AIMS

Our aims were: (a) to validate faecal calprotectin as a method for diagnosing NSAID enteropathy against the use of the faecal excretion of ¹¹¹In labelled white cells, which is currently the most frequently used technique to diagnose NSAID enteropathy; (b) to assess the day to day variation of faecal calprotectin in patients taking different NSAIDs, as a marker of method reproducibility; and (c) to assess the prevalence and severity of NSAID enteropathy in a cross sectional study of patients on NSAIDs using the calprotectin method.

PATIENTS

Recruited patients were from general rheumatology clinics at King’s College Hospital and Bromley District General Hospital over a 12 month period. The male to female ratio represented the sex distribution of clinic attendance while the age criterion was 18–80 years. All patients were seen in the clinics for rheumatological conditions requiring treatment with an NSAID, and had been on a daily dose for at least one month.

Patients excluded were those with concomitant known intestinal or significant hepatic, respiratory, cardiovascular, or neurological disease, or those taking other medications which are associated with intestinal inflammation.23 Data acquired on patients included the type of NSAID, length of regular usage, dose, disease for which NSAIDs were being used, other current medication, and other medical illnesses.

Forth eight healthy volunteers (15 men, 33 women; mean age 56 years, range 18–78) provided single stool samples to obtain a range of normality for faecal calprotectin.

As part of the validation procedure 47 patients taking NSAIDs were assessed for intestinal inflammation with four day faecal excretion of¹¹¹In labelled white cells, which was compared with the faecal calprotectin concentration in the first stool sample (obtained on day 2 of the study) to assess the correlation between the two techniques.

A different group of 312 patients (102 men, 210 women; mean age 58 years, range 20–80), taking 18 different NSAIDs provided single stool samples for assessment of faecal calprotectin by ELISA; 192 had rheumatoid arthritis, 65 osteoarthritis, and 55 other rheumatological conditions such as fibromyalgia and non-specific lower back pain, not associated with gastrointestinal disease.23 A total of 146 patients was using second line drugs (74 methotrexate, 32 sulphasalazine, 17 gold, nine azathioprine, eight penicillamine, six other), 30 were using prednisolone, and 60 were using a gastroprotective agent (12 proton pump inhibitors (PPI), 40 histamine H₂ receptor antagonists, and eight misoprostol). Table 1details all medications used. Day to day variability in faecal calprotectin was assessed in 98 paired samples from these patients, taken on consecutive days. The study was approved by the King’s Healthcare and Bromley Local Research Ethics Committees and all patients gave informed consent.

INDIUM-111 WHITE CELL LABELLING

Neutrophils were isolated by sedimentation and centrifugation and labelled with ¹¹¹In (30–300 μCi; 1–11 mBq) using tropolone as an ionophore as previously described,23 and injected intravenously. The estimated radiation dose received was 0.85–8.5 milli Sieverts (effective dose equivalent) depending on the dose received. Complete individual stools were collected over a four day period after injection of the labelled cells and counted in a high resolution bulk sample counter along with standards for 20–60 seconds which allows measurement of 0.1–0.01% (low and high dose respectively) of the injected dose with a counting accuracy of ±4%. Control data from 21 normal subjects had been established previously (median 0.41%, 95% confidence interval (CI) 0.05 to 0.94%).

FAECAL CALPROTECTIN ASSESSMENT

Stool samples were frozen on receipt at −20°C. After thawing, 5 g aliquots were suspended in 10 ml of faecal extraction buffer (Tris buffered isotonic (150 mM) saline, with 10 mM CaCl₂ and 0.25 mM thiomersol as an antimicrobial agent, pH 8.4) and homogenised for 45 seconds at 20 000 rpm with an Ultra Turrax (Ika Werke, Germany) mechanical homogeniser. The homogenates were centrifuged for 15 minutes at 10 000 g at room temperature. The top halves of the supernatants were pipetted off, frozen, and stored at −20°C until quantitation by ELISA.

Microtitre plates were coated by adding 200 μl of an IgG fraction of a rabbit anticalprotectin15 diluted 1/2000 in phosphate buffered saline to each well. The plates were covered with mylar foil and kept at +4°C until use. Calprotectin standards15 ,173.75–60 mg/l, were prepared by diluting purified calprotectin in an assay buffer: Tris (50 mM) buffer containing 150 mM NaCl, 0.5 mM MgCl₂, 2.5 mM KCl, 0.25 mM thiomersol, 0.05% Tween 20, and 10 g/l bovine serum albumin pH 8.0. Before use the microtitre plates were washed four times in buffer, the assay buffer less the bovine serum albumin. The frozen faecal extracts were thawed and diluted 1/20 and 1/200 (and dilutions of 1/400–6400 when required) in the assay buffer. Standards and diluted samples (100 μl) were added to the plates (all done in duplicate) which were covered and incubated at room temperature for 45 minutes on a plate shaker with an agitating speed of 600/min. The wells were then washed four times with washing buffer. Alkaline phosphatase (100 μl) conjugated anticalprotectin (dilution 1/1600) was added to each well, the plate covered, and incubated at room temperature for 45 minutes on the plate shaker with an agitating speed of 600/min. Thereafter the wells were washed four times and 100 μl substrate solution was added (2 × 5 mgp-nitrophenol phosphate tablets dissolved in 10 ml substrate buffer which contained 10% diethanolamine with 0.5 mM MgCl₂, 0.25 mM thiomersal, pH adjusted to 9.6 with HCl). The optical density (405 nm; measured on Micro Tracer plate reader, Syva, Milton Keynes, UK) of the highest standard was monitored and the reaction stopped by adding 50 μl of a 1 M NaOH solution to each well when its optical density read between 1.2 and 1.5, similar to that previously described.17

STATISTICS

Spearman’s correlation coefficient was used to assess the correlation between ¹¹¹In white cell labelling and faecal calprotectin concentration. Statistical comparison between controls and the NSAID treated group was performed using the Mann-Whitney U test. Wilcoxon matched pairs test, Altman Bland plot, Spearman’s correlation, and mean coefficient of variation were calculated for the paired samples. Analysis of variance (ANOVA) and multiple regression analysis were used for assessing differences between subgroups of patients taking different NSAIDs, second line agents, gastroprotective agents, and prednisolone.

VALIDATION OF FAECAL CALPROTECTIN AS A MARKER OF NSAID ENTEROPATHY

The median calprotectin concentration in the 48 normal controls was 2.0 mg/l (range 0.2–10.9 mg/l).

The four day faecal excretion of ¹¹¹In white cells (median 1.85%, range 0.05–4.89%; normal <1%, 95% CI 0.05 to 0.94%) in the 47 patients on NSAIDs differed significantly from controls (p<0.0001) and correlated significantly (r=0.83, p<0.0001, Spearman) with the faecal calprotectin concentrations (median 11.0 mg/l, range 1.0–89 mg/l; reference range <8.9 mg/l) in patients on NSAIDs. Of the 47 patients studied using both techniques, 70% had evidence of intestinal inflammation as assessed by the indium white cells and 53% as assessed by the calprotectin method. Only one patient with an elevated faecal calprotectin had a normal four day faecal excretion of¹¹¹In white cells.

There was no significant difference (p>0.1, Wilcoxon matched pairs sign rank) between calprotectin concentrations in the paired samples (bias 1.54; 95% CI −4.94 to 1.85 by Altman Bland plot;r=0.8, Spearman). The mean coefficient of variation for all paired samples was 42%. However, if we use a reference range of normality as the 95th centile of the normal control samples we obtain an upper reference limit for faecal calprotectin of 8.9 mg/l. The mean coefficients of variation for paired samples were 63% and 27% where either of the two calprotectin concentrations was less or greater than 8.9 mg/l respectively.

PREVALENCE AND SEVERITY OF NSAID ENTEROPATHY USING FAECAL CALPROTECTIN

Figure 1 shows the calprotectin concentrations from the 312 patients on NSAIDs (median 7.3 mg/l, 95% CI 6.5 to 8.0) which differed significantly (p<0.0001, Mann-Whitney U test) from the 48 controls (median 2.0 mg/l, 95% CI 1.6 to 5.0). There was no significant difference (p=0.07, Mann-Whitney) in faecal calprotectin levels between the 312 patients studied and the 47 patients studied using both the calprotectin (median 11 mg/l, 95% CI 6 to 20) and ¹¹¹In white cell excretion technique.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Box and whisker diagram showing median faecal calprotectin concentration (horizontal line), 5th and 95th centiles (box), and range (whiskers) in controls (n=48) and patients taking non-steroidal anti-inflammatory drugs (NSAIDs) (n=312).

There was no significant difference (p>0.1, ANOVA) in faecal calprotectin concentration in patients receiving different types or doses of NSAIDs or between those patients using or not using a gastroprotective agent (including those taking misoprostol as part of a combined formulation) in addition to their NSAID (table 1). Furthermore, multiple linear regression analysis (table 2) did not show any significant effect (p>0.05) of age, sex, type of NSAID used, diagnosis, or treatment with second line agent, gastroprotective agent, or prednisolone on the level of faecal calprotectin when all variables were taken into account.