Assessment of a dried blood spot C-reactive protein method to identify disease flares in rheumatoid arthritis patients

Abstract

Rheumatoid arthritis (RA) is characterised by painful, stiff and swollen joints. RA features sporadic 'flares' or inflammatory episodes-mostly occurring outside clinics-where symptoms worsen and plasma C-reactive protein (CRP) becomes elevated. Poor control of inflammation results in higher rates of irreversible joint damage, increased disability, and poorer quality of life. Flares need to be accurately identified and managed. A method comparison study was designed to assess agreement between CRP values obtained by dried blood spot (DBS) versus conventional venepuncture sampling. The ability of a weekly DBS sampling and CRP test regime to detect flare outside the clinic was also assessed. Matched venepuncture and finger lancet DBS samples were collected from n = 100 RA patients with active disease at baseline and 6 weeks (NCT02809547). A subset of n = 30 RA patients submitted weekly DBS samples over the study period. Patient demographics, including self-reported flares were recorded. DBS sample CRP measurements were made by enzyme-linked immunosorbent assay, and venepuncture samples by a reference immunoturbometric assay. Data was compared between sample types by Bland-Altman and weighted Deming regression analyses. Flare detection sensitivity and specificity were compared between 'minimal' baseline and 6 week sample CRP data and the 'continuous' weekly CRP data. Baseline DBS ELISA assay CRP measures yielded a mean positive bias of 2.693 ± 8.640 (95% limits of agreement - 14.24 to 19.63%), when compared to reference assay data. Deming regression revealed good agreement between the DBS ELISA method and reference assay data, with baseline data slope of 0.978 and intercept -0.153. The specificity of 'continuous' area under the curve (AUC) CRP data (72.7%) to identify flares, was greater than 'minimal' AUC CRP data (54.5%). This study indicates reasonable agreement between DBS and the reference method, especially at low to mid-range CRP values. Importantly, longitudinal CRP measurement in RA patients helps to clearly identify flare and thus could assist in remote monitoring strategies and may facilitate timely therapeutic intervention.Trial registration: The Remote Arthritis Disease Activity MonitoR (RADAR) study was registered on 22/06/2016 at ClinicalTrials.gov Identifier: NCT02809547. https://ichgcp.net/clinical-trials-registry/NCT02809547 .

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Distribution of CRP samples values recorded. The distribution of CRP measurements by immunoturbidimetry analysis of whole blood (Hosp. Ref.) and ELISA testing of dried blood spot (DBS EL) and plasma (Plasma EL), in (A) baseline and (B) 6 week samples for n = 100 RADAR study participants. Log10 scale of C-reactive protein concentration in mg per ml. Error bars represent the 25th and 75th percentile with median indicated at centre line.

Figure 2

Bland–Altman method comparison plots. Bland–Altman analysis comparing the measurement of CRP using the two different sampling methods across n = 100 RADAR study participants. (A) and (B) compare agreement between immunoturbidimetry analysis of whole blood (Hosp. Ref.) and ELISA testing of dried blood spot (DBS EL), at baseline and 6 weeks as labelled. (C) and (D) compare agreement between immunoturbidimetry analysis of whole blood (Hosp. Ref.) and plasma (Plasma EL), at baseline and 6 weeks as labelled. The black dashed line represents the mean, the red dashed line represents the average bias (or the average of the differences), while the upper green and lower blue lines represent ± 1.96 standard deviation. (E) The statistical parameters of the Bland–Altman plots, comparing levels of agreement and bias between sample methods relative to the reference hospital immunoturbidometry method are shown. The level of agreement (LOA) line is calculated as mean difference ± 1.96 multiplied by standard deviation. Points contained within the LOA lines denote good agreement between the two methods.

Figure 3

Deming regression method comparison analysis. Weighted Deming regression analysis comparing the measurement of CRP using the two different sampling methods across n = 100 RADAR study participants. Graphs compare systematic differences between immunoturbidimetry analysis of whole blood (Hosp. Ref.) and ELISA testing of dried blood spot (A,B; DBS EL) and plasma (C,D; Plasma EL), at baseline and 6 weeks. The red line represents the Deming regression line, the black line represents a simple linear regression line and the red shaded area the 95% confidence intervals. (E) The statistical parameters summarised from the Deming regression analysis compare systematic differences between immunoturbidimetry analysis of whole blood (Hosp. Ref.) and ELISA testing of dried blood spot (DBS EL) and plasma (Plasma EL), at baseline and 6 weeks. (F) Spearman correlation coefficients for each sample data comparison. Cl confidence limits of mean difference; SE standard error.

Figure 4

Longitudinal DBS CRP measures in home based arthritis patients. Tables summarising area under the curve (AUC) measures and change (ΔCRPt6–t0; 6 week [CRP]—baseline [CRP]) in DBS CRP concentration over the 6 week monitoring period for a subcohort of: (A) 11 participants who did not report a ‘flare’ and (B) 18 participants who did report a flare (1 individual did not provide their flare status). AUC was calculated by two different methods: in column (i) by using all weekly data points to construct an accurate continuous data AUC value, or in column (ii) by using baseline and 6 week data points to estimate a minimal data AUC value. Sparklines indicate the DBS CRP concentration of each participant over the 6 week period, such that (iii) CRP concentrations from weekly DBS are indicated by individual data points plotted on the same scale (with red data point indicating high point), or (iv) with only DBS CRP concentrations above 10 mg/L indicated. The week in which patients reported flare is listed and indicated by a red arrow in column (iii) sparklines. A threshold of 35 mg week/L was used assign positive ‘flare’ status, indicated in bold in columns (i) and (ii). Only participants with sparkline data visible in column (iv) were assigned positive flare status.