Freeze

This documentation pertains to the May 2025 Freeze of the plasma Alamar Argo NULISAseq CNS 120 - Madison dataset.

Origin

These multiplex assay results come from University of Wisconsin ADRC Biomarker Lab and were analyzed using the NULISAseq CNS Disease Panel on the Alamar Argo HT platform, and sequenced using the Illumina NextSeq1000.

Sample Selection

The cross-sectional samples used in this study were collected between July 17, 2017 and Feb 2, 2024. Sample selection was based on the availability of amyloid and tau PET imaging within one year of the blood draw (n = 239). To enrich the dataset with A+ individuals, we strategically over-sampled participants who were PET A+. To enhance representation of underrepresented groups, we also included 13 additional participants who lacked both amyloid and tau PET scans but had existing Simoa pTau217 plasma results, which were used to approximate A and T status. For detailed information, refer to the WRAP and/or ADRC pre-analytic protocols for CSF and plasma collection, available on the Wisconsin Alzheimer's Program KnowledgeBase. Additional methodological details can be found in the publication by Rea Reyes et al., 2025.

Sample and Participant Characteristics

This dataset includes 240 observations on 240 participants (72 ADRC; 168 WRAP; 162 female; 31 URG; 23 MCI and 6 Dementia; <5 participant(s) are aged 90+, of those not: mean age of 69.6; mean days since baseline of 4519).

Per data sharing agreements with the Oneida nation, data from Native American participants are not included in this dataset. 

Analytes

The following is a list of analytes included in the CNS120 panel at the time of the study. The categories are provided for reference and follow the classification used on Alamar's website. NOTE: Alamar has since updated the targets in this panel. As a result, there may be discrepancies between the list below and the current information available on their website.

Analyte concentrations are reported in NULISA Protein Quantification (NPQ) units. These values are derived by normalizing the relative concentration of each analyte using internal and inter-plate controls, followed by a log_2 transformation. For detailed methodology, refer to the "NULISAseq data processing and normalization" section in Feng et al., 2023.

Amyloid and Tau Pathologies

• ACHE: Acetylcholinesterase.
• APOE: Apolipoprotein E.
• APOE4: Apolipoprotein E isoform 4.
• AB38: Amyloid beta peptide 38.
• AB40: Amyloid beta peptide 40.
• AB42: Amyloid beta peptide 42.
• BACE1: Beta-site APP-cleaving enzyme 1.
• BASP1: Brain abundant membrane-attached signal protein 1.
• CD63: Tetraspanin protein. 
• CST3: Cystatin C. I
• GFBP7: Insulin-like growth factor-binding protein 7.
• KLK6: Kallikrein-6.
• PSEN1: Presenilin-1 - catalytic subunit of -secretase.
• pTau_181: Tau phosphorylated at threonine 181.
• pTau_217: Tau phosphorylated at threonine 217.
• pTau_231: Tau phosphorylated at threonine 231.
• SFRP1: Secreted frizzled-related protein 1. 
• MAPT: Microtubule-associated protein tau.

Inflammation

• CCL11: C-C Motif Chemokine Ligand 11.
• CCL13: C-C Motif Chemokine Ligand 13.
• CCL17: C-C Motif Chemokine Ligand 17.
• CCL2: C-C Motif Chemokine Ligand 2.
• CCL22: C-C Motif Chemokine Ligand 22.
• CCL26: C-C Motif Chemokine Ligand 26.
• CCL3: C-C Motif Chemokine Ligand 3.
• CCL4: C-C Motif Chemokine Ligand 4.
• CD40LG: CD40 Ligand.
• CHI3L1: Chitinase 3 Like 1.
• CHIT1: Chitinase 1. 
• CRH: Corticotropin Releasing Hormone. 
• CRP: C-Reactive Protein.
• CSF2: Colony Stimulating Factor 2.
• CX3CL1: C-X3-C Motif Chemokine Ligand 1.
• CXCL1: C-X-C Motif Chemokine Ligand 1.
• CXCL10: C-X-C Motif Chemokine Ligand 10.
• CXCL8: C-X-C Motif Chemokine Ligand 8.
• FCN2: Ficolin 2.
• GDF15: Growth Differentiation Factor 15. 
• GFAP: Glial Fibrillary Acidic Protein.
• ICAM1: Intercellular Adhesion Molecule 1. 
• IFNG: Interferon Gamma.
• IGF1R: Insulin Like Growth Factor 1 Receptor.
• IL10: Interleukin 10. 
• IL12p70: Interleukin 12 Subunit p70. 
• IL13: Interleukin 13.
• IL15: Interleukin 15.
• IL16: Interleukin 16.
• IL17A: Interleukin 17A.
• IL18: Interleukin 18.
• IL1B: Interleukin 1 Beta.
• IL2: Interleukin 2.
• IL33: Interleukin 33.
• IL4: Interleukin 4.
• IL5: Interleukin 5.
• IL6: Interleukin 6.
• IL6R: Interleukin 6 Receptor.
• IL7: Interleukin 7.
• IL9: Interleukin 9.
• PRDX6: Peroxiredoxin 6. 2
• RUVBL2: RuvB Like AAA ATPase 2.
• S100A12: S100 Calcium Binding Protein A12.
• S100B: S100 Calcium Binding Protein B.
• SAA1: Serum Amyloid A1. 
• SFTPD: Pulmonary Surfactant-Associated Protein.
• SLIT2: Slit Guidance Ligand 2.
• TAFA5: TAFA Chemokine Like Family Member 5. 
• TEK: TEK Receptor Tyrosine Kinase.
• TIMP3: TIMP Metallopeptidase Inhibitor 3. 
• TNF: Tumor Necrosis Factor.
• TREM1: Triggering Receptor Expressed On Myeloid Cells 1.
• TREM2: Triggering Receptor Expressed On Myeloid Cells 2.
• VCAM1: Vascular Cell Adhesion Molecule 1.

Neurodegeneration

• ANXA5: Annexin A5.
• CALB2: Calbindin 2.
• CNTN2: Contactin 2.
•  ENO2: Enolase 2.
• GDI1: Rab GDP Dissociation Inhibitor Alpha. 
• GDNF: Glial Cell Derived Neurotrophic Factor.
• GOT1: Glutamic-Oxaloacetic Transaminase 1. 
• MSLN: Mesothelin. 
• NEFH: Neurofilament Heavy Chain. 
• NEFL: Neurofilament Light Chain.
• NPTX1: Neuronal Pentraxin 1.
• NPTX2: Neuronal Pentraxin 2. 
• NPTXR: Neuronal Pentraxin Receptor. 
• NPY: Neuropeptide Y. 
• NRGN: Neurogranin.
• PDLIM5: PDZ And LIM Domain 5.
• REST: RE1 Silencing Transcription Factor.
• SMOC1: SPARC Related Modular Calcium Binding 1.
• SNAP25: Synaptosome Associated Protein 25.
• SQSTM1: Sequestosome 1.
• VSNL1: Visinin Like 1. 
• YWHAZ: Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta.

Synuclein and Synaptic Disorders 

• AGRN: Agrin. 
• ARSA: Arylsulfatase A.
• FABP3: Fatty Acid Binding Protein 3.
• FOLR1: Folate Receptor Alpha. 
• HTT: Huntingtin.
• MDH1: Malate Dehydrogenase 1. 
• NGF: Nerve Growth Factor. 
• Oligo_SNCA: Oligomeric Alpha Synuclein.
• PARK7: Parkinsonism Associated Deglycase. 
• SNCA: Alpha-synuclein. 
• SNCB: Beta-synuclein.
• SOD1: Superoxide Dismutase 1. 
• TARDBP: TAR DNA Binding Protein.
• UCHL1: Ubiquitin C-Terminal Hydrolase L1. 
• VGF: Neurosecretory protein VGF.
• pTDP43_409: TDP-43 with phosphorylation on serine 409.

Vascular

• HBA1: Hemoglobin subunit alpha 1. 
• PGF: Placental growth factor. 
• VEGFA: Vascular endothelial growth factor A. 
• VEGFD: Vascular endothelial growth factor D.

Vascular and Metabolism

• FGF2: Fibroblast Growth Factor 2.
• FLT1: Fms Related Receptor Tyrosine Kinase 1. 
• KDR: Kinase Insert Domain Receptor.
• MME: Membrane Metalloendopeptidase. 
• PDGFRB: Platelet Derived Growth Factor Receptor Beta.
• PGK1: Phosphoglycerate Kinase 1. 
• POSTN: Periostin. 
• PTN: Pleiotrophin.

Methods

Sample analysis was performed between May 16, 2024 and June 13, 2024 using the NULISAseq CNS Disease Panel (Alamar Part# 800104, Lot# 2407305) following the manufacturer's protocol. Briefly, plasma samples were thawed at room temperature, centrifuged for 5 minutes at 10,000 Ã— g at 4°C, and transferred to the 96-well sample plate provided in the reagent kit. The plate was sealed and centrifuged again for 1 minute at 1,000 × g at 4°C. Using the automated Alamar Argo HT workflow, target analytes were captured through multiple binding, washing, and release steps. This process generated immunocomplexes tagged with both sample-specific and target-specific DNA barcodes, which were pooled into a single library for next-generation sequencing. Sequencing was performed on an Illumina NextSeq1000 using a P2 100-cycle flow cell (Illumina Part# 20046811, Reagent Lot# 20841078, Flow Cell Lot# 20846922). The resulting FASTQ files were uploaded to the Argo Command Center, where data were automatically normalized and converted to NULISA Protein Quantification (NPQ) units. Samples were processed in three batches of 84 samples each. A pooled plasma control (prepared in-house) was included at the beginning and end of each plate to assess reproducibility. This allowed for evaluation of both within-plate (n = 2) and between-plate (n = 6) variability. Within-plate coefficient of variation (%CV) ranged from 0.01% to 7.5% for most biomarkers, with the exception of hemoglobin subunit alpha 1 (HBA1), which exhibited higher variability, ranging from 10.8% to 31.8%. Between-plate %CV values were similarly low for the majority of targets, falling between 0.29% and 5.6%, again with HBA1 showing elevated variability at 19.1%. Across all three assay batches, 90% of targets demonstrated a within-plate %CV of 2.0% or less.

Code Availability

The entire processing pipeline is available on the SMPH GitLab. For any questions, please contact Ramiro Rea Reyes(rreareyes@medicine.wisc.edu).

Funding/Acknowledgments

Papers including data obtained from these assays should acknowledge NIH R01 AG027161 (the Wisconsin Registry for Alzheimer's Prevention) and/or NIH P30 AG062715 (the Wisconsin Alzheimer's Disease Research Center).

A manuscript based on this work has been published: Rea Reyes et al., 2025. This paper looked into differential expression of biomarkers among cognitively impaired individuals, amyloid and tau PET status, and the relationship of pTDP43 with cognitive scores and hippocampal volume.

For further details or collaboration inquiries, please contact Rachael Wilson or Ramiro Rea Reyes

See Also

Biofluids Data Documentation

Document History