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FERRING-NOV27-2014

Quantitative measurement of calprotectin is often helpful in the diagnosis and treatment of inflammatory conditions. Neutrophils, which are vulnerable and short-lived cells, are produced in the bone marrow and mobilized to the blood in large amounts in inflammatory conditions, migrating into inflamed tissue where active enzymes and also calprotectin are released from the cells. In IBD neutrophils migrate through the wall of the intestine and calprotectin is consequently found in increased amounts in stools ; feces concentrations more than hundred times the upper cut off value can be found in IBD. Calpro AS was established in 1993, and based on the technology of Calpro the first commercial ELISA kit, Nycomed ELISA, for determination of calprotectin in faeces was launched in 1994. A patented buffer for extraction of calprotectin from feces, which had been developed by Calpro, was included in the test kit. In 2000 an improved assay was developed by Tøn et al. Whereas the initial cut off value for fecal calprotectin between normal and patients with organic bowel disease was 10 mg/kg, it was now increased to 50 mg/kg, which is still recommended for the Calpro kits. Samples with high calprotectin values were increased to a slightly higher degree than low calprotectin samples, thus improving the separation between high and low calprotectin levels.

In the 20th century there was no commercial test kit available except Nycomed ELISA based on Calpro technology, but after standardized extraction methods had been developed and some gastroenterologists described  faecal calprotectin as a promising noninvasive marker to differentiate IBD from IBS, the interest “exploded”  around 2000.  Up to now more than 2000 studies on calprotectin are published in peer review journals. Several issues regarding the use of fecal calprotectin testing in clinical medicine are discussed, included methodological and economic aspects.

The main topics and recommendations are

  1. Fecal calprotectin testing is widely used as a diagnostic tool to distinguish between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, it is not a tool for diagnosing IBD alone as elevated calprotectin levels could be attributed to other causes such as colorectal cancer ).
  2. There is a strong positive correlation between the disease activity (grade and extent of inflammation) in IBD and the fecal calprotectin level (8,18). The test can therefore be successfully used to monitor the effect of treatment.         
  3. The test is able to identify patients who do not respond to a specific treatment, thereby helping the doctor to make optimal therapeutic decisions.
  4. When the calprotectin values decrease to a low and persistent stable level a “true” or sustained remission is achieved, and the medication can be adjusted accordingly.
  5. Increasing calprotectin concentrations from the stable level mentioned above indicates relapse which in this way is revealed at an early stage. This is favorable for the patient and his quality of life, making early and appropriate treatment available, thereby shortening the period of fulminant symptoms.
  6. The ability of fecal calprotectin to differentiate between organic and functional disorders makes it a useful and cost effective tool for the clinician , identifying patients with GI symptoms who may or may not require endoscopy or other invasive analyses. Many patients are spared for an uncomfortable examination! This is particularly important in endoscopy of children; general anesthesia is required for this procedure.
  7. Fecal calprotectin is a very convenient marker in the diagnosis of necrotizing enterocolitis in newborn children.
  8. Calprotectin in bile is a promising disease marker in patients with primary sclerosing cholangitis with possible prognostic value.
  9. The diagnosis gastrointestinal graft versus host disease (GI-GVHD), which is a serious complication after allogeneic stem cell transplantation, is based on clinical symptoms and histological findings. No biomarkers are routinely used to predict GVHD. However, recent data demonstrate that elevated fecal calprotectin levels were significantly associated with presence of GI- GVHD, making this marker a promising tool in early diagnosis.

As the original inventor of faecal calprotetin assays the cut off value 50 mg/kg between normal and pathological levels were established by Calpro, based on ground breaking scientific publications. Calpro has kept this critical cut off unchanged since 2000, and this cut off value was confirmed in a study comparing different ELISAs by Labaere. Since 2005 several manufacturers have launched calprotectin ELISAs; each company does their own independent calibration. However, despite values between assays differ considerably and ELISAs from different manufacturers cannot be used interchangeably, they have all adopted 50 mg/kg as cut off.

Sampling of faecal calprotectin; general advice

In proctitis the area of inflamed mucosa is very limited and the contact time between stool and tissue may be short, especially if the sample is collected late in the day and not from the first defecation. In this situation the intestinal content may pass the short inflamed area very rapidly, not allowing “the incubation time” in the gut to be sufficient for delivery of calprotectin into the stool. This is the reason why it is recommended that samples for calprotectin determination should be collected from the first defecation in the morning, thereby ensuring that “the incubation time” is sufficient. Lasson et al. (31) studied fecal calprotectin in patients with active ulcerative colitis, samples were collected from consecutive defecations during the day. They found that the levels are highest in samples collected when there is long time between the defecations. It is concluded that the sampling should be the same from day to day, preferably in the morning.

How should different calprotectin values be interpreted by the clinician?

Many European gastroenterologists have used our ELISAs for many years, and I think the recommendations below are representative for a clinical practice that is adopted by many clinicians. The patients are divided into two groups; the evaluation of test results is different in the two groups.

Group 1: This is undiagnosed patients with stomach problems; clinical examination cannot exclude IBD. The cut-off value is 50 mg/kg, but values between 50 and 100 are often seen without endoscopic findings. They can be characterized as false positives or lying in a grey zone. Fecal calprotectin should be repeated and clinical parameters considered. Values higher than 100 are always taken as positive.

Group 2: This is patients with IBD. Calprotectin values of several hundreds or thousands are seen in patients with active IBD, reflecting the disease activity objectively. By repeated testing, every week or month, the marker is very useful in monitoring the effect of treatment in active iBD. If high calprotectin levels are not reduced during treatment, non-responders can be identified and the medication adjusted accordingly. During effective treatment a steep fall in the calprotectin curve is usually seen; when values pass 200 or possibly 150 mg/kg and persist below that level, this indicates mucosal healing, which can be verified by endoscopy, and the treatment can be stopped. Increasing values passing 150/200 mg/kg indicate relapse and treatment should again be considered.

Calprotectin levels between 150/200 and 500 mg/kg indicate “moderate disease activity” and values above that “high disease activity”.

The conclusion so far is that in patients with clinical quiescent disease and calprotectin levels below 150/200 treatment is not necessarily needed. However, it is important to bear in mind that the cut off values mentioned above are only indicative, and hopefully ongoing studies will elucidate this important issue more precisely.  Fecal calprotectin is an objective marker of disease activity in IBD, but although not decisive, it should be taken into consideration as an important factor to achieve optimal decisions on treatment of patients with IBD.

In most biological quantitative tests there are “grey zones”  or “border zones” in which the interpretation of  results are  questionable; it is favourable to make these zones as narrow as possible. In Calpro Calprotectin ELISA (CalproLab) this objective is achieved; the grey zone in group 1 is in fact only from 50 to 100 mg/kg. In contrast, test kits from manufacturers that measure 2 or 3 times higher than that of CalproLab and still maintain 50 mg/kg as the cut off value, have a problem: the wide grey zone makes the interpretation of results difficult, necessitating retesting of many samples.

Our CalproLab assay has one of the broadest measuring ranges, from 25 to 2500 mg/kg with only one dilution of the fecal extracts, in the market. Retesting using alternative dilutions is therefore seldom necessary.

By using our prefilled faecal extraction device, EasyExtract, we can also offer a most convenient, easy and hygienic sample preparation. In the future, the patients will be able to make their own fecal extract and send it by postal service directly to the laboratories by use of this device. EasyExtraxt with fecal extract can be analyzed directly without any centrifugation. By use of an ELISA automate (we recommend DS 2 by Dynex, and our CalproLab kit is especially adapted to this instrument) the EasyExtract tube can be placed directly into the instrument. This combined solution with practical and hygienic sample handling and an automated calprotectin assay is probably one of the most time-saving and convenient calprotectin assays on the market.