Point-of-care measurements reveal release of purines into venous blood of stroke patients

Abstract

Stroke is a leading cause of death and disability. Here, we examine whether point-of-care measurement of the purines, adenosine, inosine and hypoxanthine, which are downstream metabolites of ATP, has potential to assist the diagnosis of stroke. In a prospective observational study, patients who were suspected of having had a stroke, within 4.5 h of symptom onset and still displaying focal neurological symptoms at admission, were recruited. Clinical research staff in the Emergency Departments of two hospitals used a prototype biosensor array, SMARTCap, to measure the purines in the venous blood of stroke patients and healthy controls. In controls, the baseline purines were 7.1 ± (SD) 4.2 μM (n = 52), while in stroke patients, they were 11.6 ± 8.9 μM (n = 76). Using the National Institutes for Stoke Scale (NIHSS) to band the severity of stroke, we found that minor, moderate and severe strokes all gave significant elevation of blood purines above the controls. The purine levels fall over 24 h. This was most marked for patients with haemorrhagic strokes (5.1 ± 3.6 μM, n = 9 after 24 h). The purine levels measured on admission show a significant correlation with the volume of affected brain tissue determined by medical imaging in patients who had not received thrombolysis or mechanical thrombectomy. ClinicalTrials.gov Identifier: NCT02308605.

Keywords: Biosensor; Cerebral ischemia; Point of care; Purines; Stroke.

Conflict of interest statement

ND, FT and CI declare the following conflicts of interest:

ND is a Founder and Director of Sarissa Biomedical Ltd. and has equity in this company. FT is employed by Sarissa Biomedical Ltd., the manufacturer of the biosensors used in the study and related in vitro diagnostic devices. FT, ND and CI are co-inventors on two patent applications relating to the measurement of purines as a diagnostic tool for brain ischaemia.

RS, NP and CR declare that they have no conflict of interest.

Figures

Fig. 1

Consort diagram for the SMARTCap trial. Purine measurements on admission and at 24 h were not obtained because the measurement procedure ran into a problem and one of the steps could not be completed; or the measurement was completed but the resulting electrochemical data was spurious (see “Materials and methods”); or, for the 24-h purine level, the patient was repatriated before the measurement could be made. Twenty-four- to 48-h scans were not made because the MR scanner was not available; or the patient did not consent for an MR scan; or the patient was too ill for the MR scan; or the patient was repatriated before the scan could be made

Fig. 2

Design and operational principles of SMARTCap. a Diagram of SMARTCap showing the four working electrodes (black) and two Ag/AgCl pseudo-reference electrodes (grey). Two of the working electrodes were coated with a gel layer containing the enzymes for a purine biosensor. The other two working electrodes were coated with a gel layer lacking enzymes to comprise null biosensors). b Enzymatic cascade used on the working electrodes for detection of purines

Fig. 3

Purine levels in venous blood of stroke patients are elevated relative to those measured of healthy controls. Dot plots showing the values of purines measure with SMARTCap in healthy controls, stroke patients, and patients identified as stroke mimics. Box plots show mean and SD, whiskers are 5–95 percentiles. Comparison of three groups with three-level single factor ANOVA, followed by pairwise t tests

Fig. 4

Purine levels measured in stroke patients grade with severity of stroke classified by NIHSS. A five-level single factor ANOVA shows that the purine levels in controls and stroke groups are significantly different. Pairwise t tests show that purine levels in minor, moderate and very severe stroke patients are significantly elevated relative to controls. Box plots show mean and SD, whiskers are 5–95 percentiles

Fig. 5

Purine levels in venous blood of stroke patients plotted against time from symptom onset, separated according to NIHSS. For minor strokes (NIHSS 1–4), there is a statistically significant positive correlation, i.e. the longer the delay in measurement, the higher the purine level in blood. For moderate strokes (NIHSS 5–15), there is a slight but statistically significant negative correlation, i.e. with symptom onset time to measurement delay, there is a slight fall in purine levels. With strokes of greater severity, a similar trend cannot be excluded, but the numbers are too small to interpret reliably. RS Spearman’s rank correlation coefficient

Fig. 6

Purines measured at time of admission correlate with volume of affected brain tissue measured by CT or MR imaging. The top panel shows non-thrombolysed ischaemic and haemorrhagic patients; the bottom panel shows only haemorrhagic patients. The inset shows lack of correlation between purines measured on admission and infarct size measured by MRI in ischaemic stroke patients who had undergone thrombolysis/thrombectomy. CT scans performed the same day as admission for haemorrhagic patients, one ischemic stroke patient had CT scan performed at 24–48 h after admission, and the MRI scans performed 24–48 h after admission. RS Spearman’s rank correlation coefficient

Fig. 7

Purine levels show a trend to normalisation 24 h after admission. The purines measured at time of admission and at 24 h shown for ischaemic and haemorrhagic strokes. Comparison of 0 h and 24 h via t tests. The difference is not significant for ischaemic strokes

Fig. 8

ROC analysis of venous purine levels in stroke patients versus healthy controls and mimics. The purines measured at time of admission show some discrimination of stroke versus controls and mimics, but the performance is not adequate for a diagnostic test. AUC area under the ROC curve