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Clinical Trial
. 2025 Mar 3;135(5):e185463.
doi: 10.1172/JCI185463.

A multi-analyte blood test for acute spinal cord injury

Tej D Azad  1 , Kathleen R Ran  1 , Joshua D Materi  1 , Divyaansh Raj  1 , Timour Al-Khindi  1 , Sameer Gabbita  1 , Marvin Li  1 , Elizabeth T Wang  1 , A Karim Ahmed  1 , Megan Parker  1 , Anita L Kalluri  1 , Daniel Lubelski  1 , Christopher M Jackson  1 , Daniel M Sciubba  2 , Jon D Weingart  1 , Ali Bydon  1 , Timothy F Witham  1 , David W Nauen  3 , Srinivasan Yegnasubramanian  3   4 , Nicholas Theodore  1 , Chetan Bettegowda  1
Affiliations
Clinical Trial

A multi-analyte blood test for acute spinal cord injury

Tej D Azad et al. J Clin Invest. .

Abstract

BACKGROUNDRapid diagnosis to facilitate urgent intervention is critical for treatment of acute spinal cord injury (SCI). We hypothesized that a multi-analyte blood biomarker would support point-of-care SCI diagnosis, correlate with injury severity, and predict long-term neurologic outcomes.METHODSDroplet digital PCR (ddPCR) assays were designed to amplify differentially hypomethylated genomic loci in spinal cord tissue. An optimized ddPCR assay was applied to cell-free DNA (cfDNA) from plasma samples collected from prospectively enrolled acute SCI patients. Targeted proteomic profiling was also performed. Spinal cord-derived cfDNA and plasma proteins were tested for their association with SCI and ability to predict conversion in American Spinal Injury Association (ASIA) score at 6 months.RESULTSA bespoke ddPCR assay detected spinal cord-derived cfDNA in plasma of 50 patients with acute SCI (AUC: 0.89, 95% CI 0.83-0.95, P < 0.0001). Levels of cfDNA were highest in patients with the most severe injury, i.e., ASIA A, compared with those with ASIA B (P = 0.04), ASIA C (P = 0.009), and ASIA D injuries (P < 0.001). Dimensionality reduction identified 4 candidate proteins (FABP3, REST, IL-6, NF-H) that were integrated with spinal cord-derived cfDNA to derive the Spinal Cord Injury Index (SCII), which has high sensitivity and specificity for SCI diagnosis (AUC: 0.91, 95% CI 0.82-0.99, P < 0.0001), correlates with injury severity (P < 0.0001), and predicts 6-month neurologic improvement (AUC: 0.77, 95% CI 0.61-0.93, P = 0.006).CONCLUSIONThe detection of spinal cord-derived cfDNA and plasma protein alterations as part of a multi-analyte blood test can inform SCI diagnosis and prognosis.FUNDINGNorth American Spine Society Young Investigator Award; Morton Cure Paralysis Fund.

Keywords: Neurological disorders; Neuroscience.

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Figures

Figure 1
Figure 1. Overview of study for detection of acute SCI via a multi-analyte assay of peripheral blood.
Figure 2
Figure 2. Cohort overview with patient, operative, and injury characteristics.
NA, not applicable.
Figure 3
Figure 3. Methylation-based cfDNA markers of SCI.
(A) Structure at a genomic locus identified as spinal cord–specific biomarker. Lollipops represent CpG sites. Red highlights hypomethylated CpG sites identified in the Illumina MethylationEPIC 850K array. Arrows mark positions of PCR primers. (B) Spike-in experiment demonstrating the ddPCR assay sensitivity for detection of spinal cord–derived biomarkers. Human spinal cord gDNA was mixed with human leukocyte gDNA in the indicated proportions (0 to 5%), and the concentration (copies/uL) of fully unmethylated spinal cord–derived markers was determined. Data represent mean ± SEM. (C) Receiver operating curve demonstrating the ability of our hypomethylation-based ddPCR assay to discriminate between acute SCI patients and healthy controls. (D) Plasma concentration in genome equivalents (hGE/mL) of spinal cord–derived cfDNA in acute SCI patients. (E) Percentage of spinal cord–derived cfDNA out of total measured cfDNA in acute SCI patients. Mann-Whitney U tests were used to compare groups in panel E. AUC, area under the receiver operating curve.
Figure 4
Figure 4. Proteomic profiling of plasma from patients with acute SCI.
(A) PCA of protein biomarkers measured in acute SCI patients versus healthy controls. (B and C) Difference in GFAP and NF-L concentrations between acute SCI patients detected using ddPCR and healthy controls. (D–G) Difference in FABP3, REST, IL-6, and NF-H concentrations between acute SCI patients detected using ddPCR and healthy controls. Mann-Whitney U tests were used to compare groups in panels B–G.
Figure 5
Figure 5. Development of the SCII.
(A) Correlation between concentrations of spinal cord–derived cfDNA and FABP3, REST, IL-6, and NF-H. (B) Receiver operating characteristic (ROC) curve for ability of SCII to discriminate between patients with acute SCI and healthy controls. (C) Distribution of SCII across injury severity. Yellow indicates patient who achieved 6-month ASIA conversion. (D) Receiver operating characteristic curve for ability of preoperative SCII to predict 6-month ASIA conversion. Mann-Whitney U tests were used to compare groups in panel C.
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