Participants
Primary care consulters aged > 18 with low back-related leg pain were recruited as part of a longitudinal study (the ATLAS study) investigating the overall prognosis of low back-related leg pain and sciatica in primary care. The ATLAS study procedures have been described in detail elsewhere [11], here we give brief information on recruitment and assessment details. Patients consulting their GP (General Practitioner) low back and leg pain, who were potentially eligible for the ATLAS study, were sent a letter with information about the study, an invitation to attend the research clinic, and baseline questionnaires capturing sociodemographic, pain, psychological and health variables. Patients with other inflammatory conditions, such as rheumatoid arthritis were excluded. At the research clinic, patients underwent a standardised clinical assessment by one of seven experienced musculoskeletal physiotherapists, and were diagnosed as having clinically defined sciatica (spinal nerve root involvement) or referred (non-specific) leg pain, based on the examiner’s clinical opinion. Providing there were no clinical contraindications to the procedure, patients had a lumbar spine magnetic resonance imaging (MRI) scan within 2 weeks of their baseline assessment. The MRI findings were not part of the clinical diagnosis of sciatica or referred leg pain. All MRIs were scored by a single assessor, a senior consultant musculoskeletal radiologist. The assessor provided a clinical report indicating the presence/absence of definite or possible nerve root compression by lumbar spinal level (3 lower lumbar levels) and side (right/left) and the reason(s) for it if present (e.g. disc prolapse, stenosis) as per normal practice in radiological reporting.
For the purposes of this nested study (which for practical considerations, i.e. availability of phlebotomy service and storage for blood samples, could only be conducted at a single research site), patients consenting to the main study at this site, were also invited to take part in the biomarker sub study. Consenting patients had serum samples taken for biomarker analysis in addition to the standardised clinical assessment.
Participants were classified as having nerve root involvement due to suspected disc prolapse on the basis of the clinical assessment (clinically defined: yes/no).
For the purposes of analysis, we first examined levels of serum biomarkers between patients with clinically diagnosed disc-related sciatica and with referred leg pain, and secondly we examined levels of serum biomarkers between patients with clinically diagnosed disc-related nerve root involvement and evidence of concordant MRI findings, and those patients without evidence of concordant MRI findings.
All patients provided written informed consent. The biomarker study was approved by the North West- Greater Manchester South Research Ethics Committee (REC reference number: 12/NW/0173).
Measurement of cytokines, chemokines and MMPs
Sera were separated from bloods collected in plain BD Vacutainer® tubes at study entry. All sera were stored at -70o C until required. Key cytokines, chemokines and matrix metalloproteinases (MMPs) implicated in sciatica pathogenesis including TNFα, IL-1, IL-6, MMP1,3,8, aggregan and monocyte chemoattractant protein-1 (MCP-1/CCL-2) were measured in duplicate on a Bio-Plex™ 200 suspension array system using commercially available multiplex detection kits for cytokines (Milliplex multi-analyte profiling (MAP) kits, Millipore (UK) Ltd., Walford, Hertfordshire, UK), or Fluorokine MAP kits for MMPs (R&D Systems Europe, Abingdon, Oxfordshire, UK). Further details on the kits used are available in the Appendix. All assays were carried out according to the manufacturers’ instructions. High and low control samples were used in each assay.
Statistical analysis
All data were tested for normality and the appropriate parametric or non-parametric test was selected for analysis. Initial univariable analysis was carried out using the Mann Whitney U test to compare median cytokine levels between groups. A multivariate variable selection procedure using the algorithm of McHenry [12] was used to select biomarker variables which showed the strongest association with either clinically determined sciatica, with/without MRI evidence of nerve root compression. Multivariate logistic regression analysis was used to investigate the association between clinical measures and biomarker levels while adjusting for possible confounders such as age, sex, and symptom duration.
Statistical analyses were carried out using Number Cruncher Statistical Software package for Windows (NCSS 2000, NCSS Statistical Software, Kaysville, Utah, USA). The significance level was set at a p value of 0.05.