Multi-Biomarker Panels in ALS and MND — NfL, pNfH, GFAP, and TDP-43
No single biomarker tells the complete story of motor neuron disease. NfL reflects axonal injury rate. pNfH may capture large-fiber motor neuron degeneration more specifically. GFAP reflects glial activation and non-motor pathology. TDP-43 pathology is a disease-defining feature of most ALS and some related conditions. The emerging consensus in the field — documented in multiple 2025 reviews — is that panels of biomarkers will outperform any single marker for diagnosis, prognosis, and trial stratification. This page explains why, what the evidence currently shows, and what it means for PLS.
Why single biomarkers are not enough
NfL is the most validated fluid biomarker for motor neuron disease, but it has known limitations. It rises with any cause of axonal injury — stroke, traumatic brain injury, multiple sclerosis, Alzheimer's disease, and even vigorous exercise transiently. It is elevated with age. And it cannot distinguish PLS from ALS, or ALS from other conditions with motor neuron involvement. In a diagnostic context, a high NfL in a patient with a motor syndrome tells you that neurodegeneration is occurring at a rate above baseline — but not what is causing it.
pNfH adds information about the heavy chain component — potentially more specific to large myelinated motor fibers — and its MIROCALS trial data show it can stratify patients in ways that predict treatment response. But pNfH has its own limitations: it is lower in PLS than ALS (due to slower degeneration), raising questions about assay sensitivity in slowly progressive patients.
GFAP captures a different biological process — glial activation rather than axonal loss — and is particularly informative for non-motor features. But elevated GFAP can reflect astrogliosis from many causes, limiting its disease specificity.
Combining these markers — measuring the pattern rather than any single value — is more discriminating than using any one in isolation. A patient with high NfL, high pNfH, and high GFAP has a very different biological profile from a patient with high NfL but normal pNfH and GFAP. Panels can encode more information.
Current evidence on multi-biomarker panels in ALS
NfL + pNfH combination
Several studies have evaluated NfL and pNfH together in ALS cohorts and found that combining the two provides better characterization of disease severity than either alone. The 2022 Journal of Neurophysiology study on serum pNfH specifically documented its complementarity to NfL — the two markers were correlated but not redundant, each adding predictive value for disease severity characterization. In the context of MIROCALS, pNfH stratification revealed a treatment effect hidden in the overall trial population — a finding NfL stratification alone would not have produced.
NfL + GFAP combination
The 2025 Scientific Reports study that examined serum NfL and GFAP simultaneously found that both were significantly elevated in ALS versus controls (NfL: 81.49 ± 47.06 vs. 9.21 ± 3.05 pg/mL; GFAP: 104.42 ± 37.31 vs. 57.71 ± 11.64 pg/mL). NfL correlated with disease progression rate; GFAP correlated with disease duration and non-motor features. The two markers captured different dimensions of disease biology and were more informative together than separately.
Three-marker and four-marker panels
The 2025 Frontiers Molecular Biosciences review synthesized the evidence for a multi-marker approach, concluding that NfL + pNfH + GFAP together provide a more complete picture of ALS disease activity than any single marker. The review positions this combination as the direction the field is moving toward, acknowledging that no single validated panel has yet been formally established for clinical use.
The 2023 ScienceDirect protein biomarkers review took a broader view, examining the full landscape of protein biomarkers across diagnosis and prognosis in ALS. Beyond NfL, pNfH, and GFAP, the review covered TDP-43 protein quantification as a disease-specific marker.
TDP-43: the disease-specific biomarker candidate
TDP-43 (TAR DNA-binding protein 43) is a nuclear protein that, when mislocalized and aggregated in the cytoplasm of motor neurons, is the pathological hallmark of most ALS (approximately 97% of ALS cases show TDP-43 pathology) and of the related condition FTLD-TDP. Unlike NfL, pNfH, and GFAP — which reflect disease severity and progression rate but are not specific to ALS — TDP-43 pathology is close to disease-defining.
The challenge has been measuring TDP-43 in accessible biological fluids. TDP-43 in CSF and blood has been technically difficult to quantify in ways that reliably reflect the pathological burden in motor neurons. However, progress is being made:
- Misfolded or phosphorylated TDP-43 fragments (pTDP-43) have been detected in CSF of ALS patients at levels above controls in some studies.
- Ultrasensitive assays (Simoa, proximity extension assay) are beginning to enable reliable blood-based detection.
- Treatments specifically targeting TDP-43 pathology (including some antisense oligonucleotide programs) would require TDP-43 as a pharmacodynamic biomarker — creating strong development incentives for validated TDP-43 assays.
For PLS specifically, TDP-43 relevance is less clear. The pathological hallmark of PLS is corticospinal tract degeneration, and TDP-43 pathology — while present in some pathological PLS studies — is not as consistently documented as in ALS. Whether TDP-43 measurement would add to an NfL + pNfH + GFAP panel in PLS is an open question.
Honest assessment of where the field stands
No validated multi-biomarker panel exists for ALS or PLS as of 2026. The evidence base shows that panels outperform single markers in principle, that NfL + pNfH + GFAP are the leading candidates for inclusion, and that TDP-43 adds disease-specific potential for ALS particularly. But no formal panel has been prospectively validated, calibrated with reference ranges, and approved by any regulatory agency.
In ALS, NfL alone is the closest to clinical readiness — its prognostic value in individual patients is now documented in population-based studies, and regulatory acceptance (tofersen) establishes it as a pharmacodynamic endpoint. The panel approach is where the field is headed, but it remains a research aspiration rather than a clinical standard.
For PLS, the biomarker field is at an earlier stage. NfL is the best-evidenced single marker (PNHS p=0.001 finding). pNfH and GFAP have not been studied in dedicated PLS cohorts. A PLS-specific panel does not yet exist. The PNHS biorepository is the most likely source of the samples needed to develop one.
Why it matters for PLS patients
The practical significance of panel biomarker development for PLS patients is in trial design. A validated panel that can stratify patients by biological disease activity — identifying faster progressors, confirming that a treatment is having a biological effect, detecting changes earlier than functional scales — would make PLS trials smaller, shorter, and more likely to succeed. The field's ability to run a meaningful treatment trial in a rare, slowly progressive disease depends significantly on how well the biomarker infrastructure can compensate for the difficulty of detecting clinical endpoints.
How this connects
The individual biomarkers in the panel have dedicated pages: NfL, pNfH, and GFAP. The trial design context in which panels would be used is covered by the Scirocco 2025 review. The PNHS biorepository that will generate PLS-specific panel data is described on the PNHS page. For the broader biomarker overview, see the Biomarker Research hub.
Key studies referenced
Emerging biomarkers in amyotrophic lateral sclerosis: from pathogenesis to clinical applications.
Frontiers in Molecular Biosciences. 2025.
Protein biomarkers for the diagnosis and prognosis of amyotrophic lateral sclerosis.
ScienceDirect. 2023.
Correlation analysis of serum neurofilament light chain and glial fibrillary acidic protein
with ALS disease parameters. Scientific Reports. 2025.
Phosphorylated neurofilament heavy chain as a potential diagnostic biomarker in ALS.
Journal of Neurophysiology. 2022.
MIROCALS trial results with CSF pNfH stratification. The Lancet.