the median value.</p

Dilution adjusted SMN Signal in whole blood and blood pellets (pg/mL blood).

<p>Whole blood and red blood cell pellets were evaluated for SMN signals with the four individuals from Study 3. SMN protein was detectable in both matrices. Dilutional linearity was not observed across all samples or at the same dilutions across samples, suggesting there was biological interference in blood pellet and whole blood.</p

Study SMAF-001: SMN in SMA patients and carriers.

<p>SMN protein (normalized by total protein) was evaluated in Type 2 and 3 SMA patients and Carriers for differences by motor function and stability of signal with lysate storage. <b>A</b>: SMN protein classified by Type and Carrier status completely overlapped between groups. When adjusted by age there was a significant difference in SMN between patients and adult carriers (p<0.001) but not between SMA Types (p = 0.75). <b>B</b>: SMN levels differentiated by current motor function appeared to distinguish between sitters and ambulatory patients (P<0.05), with the exception of a 1.4 year old recently diagnosed child who could sit with assistance. When controlling for age this trend was not statistically significant (p = 0.97). <b>C</b>: SMN levels were plotted against subject age and there was a trend towards lower SMN levels in older individuals. The correlations for age-related decline were different between SMA and Carriers, with R²-values at 0.65 (p<0.005) and 0.30 (p<0.05) respectively. Arrows depict subjects taking valproic acid, a drug with putative SMN-upregulating effects. <b>D</b>: Samples from each subject were processed and frozen as lysates for storage for 48 h, 1 month, 3 months, and 6 months. Comparability of signals between the 48 h timepoint and subsequent timepoints was generally high, with R²-values of 0.79–0.94 for each timepoints. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g008" target="_blank">Figure 8A–B</a> error bars indicate the minimum and maximum values while the horizontal bar indicates the median value. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g008" target="_blank">Figure 8D</a> the trendlines depict R²-values.</p

Study 2: Impact of post-isolation delay and cell freezing.

<p>SMN signals were evaluated in PBMCs from 4 individuals that were analyzed with 45 minute, 2 h and 24 h delays after cell isolation. A subsample from each timepoint was frozen to assess post-freezing viability and SMN signals. <b>A:</b> Cell viability was generally lower in PBMCs that had been frozen, ranging from 88–95% viability compared to 93–98% in unfrozen cells. Statistical comparisons were made to the 0 h timepoint. <b>B</b>: The comparative recovery of viable PBMCs after freezing relative to fresh samples was only ∼40–60% at all timepoints, suggesting a major loss of cells in the freezing process. <b>C</b>: Delaying the processing of isolated PBMCs to lysates had no impact on protein concentrations through delays of 2 h, however there was again a trend for increased protein concentrations in samples left for 24 h. <b>D</b>: SMN levels (normalized to protein concentrations) in unfrozen PBMCs were generally similar across all timepoints, despite wide variability in signals. <b>E</b>: Analysis of SMN by cell counts revealed that SMN signals in unfrozen cells tended to increase with post-isolation delays. Frozen cell SMN signals generally seemed to decrease over time compared to both frozen cells processed with minimal delays or compared to unfrozen cells. Signals from frozen cells with 24 h post-isolation delays were lower than unfrozen cells. Error bars represent minimum and maximum values. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g002" target="_blank">Figure 2</a> the bodies of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median.</p

Optimizing cell densities and reagents for lysis and SMN extraction.

<p>To examine the impact of lysates and lysis reagents on SMN signal a number of factors were tested. <b>A</b>: SMN signal was evaluated in lysates created from a single sample purchased from AllCells. PBMCs were processed in ER4 at densities of 10⁶, 10⁷ and 5×10⁷ cells/mL and the resulting lysates diluted from 1∶2 to 1∶32 for analysis in the SMN ELISA. At the highest concentration SMN signal was linear with adjusted dilutions until after 1∶16, suggesting interference at this density. Concentrations of 10⁶ and 10⁷ cells/mL had parallel linear increases in signal between 1∶2 and 1∶8, with the 10⁷ dilution continuing to rise at the 1∶32 dilution. <b>B</b>: Treatment of PBMCs in erythrocyte buffer for 5 minutes increased the SMN signal by roughly twofold over the untreated cells. <b>C</b>: SMN protein standard was formulated in different dilutions of ER4 at different concentrations to determine the impact on spiked SMN signal recovery. Use of 1∶4 to 1∶16 ER4 reagent allowed for >90% signal recovery across several SMN levels. Error bars represent standard deviations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g004" target="_blank">Figures 4A–B</a>. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g004" target="_blank">Figure 4C</a> error bars indicate the minimum and maximum values while the horizontal bar indicates the median value.</p

Study SMAF-001 demographics.

<p>SMA Type and Carrier status was self-reported by subjects or their consenting parents. Subjects identified as Type 2/3 were included in the Type 3 group.</p

Study 4: Subcellular PBMC populations and SMN signal.

<p>SMN levels were analyzed by CD+ cell subtype at the 7d timepoint from Study 4. <b>A</b>: Analysis of SMN by PBMC cell subpopulation revealed that the CD14+ population had statistically significant reductions in SMN levels. <b>B</b>: Fractionation of PBMCs with normalization by cell count showed no differences in SMN signal in group analysis. <b>C</b>: Evaluation of total soluble protein levels by CD+ population revealed that CD14+ cells had double the protein concentrations of CD4+, CD8+, CD19+, and CD56+ cells. This differential is sufficient to drive variability in situations that cause CD14+ populations to fluctuate. <b>D</b>: SMN levels (normalized by protein) show consistently lower levels in CD14+ fractions compared to all other fractions, with differences up to 7x within individual PBMC subpopulations. <b>E</b>: SMN in individuals as measured by cell counts were also variable ranging up to 3.5x between individuals’ subcellular populations, but was overall less variable than protein normalized SMN measures. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g005" target="_blank">Figures 5A–C</a> the bodies of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g005" target="_blank">Figures 5D–E</a> error bars depict standard deviation.</p

SMAF-001 SMA patient characteristics.

*<p>Subject 3004 is the sibling of Subject 3002, Subject 3026 is the sibling of Subject 3025, and Subject 3029 is the sibling of Subject 3028.</p>**<p>Subject 3008 was not diagnosed until age 50.</p

Study 3: Comparison of PBMC isolation methods and SMN measures from various blood fractions.

<p>Due to the variability in SMN signal and protein concentrations seen with CPT tube PBMC isolation, other methods were explored using the four subjects from Study 2. <b>A</b>: PBMC yield was greatest in samples collected with EDTA tubes and subsequent Lymphoprep gradient separation, and showed no statistically significant changes with isolation delays of up to 24 h. <b>B</b>: Total soluble protein tended to increase with 24 h delays in samples collected by EDTA tube and isolated by CPT tubes, while there was no obvious change in protein concentrations in EDTA/Lymphoprep processing. <b>C</b>: SMN as measured by total protein tended to decrease with isolation delays with EDTA/CPT processing. SMN signals were similar with delays up to 24 h with EDTA/Lymphoprep processing. <b>D</b>: SMN by PBMC counts was variable for both EDTA/CPT and EDTA/Lymphoprep processing methods. However, the EDTA/Lymphoprep values were generally overlapping and did not appear to decrease from the no-delay timepoint (t = 0). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050763#pone-0050763-g003" target="_blank">Figure 3</a> body of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median.</p