SMN only detected in cerebral spinal fluid samples containing hemoglobin.

<p>The CSF samples obtained from healthy volunteers were concentrated prior to analysis, and the sensitivity of the SMN-ECL immunoassay was 0.3 pg/mL. Hemoglobin was measured using a hemoglobin immunoassay (Bethyl Laboratories E88-135). Approximately 3 pg of SMN correspond to 10,000 ng of hemoglobin in 1 mL of whole blood from a healthy adult. LLQ: lower limit of quantification.</p

SMN protein was found in various cell types in whole blood.

<p>Platelets, red blood cells, PBMCs and reticulocytes were separated by a fractionation procedure.</p

Correlation of SMN protein levels between tissues.

<p>SMN protein levels were correlated in WT and C/C-allele SMA mice between (A) whole blood and brain and (B) whole blood and spinal cord.</p

SMN protein levels in capillary and venous blood obtained over time from healthy volunteers did not vary significantly.

<p>Venous (A, B) and capillary (C) whole blood samples were obtained at 0, 4, 6, 24, 48, 72 hours and 1, 2, 3, 4 weeks from five healthy individuals. Fig 2B is an expanded version of Fig 2A. (D) SMN protein levels in capillary blood correlated significantly with SMN levels in venous blood (r² = 0.76, p < 0.0001).</p

SMN protein levels in tissues of C/C-allele and WT mice measured by SMN-ECL and SMN-ELISA.

<p>Protein levels were measured in the spinal cord of C/C-allele and WT mice using (A) SMN-ECL and (B) SMN-ELISA. Both assays showed a statistically significant difference in SMN levels between WT and C/C-allele mice (p < 0.0001). (C) SMN protein levels in the whole blood of C/C-allele, WT and heterozygous mice measured by SMN-ECL.</p

SMN protein levels in WT and C/C-allele SMA mice of different ages.

<p>SMN protein levels were measured by SMN-ECL at various days post-birth in (A) brain, (B) whole blood, (C) spinal cord, and (D) gastrocnemius muscle.</p

SMN protein stability in whole blood: short term, long term, and freeze / thaw events.

<p>Whole blood of healthy subjects was used in the study. (A) SMN protein was measured in previously frozen, undiluted whole blood samples incubated at 4°C or at room temperature. (B) SMN protein was measured in undiluted whole blood samples of two subjects stored at -80°C or at -20°C. (C) SMN protein levels were measured in samples of two subjects that went through freeze-thaw cycles. *FDA acceptance criteria (below 85%).</p

SMN protein levels in SMA patient and control whole blood samples.

<p>(A) SMN levels with respect to age in all subjects. (B) SMN protein levels were measured in SMA patients with 2, 3 and 4 copies of <i>SMN2</i>. In patients over 2 months of age, SMN levels were significantly greater in SMA patient samples with 4 <i>SMN2</i> copies relative to those with 2 and 3 <i>SMN2</i> copies (p = 0.0001). (C) SMN was also measured in three control samples and SMN levels were found to be significantly greater in the control samples relative to levels in SMA patients over 2 months of age (p < 0.0001).</p

Clinical data of SMA and Control Subjects.

*<p>ANOVA for continuous variables; Fisher exact test for categorical variables.</p><p>There were no significant differences in age or gender across the recruitment cohorts. A key goal of this study was to minimize the confounding correlation between present age and functional status. This goal was largely achieved, both overall and within SMA groups Type II and III, and to a partial extent, SMA Type I through a competitive recruitment plan managed through the data coordinating center at the New England Research Institutes (NERI). The Modified Hammersmith Motor Function Scale differentiated between SMA subjects and controls and between Type I, II and III subjects, as did respiratory support, reflecting current level of function. FVC and the nutritional assessment score significantly distinguished between SMA type; however, BMI proved to be far less discriminatory.</p

Top 20 univariate markers across all outcome measures.

<p>Each numeric entry indicates the number of statistical tests in which the described analyte was found to be a statistically significant biomarker when evaluated for regression against the given outcome measures or categorical characteristics: SMA Types, Disease Onset, Current Level of Function and Respiratory Support. Each outcome measure or characteristic inherently has a certain number of possible sub-categories available for pairwise statistical testing (in parentheses for each outcome or characteristic): 12 for SMA Types, 5 for Disease Onset, 15 for current level of function and 4 for Respiratory Support (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035462#pone.0035462.s001" target="_blank">Table S1</a> for details). The Total denotes the sum of all outcomes for which the protein is a marker and indicates the overall strength of the relationship between the analyte and SMA values; the maximum possible number for Total is 36, as all the analytes above are also statistical significant regressors with the MHMS outcome. AAA – Amino acid analysis; FFA – Free fatty acid; Q-VALUE – significance corrected for the effect of multiple comparisons; STD – Standard deviation; UCL – Upper 95% confidence limit; LCL – lower 95% confidence limit (on the value of slope). Slope values listed in red are positive values and those in green negative values.*These are distinct isoforms detected by the LC/MS method.</p