Harvard researchers at Children’s Hospital Boston have identified a protein in the urine of appendicitis patients that they believe may provide the basis of a quick, noninvasive, accurate, and inexpensive test for the common condition.
Acute inflammation of the vermiform appendix, commonly known as appendicitis, is one of the oldest emergencies in the annals of medicine, but its diagnosis still can be challenging, leading to delays and mistakes that can result in complications and death. But all that may be changing.
In a report released today in the online edition of Annals of Emergency Medicine, the researchers at Children’s Proteomics Center say the marker they found is 500 times more abundant in the urine and tissue of those with appendicitis than in healthy individuals.
The little worm-shaped organ featured in Leonardo da Vinci’s anatomical drawings, and whose inflammation may have been forever preserved in an Egyptian mummy of the Byzantine era, continues to be the cause of one of the most common diseases in children and adults in this century, and remains among the most urgent surgical emergencies worldwide.
In the United States, approximately one in 1,000 people are afflicted by the disease yearly. About 3 percent to 30 percent of children arriving at the emergency room with abdominal pain are subject to unnecessary appendectomies, and a larger percentage, about 30 percent to 45 percent of those who are suffering from the disease, are diagnosed when the appendix has already ruptured.
“We wanted to develop ways to identify markers and to establish protocols for early diagnosis of disease,” says clinical fellow Alex Kentsis. “As a first step in the process, we thought we could use appendicitis as a test case because it is still a tremendously important problem in medicine for both kids and adults.”
“People come to the emergency room with abdominal pain; they can spend hours there before they get a slot for the CT, and while being there their appendix bursts because it just took so long,” says Hanno Steen, director of the Proteomics Center and assistant professor of pathology. “We wanted to come up with something that’s much faster and much easier; and what could be easier than peeing into a cup and putting a dipstick in and waiting a few minutes to see whether the color changes? That’s basically the motivation to take the technology out; because technology is always associated with significant costs and also takes time.”
Using proteomics, the systematic study of the proteins in tissues, cells, or body fluids, Kentsis, Steen, and Richard Bachur, associate professor of pediatrics and chief of emergency medicine at Children’s Hospital, analyzed the urine samples of 12 children: six healthy and six with appendicitis, the latter both before and after the removal of their appendixes. “We were looking for proteins that were significantly more abundant in urine,” recounts Steen.
“Out of this study and other means we came up with a list of 57 proteins. Then we took 67 urine samples from children with suspected appendicitis. This time we only looked at those proteins that were both the most abundant in some samples and less abundant in others.”
This was possible using state-of-the-art mass spectrometry, what Steen calls “a very fine balance” that allows researchers to measure and detect a wide range between the most abundant proteins and the least abundant proteins of about nine to 12 orders of magnitude. “That’s equivalent to the difference between the height of an ant and the distance from the Earth to the moon,” says Steen.
The team identified seven diagnostic marker candidates. “Out of those, we found this one protein, LRG, which was 500 times more abundant in urine from patients with appendicitis compared to controls,” says Steen. “That’s much more than we expected. It was a dream situation from our perspective. It made the study very easy.”
The protein, LRG, or leucine-rich alpha-2-glycoprotein, appears to be a specific marker of local inflammation. “During the second phase of the study, we attempted to validate this candidate blindly to determine how well it identified patients with appendicitis,” explains Kentsis. The researchers saw that LRG had the best performance in terms of specificity (how much it can distinguish those patients with appendicitis from those without it) and sensitivity (how well it serves as a marker to detect the disease).
The team also looked at the extracted appendix tissue, finding a correlation between the abundance of LRG in the tissue and its abundance in the urine samples. “Patients with very high concentration of the protein in the urine also had very high concentration of the protein in their appendix,” adds Steen.
If the observed performance of LRG is confirmed in studies with a larger number of patients, the use of the protein as a diagnostic marker could be implemented in the emergency departments of hospitals, says Kentsis. “We hope using LRG will improve the accuracy in diagnostic evaluations for appendicitis, and hopefully make them faster, reducing the complications from delayed or wrong diagnosis, and the costs and mortality associated with that.”
It’s unlikely that the new diagnostic marker will be used in the form of a home test anytime soon.
“Having the test at the pharmacy is a dream,” says Steen. “But we first have to confirm our findings doing further and larger patient studies, and we also have to validate it in adults.”
“In a way, you could think of having this test available at any pharmacy as a dipstick, like you buy a pregnancy test,” says Steen, “but you have to be very careful because when you do the pregnancy test, and you get one result or the other, it doesn’t matter much what you do next. But if you have a dipstick test for appendicitis, and you don’t have the specificity or the sensitivity you need, if the test is negative, but it turns out you really do have the disease, you probably won’t go to the doctor, and in the case of appendicitis this would be very dangerous.”
The biggest contribution to the diagnosis of appendicitis in this country, says Kentsis, has been the use of high-resolution imagery such as CT scanners. “Unfortunately, the technology is available in a more limited way in most of the rest of world, and in some places it is not available at all,” he says. “The diagnosis of appendicitis there really depends on the experience and skill of individual doctors, and it remains a challenging diagnosis to make.”
The scientists hope that testing for LRG will make that easier.