Prospective clinical study

To compare the diagnostic accuracy of the blood test, including hardware and software, a prospective validation study was established at the Western General Hospital, Edinburgh, with local ethical approval (2017/0320/SR938, 15/ES/0094). Informed consent was obtained from all patients prior to serum collection. Lothian region patients were eligible if suspected of having a brain tumour by their GP and referred directly for brain imaging through the local open access CT (OACT) service. This is most closely representative of the target patient cohort for our triage blood test. The criteria for GPs to access the OACT pathway is simply that their assessment that a patient’s symptoms necessitate urgent brain imaging; patients requiring emergency imaging are referred to the Emergency Department or indeed via a stroke referral pathway. As such these patients represent a cohort of patients the GP has selected for referral brain imaging. Patients referred via the OACT route for brain imaging were eligible to take part in this validation study provided they were able to give informed consent. Patients with a new brain tumour diagnosis referred to the neurosurgical unit for assessment were also eligible for inclusion. Analysis of the blood samples with our strategy was blinded to the whether the patient had a brain tumour or not. Sample handling protocols were consistent with those from the retrospective patient cohort previously mentioned.

The study was developed to have an adaptive design, to ensure that the optimum number of patients was recruited to enable assessment of test sensitivity and specificity. For initial sample size estimates (80% power, 5% significance level), to estimate specificity within 5% required a sample size between 200 and 600 assuming 2% prevalence (or 98% without brain tumour) for an assumed specificity in the range of 95% (n = 188) to 75% (n = 622). For the sensitivity, to demonstrate 90% sensitivity to within 30% requires 400 subjects (3% prevalence) or 600 (2% prevalence); note specificity was 91% in our previous study. The sample size was planned to be calibrated throughout the study by scheduled interim sample analysis and the cohort presented here is the first patient group from the larger study to be analysed. With a prevalence of 1–3% of actual brain tumours amongst imaged patients, we expected to generate evidence on specificity (true negatives and false positives) far more quickly than for sensitivity (true positives and false negatives). An estimate of the overall specificity of the blood test was made by correlating the predicted diagnosis with the gold standard (brain imaging, and biopsy where necessary). This interim analysis was made within the adaptive element of the design to assess how many additional samples would be needed in the full study to estimate specificity. Supplementary Fig. ¹ shows a consort flow diagram of the validation study and Supplementary Table ² shows the demographic data for patients that either accepter or declined to be part of the study to highlight the unbiased approached to patient recruitment.