Three Biopsy Protocol

The three-biopsy protocol is now out of date – it has been made obsolete by the modern blood tests for celiac disease.

This biopsy focus on coeliac disease was taken a step forward in 1969, in Interlaken, Switzerland. A group of prominent gastroenterologists in paediatrics worked out a scheme to reliably diagnose coeliac disease. Remember, this was well before any blood tests came into the picture. It was also before it was understood that gluten could cause disorders outside the gut.

Three Biopsy Protocol

The object of this 1969 exercise was to unambiguously diagnose coeliac disease, also at that time called gluten-sensitive-enteropathy (enteropathy is the damage to the small bowel mucosa). They deemed that to diagnose coeliac disease with certainty it was necessary to do three biopsies: the three biopsy protocol. Remember that when this protocol was devised there were no useful screening blood tests for coeliac disease.


First, you needed to suspect that a child might have coeliac disease. The child would usually present with lots of pale sloppy poos, with a big tummy and not growing well. Also this child might be irritable and cranky. Often there would be coexisting nutritional deficiency. They would undergo their first small bowel biopsy to see whether the small bowel tissue was normal or abnormal.

If this biopsy was normal, then this child categorically did not have coeliac disease.

If the biopsy tissue was abnormal, then the child “might” have coeliac disease. This is because there are possible causes of abnormal small bowel tissue other than gluten toxicity.


If the first biopsy was abnormal, this child was then put onto a gluten-free diet. After a period of between six months to two years this child then had the second biopsy. This was to see if the gut had completely recovered on the gluten-free diet.

If the child had not got better, then the child was considered not to have coeliac disease. Therefore, gluten was allowed back into the diet.


If the child had got better on a gluten-free diet (that means that biopsy-two was normal), then unfortunately, the diagnosis of coeliac disease could still not be made. Even more evidence was required.

Therefore, this child was then put back onto gluten-containing foods again (a gluten challenge). The child would then be fed with gluten for three to six months (or less if the child got symptoms). Finally, they would have their third small bowel biopsy.

If biopsy-three was abnormal, then at last this child could be given the definite diagnosis of coeliac disease.

To summarise, if this child had gone through that series of three biopsies, with the results: abnormal-normal-abnormal, then this child could be given the diagnosis of coeliac disease. This is because the first abnormal biopsy could be due to other conditions; it does not always come from gluten toxicity. However, in some children it was shown that it can take more than two years before definitive changes could develop in the small bowel tissue again.

But the situation was not so straight forward. There is often a “maybe” result with a small bowel biopsy.

Do you need an endoscopy?

See this link to “Who needs an endoscopy

See this link to “Biopsy: not the “gold standard

Read “The Gluten Syndrome

Dr Rodney Ford MD. MB.BS. FRACP.