Is there an added value of faecal calprotectin and haemoglobin in the diagnostic work-up for primary care patients suspected of significant colorectal disease? A cross-sectional diagnostic study

We are the first to develop a diagnostic strategy in primary care patients suspected of SCD, considering signs, symptoms, simple blood analyses, and both faecal calprotectin and Hb levels. This study showed that especially a POC FIT, and to a much lesser extent calprotectin tests, have incremental value beyond patient history, physical examination, and CRP in ruling out SCD in primary care patients with persistent lower abdominal complaints. Use of a simple diagnostic model including calprotectin POC and POC FIT test results could safely rule out SCD and prevent endoscopy referral in about 30 % of patients with 96.4 % NPV (at a 5.0 % SCD probability referral threshold). Excluding the calprotectin test from this model yielded similar results, missing one additional AA patient (of the 49 present in our study). Substituting the calprotectin POC test by an ELISA did not substantially change these results.

A perfect strategy would not miss any SCD patients. A substantial reduction of the number of unnecessary endoscopy referrals – as we show is feasible – will, however, inevitably result in a small risk of missing serious SCD. In our study, one patient with stage 1 CRC was not selected for referral by any of the POC FIT extended models at the???5.0 % SCD probability threshold (this patient tested negative on both the calprotectin POC test and the POC FIT). With keen attention in case of non-referral at first consultation to persisting symptoms over a time frame of 2–3 weeks, we think this will result in delaying, but not missing, such diagnoses. Such a limited delay will also not likely advance the disease stage substantially for CRC patients who were initially non-referred [29].

Notwithstanding the 2013 NICE recommendation for use in diagnosing IBD [10], calprotectin has so far only been studied in absence of other diagnostic information [11–13]. One retrospective study investigating the use of calprotectin in irritable bowel syndrome-suspected primary care patients from the United Kingdom reported an AUC for SCD of 0.89 (95 % CI, 0.85–0.93), much higher than we report here (0.68; 95 % CI, 0.63–0.73 [POC], 0.66; 95 % CI, 0.61–0.72 [ELISA]) [12]. Besides the different patient populations, adenomas were not considered SCD in that study, as they were in ours. As calprotectin levels are low in (advanced) adenoma patients [11], this partly explains the observed difference between the studies (AUCs for SCD without adenomas in our data: 0.74; 95 % CI, 0.69–0.80 [POC], 0.73; 95 % CI, 0.67–0.80 [ELISA]). Related to this, the prevalence of AAs in our study almost doubled from February 2011 onwards (from 4.2 to 7.7 %, comprising 25.8 % versus 41.8 % of SCD cases – an increase that could not be explained by changes in patient mix throughout the study period, nor by differences in detection rates between endoscopy centres, but may have been introduced by increased awareness of gastroenterologists who around that time started preparing for the introduction of the CRC screening program in 2014). This increase in AA prevalence likely explains why our current results are less favourable compared to our previous (interim) analysis of patients enrolled through January 2011 (AUC: 0.75; 95 % CI, 0.67–0.82 [POC], 0.73; 95 % CI, 0.66–0.81 [ELISA]) [11]. Still, calprotectin did not show as much incremental diagnostic value as expected. This observation remained when analysing the data for IBD instead of SCD, and when considering adenomas non-SCD (data not shown).

Faecal Hb testing for CRC screening is widely accepted. Here, we showed that a qualitative POC FIT also has large incremental value for ruling out SCD in primary care. Our data further suggests that the POC FIT has value even in patients with overt rectal bleeding, equally so as in those without (Additional file 1). Additional analysis showed that the POC FIT was negative in 65.6 % of our patients with overt rectal bleeding. It may be more specific for blood mixed with faeces, thereby better reflecting the generally higher gastrointestinal location of SCD compared to other causes of rectal bleeding (e.g. haemorrhoids).

In a recent United Kingdom-based primary care study that ran between 2013–2014, 755 patients referred for bowel examination had available data on both faecal calprotectin (same ELISA as in our study) as well as Hb levels (using the quantitative EIKEN OC-Sensor assay) [16]. The authors concluded that undetectable faecal Hb may be sufficient to exclude CRC/IBD/higher-risk adenomas with 41.7 % test negatives, 96.2 % NPV and 88.2 % sensitivity – thereby questioning the added value of calprotectin, as in our study. Other studies have also advocated quantitative faecal Hb testing for ruling out SCD [30, 31], or advanced neoplasia [32–34], in symptomatic patients. We could not confirm these promising results of faecal Hb by itself (Table 1), which is possibly because of the higher threshold of our POC FIT (with a detection limit of 6 ?g/g), and it being a qualitative and not a quantitative test. Previous results suggest that using a single test could, in fact, be sufficient in deciding whom to refer for endoscopy. Indeed, our results also underscore that a positive POC FIT already implies the need for referral by itself (at the???5.0 % SCD probability threshold; see nomogram in Additional file 1). Here, the clinical data do not add much, but they do when the POC FIT returns negative. Also, in daily clinical practice, and certainly in primary care, it is rare that – except in a screening situation – physicians would immediately apply such test in suspected patients presenting with symptoms and signs of SCD without even considering any other pre-test diagnostic information from history taking and physical examination. The diagnostic process in primary care is sequential, starting with history taking and physical examination, and follow-up testing only in cases where the first provide indications that legitimates additional testing. To adhere as much as possible to primary care practice, we therefore explicitly first evaluated the diagnostic value of history taking, physical examination, and simple blood analysis, and subsequently the added value of the POC FIT test, rather than the other way around. Obviously, in unsuspected people, in the realm of screening, a single-test approach using first and foremost the POC FIT test, seems a very reasonable approach, but in our view not for diagnostic work-up of clinically suspected patients, which was the focus of this paper.

A major strength of our study is its prospective conduct in a primary care setting, where results from secondary care studies may not be applicable [8]. We also took care to enrol representative patients from 266 general practices, while measuring all potentially relevant diagnostic information, including blood and faecal biomarkers, under routine conditions, enhancing the generalizability of our results. Moreover, patients underwent reference testing by the same standard, including 3 months follow-up after inconclusive endoscopy to identify any initially missed SCD, and index and reference tests were interpreted independently in each patient. Finally, we purposely developed diagnostic models for SCD, and not solely for CRC (or IBD) as commonly done. This resulted in a diagnostic strategy applicable to primary care patients with persistent lower abdominal complaints that is optimally aligned with the diagnostic challenge at hand: ruling out SCD.

When defining SCD, we only included adenomas??1 cm as AA, without taking histologic high-risk features such as the presence of high-grade dysplasia or villous components in smaller adenomas into account. However, such high-risk features are seldom present in small adenomas [35], and we estimate that about 2 to 3 of the small adenomas we have considered non-SCD are actually high-risk lesions. This amount of misclassification (i.e. only ~2 % of all SCD cases in CEDAR) will likely not have importantly influenced the results. Some other limitations of our study also need discussion. For instance, we did not enrol primary care patients urgently referred for endoscopy (e.g. for on-going bleeding or imminent obstruction) or at very low SCD-suspicion (not necessitating endoscopy). Our study population thus reflects patients at intermediate risk of SCD. These patients, however, pose the largest diagnostic dilemma, where an improved diagnostic work-up is especially urgent. Further, most diagnostic predictors had missing data despite systematic data collection, and we had to use state of the art multiple imputation of the 5.2 % missing data points to prevent selection bias and loss of information [23–25]. Furthermore, as we used all available data to optimally develop the best diagnostic strategy, and despite using bootstrapping techniques for internal validation to correct for over-optimism, formal external validation of our findings is still warranted.

Finally, the use of a qualitative POC FIT in the way that we did in this study, although easily implemented in primary care, also has limitations. First, as the qualitative POC FIT yields a positive or a negative test result (with a detection limit of 6 ?g Hb/g faeces), the diagnostic information that would be available by quantitatively assessing the amount of Hb present in faeces is lost. Second, patients collected faecal samples in regular blue-capped containers without Hb stabilizing buffer (so each patient needed to fill only one faecal container for both calprotectin and Hb analysis). Samples were kept refrigerated, and – if not frozen before further processing – 90 % were tested within 3 days of collection. Additional data-analysis showed that the chance of a positive POC FIT slightly decreased with increasing time between collection and testing (0.3 % absolute decrease per day; P?=?0.19), and that frozen samples were more likely to be POC FIT negative than non-frozen samples (absolute 8.6 % decrease in POC FIT positivity; P?=?0.017; calprotectin results seemed not to be affected). Some patients have thus likely tested falsely negative for the POC FIT because of Hb degradation in our study. However, in none of the models with POC FIT did its odds ratio for SCD significantly differ in patients whose faecal samples were and were not frozen. Furthermore, the POC FIT performed well in our study despite these limitations, and the sensitivity and discriminatory performance of faecal Hb testing in primary care will thus likely be even better when using Hb stabilizing buffers in faecal sample collection devices and using a quantitative FIT.