Phical test performance
A very useful test for the distinction between organic and functional gastro-intestinal disorders. Consider it when you suspect inflammatory bowel disease or colorectal cancer.
- Introduction
- Sampling
- Faecal extracts
- ELISA
- PhiCal Test versus isotope labelled leukocytes
- PhiCal Test and ulcerative colitis
- PhiCal Test and Crohn's disease
- PhiCal Test and irritable colon syndrome
- PhiCal Test as predictor of IBD relapse
- PhiCal Test and colorectal neoplasia
- Conclusion
- References
Introduction
PhiCalTest, or the assessment of faecal calprotectin, is a widely used method for detection of organic disease of the gastrointestinal tract. It introduces a new principle for objective determination of the inflammation in IBD (Inflammatory Bowel Disease), i.e. ulcerative colitis (UC) and Crohn´s disease (CD). Traditionally, the disease activity has been evaluated by clinical indices like the Crohn´s disease activity index (CDAI) or by the Harvey-Bradshaw "simple index" (SI). However, the use of more sophisticated methods, for instance the use of isotope labelled white cells, has shown that such indices tend to be inaccurate.
The quantitation of the neutrophil marker protein calprotectin in simple extracts of small (0.1g) stool samples is now available as an attractive alternative to the previous methods. This faecal test is non-invasive, objective and can be used repeatedly for the early detection of relapse in IBD, monitoring disease activity and document response to medical treatment. In children with chronic abdominal symptoms where IBD may be suspected, most physicians will hesitate to use isotope labelled leukocytes or endoscopy under general anaesthesia as diagnostic procedures.
The PhiCal Test has such a high negative predictive value that active IBD can be ruled out and invasive procedures avoided. The PhiCal Test is performed by an ELISA kit. Distributors and information on how to order can be found on our home page www.phical.com Back To top
Sampling
Traditionally, biological markers in stools have been assessed in 24-hour stool collections, but it is rather inconvenient for out-patients to collect and send such materials to the laboratory. Fortunately, calprotectin is so evenly distributed in stools (fig 1) that only small stool samples (typically one teaspoon full) are necessary for analysis. Furthermore, this protein is so stable that stools can be sent by ordinary mail. In fact, samples can be stored at room temperature for up to seven days. The stability of calprotectin is caused by its binding of calcium.
Faecal extracts
A new hygienic extraction procedure has been developed where stool samples of only about 0.1 g are extracted with about five ml of a new buffer in a closed tube on a rotator for about 30 minutes. The supernatant after centrifugation is run on a simple ELISA.
ELISA
Calprotectin is assessed by a simple ELISA, using polyclonal rabbit antibodies for capture and affinity purified, enzyme (alkaline phosphatase) labelled antibodies (against six different epitopes on calprotectin) for development. Using six epitopes makes the ELISA more robust and less likely to give falsely low values. In contrast, assays using monoclonal antibodies against a single epitope, may give low values due to masking or destruction of the epitope due to complex formation or enzymatic degradation in the bowel. The ELISA has between and within assay variations of 14.8% and 1.9% respectively.
The new faecal extraction is much more efficient so that both in healthy controls and patients higher values are found. For this reason the new cut off value is now 50 mg/kg in contrast to the previously reported 10 mg/l.
The PhiCal Test versus isotope labelled leukocytes
Excretion of isotope labelled leukocytes has been regarded as the "gold standard" of disease activity in IBD. However, this requires three days of hospitalisation for the collection of stools and the determination of the isotope. Furthermore, it also exposes the patient to a potentially hazardous radiation, making this method unsuited for repeated use in children and fertile women.
We have compared this method with the PhiCal Test in nine healthy controls, 10 patients with UC and 19 patients with CD. Although these were all out patients with very low disease activity, a highly significant correlation was found between the three day excretion of 111-indium labelled granulocytes and that of faecal calprotectin (r=0.87, p<0.0001) (figure 2).
In a similar study published lately, this close relationship was confirmed.This suggests that the PhiCal Test can replace the use isotope method. Its simplicity and low cost makes it an attractive alternative allowing follow-up by repeated testing.
Calprotectin in patients with Ulcerative Colitis
Data from a study of 62 outpatients referred for colonoscopy are shown in Figure 3. The disease activity was determined by endoscopy and histology using accepted criteria. There was a highly significant increased concentration of faecal calprotectin in the group of patients with active disease as determined by histological criteria when compared to patients with no/low disease activity and those with normal colonoscopy. Furthermore, calprotectin levels were better correlated to the degree of inflammation than the extension of the disease.
PhiCal Test in Crohn´s disease and irritable bowel syndrome
It is often difficult to differentiate between patients with IBS and CD by clinical parameters. This results in many unnecessary colonoscopic examinations adding an substantial economical burden on the society.
Calprotectin was determined in 220 patients referred for colonoscopy at King´s College, London. The patients were evaluated by the ROME criteria for possible IBS. The results showed that patients with CD had significantly higher faecal calprotectin levels than those with IBS (p<0.0001) (Fig 4).
A ROC plot analysis revealed that a cut-off level of 150 mg/kg (corresponding to 30 mg/l in the old method) distinguished between CD and IBS with 100% sensitivity and 97% specificity (fig 5).
Among 30 patients with other disorders, elevated calprotectin concentrations were found among 2/2 colorectal cancer, 2/3 adenomas and 6/6 with microscopic colitis. Similar results were reported in a study at Mayo Clinic USA.
The PhiCal Test as a predictor of relapse of IBD
Tibble et al. reported in Gastroenterology last year a study of 43 patients with CD and 37 with UC, and concluded that the test was useful to predict a future relapse of IBD (Fig 6). In each patient, faecal calprotectin was assessed in a single stool sample at inclusion, and changes in the disease activity were assessed by the Harvey Bradshaw "Simple Index".
The Kaplan-Meier curves show that 58% of patients with CD and 51% of those with UC had relapsed during the one-year follow-up period. ROC analysis revealed that faecal calprotectin level of 250 mg/kg (50mg/l by the old method) predicted a relapse with 90% sensitivity and 83% specificity. These results may suggest that the PhiCal Test can be helpful in adjusting the medical treatment of IBD and thereby reduce side effects and probably the development of secondary changes in the GI tract in IBD patients.
The PhiCal Test and colorectal neoplasia
Several publications have reported increased levels of faecal calprotectin in patients with colorectal cancer and adenomas.
As shown in Fig.7 levels normalise or drop dramatically after surgery, indicating a close relationship between increased calprotectin and the presence of the tumour.
Most publications agree that increased calprotectin levels are found in approximately 90% of symptomatic cancers and in 60-70% of adenomas. The mechanism behind is probably ulcerations and increased permeability of the mucosa so that large numbers of neutrophils migrate into the gut lumen towards the high concentrations of bacterial chemotactic substances there.
A negative predictive value of 99.1% indicates that patients with a normal PhiCal Test have a very low risk of having colorectal cancer.
Conclusion
Calprotectin is a non-specific marker of gastrointestinal disease of both inflammatory and neoplastic character. The high sensitivity and negative predictive values can be useful to select patients for colonoscopy. Calprotectin is also an interesting marker of disease in various organs since elevated concentrations are found in plasma/serum, cerebrospinal fluid, synovial fluid or urine during infections or inflammatory conditions.
References
1. Saverumuttu SH. Clinical remission in Crohn´s disease - assessment using faecal 111In granulocyte excretion. Digestion 1986; 33: 74-79.
2. Fagerhol MK, Nomenclature for proteins: is calprotectin a proper name for the elusive myelomonocytic protein ? J Clin Pathol 1996; 49:M74-79
3. Røseth AG, Fagerhol MK, Aadland E, Schjønsby H. Assessment of the neutrophil dominating protein calprotectin in feces. Scand J Gastroentrol 1992; 27:793-98.
4. Røseth AG, Schmidt PN, Fagerhol MK. Correlation between faecal excretion of 111-Indium labelled granulocytes and calprotectin, a granulocyte marker protein in patients with inflammatory bowel disease. Scand J Gastroentrol 1999; 34:50-54.
5. Johne B, Kronborg O, Tøn H, Kristinsson J, Fuglerud P. A new fecal calprotectin test for colorectal neoplasia. Clinical results and comparison with previous method. Scand J Gastroenterol 2001; 36:291-96.
6. Røseth AG, Aadland E, Jahnsen J, Raknerud N. Assessment of disease activity in ulcerative colitis by faecal calprotectin, a novel granulocyte marker protein. Digestion 1997; 58:176-80
7. Tibble J, Teahon K, Tjodleifsson B, Roseth A, Sigthorsson G, Bridger S, Foster R,Sherwood R, Fagerhol M, Bjarnason I. A simple method for assessing intestinal inflammation in Crohn´s disease. Gut 2000; 47:506-13.
8. Limburg PJ, Ahlquist DA, Sandborn WJ, Mahoney DW, Devens ME, Harrington JJ, Zinsmeister AR. Fecal calprotectin levels predict colorectal inflammation among patients with chronic diarrhoea referred for colonoscopy. Am J Gastroenterol 2000; 95:2831-37.
9. Tibble J, Sigthorsson G, Bridger S, Fagerhol M, Bjarnason I. Surrogate markers of intestinal inflammation are predictive of relapse in patients with inflammatory bowel disease. Gastroenterology 2000; 119:15-22.
10. Røseth AG, Kristinsson J, Fagerhol MK, Schjønsby H, Aadland E, Nygaard K, Roald B. Faecal calprotectin: A novel test for the diagnosis of colorectal cancer ? Scand J Gastroentrol 1993; 28:1073-76.
11. Kristinsson J, Røseth A, Fagerhol MK, Aadland E, Schjønsby H, Børmer OP, Raknerud N, Nygaard K. Fecal calprotectin concentration in patients with colorectal carcinoma. Dis Colon Rectum 1998; 41:316-21.
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