Laboratory
360 Amsden Avenue
Versailles, Kentucky 40383
Procedure Description:
CLOtest
Prepared by:______
Norma J. Whitworth, BSMT(ASCP) Date
Laboratory Manager / Approved by:
______
Richard Lozano, M.D. Date
Medical Director
Effective Date: / Revised:
Annual Review:
Principle:
CLOtest detects the urease enzyme of Helicobacter pylori, formerly called Campylobacter pylori, in gastric mucosal biopsies. H. pylori can cause active chronic gastritis and has been implicated as a primary etiologic factor in duodenal ulcer disease, gastric ulcer and nonulcer dyspepsia. H. pylori colonizes the mucous layer of the antrum and fundus of the stomach but does not invade the epithelium. By virture of this ability to colonize the gastric mucosa, persist despite the host immune defense, and cause host tissue damage, H. pylori is an effective and significant bacterial pathogen.
H. pylori produces large amounts of urease enzymes. Although urease primarily allows the organism to utilize urea as a nitrogen source, the breakdown of urea also produces high local concentrations of ammonia, which enable the organism to tolerate low pH.
Although H. pylori can be detected with histology or culture of gastric tissue, simple tests for the presence of urease enable more rapid and convenient diagnosis. Tests for gastric urease are specific for H. pylori because mammalian cells do not produce urease and, except for H. pylori, the stomach is usually sterile.
Materials Needed:
CLOtest test pack
Sterile needle
Storage:
CLOtest should be stored in the refrigerator at 2-8°C. The kit has a shelf life of 18 months when refrigerated.
CLOtest is a sealed plastic slide holding an agar gel, which contains urea, phenol red ( a pH indicator), buffers and bacteriostatic agents.
For In Vitro testing only.
Specimen Collection and Handling:
Patients should not have taken antibiotics or bismuth salts for at least 3 weeks prior to endoscopy. These agents can cause false negative results. A biospy specimen is needed of 1-3 mm and 0.04 – 12 inches in diameter. A sterile needle or forceps should be used for collection and insertion into agar gel.
Quality Control:
Quality control is ran per lot # change, new kit opened, and weekly on the CLOtest kit. Urease Type 3 Positive Control is used. Perform QC as follows:
1.) Peel back the label on the CLOtest slide so that you can see the yellow gel.
2.) Put “CLOtest Positive Control” tablet into gel.
3.) Re-seal the CLOtest label over gel.
4.) Set a timer for 5 minutes. Read results.
Gel should turn red of magenta indicated a positive reaction. If gel does not turn to red or magenta, call Ballard Medical Products at (800) 528-5591.
Testing Procedure:
Preparation of CLOtest:
1.) Check color and fullness of agar gel. If CLOtest is any color other than yellow, do NOT use.
2.) Warm CLOtest slide should be warmed at a temperature of 30-40°C by placing it on a warming plate, inside an incubator, or hold within hands.
Collection and Insertion of Biopsy Specimen:
Endoscopy Collection:
1.) The doctor should examine the stomach prior to biospy collection. The usual area to biopsy is the sump of the antrum, along the greater curve. The biopsy should be from a normal looking tissue area instead of an infected or ulcerated one. This is because H. pylori may be present in smaller numbers if the epithelium is eroded or the mucus layer is denuded.
2.) Using sterile forceps or needle, remove tissue specimen. The standard biopsy forceps will provide a specimen of sufficient size (1-3 mm/0.04 – 0/12 inches in diameter). Collect a second biopsy if the first specimen appears to small. Avoid blood contamination from the first biopsy site.
3.) Turn CLOtest slide so that the label is facing up and you do not see the well with the yellow gel.
4.) Peel the label away from the plastic so that you can see the yellow gel. Do NOT remove the label.
5.) With a sterile needle, take the biospy sample from the biopsy forceps and push it into the yellow CLOtest gel. Make certain that the biopsy specimen is buried in the gel so that it will have the most contact with the gel.
6.) Re-seal the CLOtest label by pressing the label back on the plastic slide so that the gel is covered.
7.) Write the patient’s name, medical record number, and the date and time of collection on the specimen label and lab requisition.
8.) Transport CLOtest to lab within 20 minutes of collection or call for pick-up, ext. 2367.
Laboratory:
1.) Read results from time of collection at 20 minutes.
2.) Place CLOtest slide into 37°C incubator.
3.) Set timer for 1 hour from time of collection. Read results.
4.) Set timer for 3 hours from time of collection. Read results.
5.) At 3 hours place CLOtest in tray on counter labeled “CLOtest.” Set timer for 24 hours after initial collection. Leave at room temperature. Read results.
6.) Document results on Clotest result form or in Meditech. Result must include date, time, and technologist’s initials.
Interpretation: Reporting Results:
Positive result: Red or magenta gel color change
Negative result: Yellow gel color(no change) or any color other than red or magenta
· If the biopsy specimen is contaminated with blood, a color other than yellow may be seen around the edge of the biopsy. This is NOT a positive test result.
· If urease is positive in the the biopsy specimen, a red color change will be seen around the embedded tissue area. If only a small number of H. pylori are present in the specimen, the entire CLOtest gel will change color.
· Any change of color of the entire gel form yellow will be a positive test. It will be the normal shades of red (magenta, pink, orange, etc.).
· Do not read test after 24 hours, it is invalid at this stage.
Limatations:
- False negatives may occur when very low numbers of H. pylori are present of the bacterium has a patchy distribution. For example, in 1 – 5 % of patients, the bacterium is present in the body of the stomach but not the antrum and vice versa.
- H. pylori does not colonize the intestinal mucosa, a false negative test can result if biopsy from this area. Therefore, two biopsies may be needed, one from the antrum and one from the gastric mucosa.
- All tests will be less sensitive if the patient has recently taken antibiotics or bismuth. Re-growth may be patchy after suppression with antibiotic. Again, an extra biopsy may be taken to avoid a false negative result.
- False positive tests are rare. The only reported cases of false positive tests were due to using gel ere slightly orange before use. Therefore, do not use any tests that are not yellow in color.
- A false positive could occur in a patient with Achlorydria. For example, patients with Pernicious Anemia, previous gastric surgery, or who have recently taken antacid, or large does of H2 receptor antagonista. When acid is absent, commensal organisms such as Proteus spp. May grow in the stomach and produce urease. False positive reactions due to other organisms will usually not react before three hours because these bacteria produce much less urease than H. pylori.
- If there are factors which may adversely effect the performance of the CLOtest, the physician is advised to consider other diagnostic measures, such as culture with gram stain and/or histology in order to confirm or disprove a diagnosis of H. pylori.
- Since therapy for H. pylori is only 50 – 75 % effective at present, it is important to reassess the patient after 28 days after completing therapy to confirm eradication. Biopsies taken before this time may give false negative results because of patchy re-growth of the organism.
Reference:
Ballard Medical: CLOtest Package Insert, Trimed Specialties, Draper, Utah, 4/98.
Lehmann, C., Saunder’s Manual of Clinical Laboratory Science, WB Saunders Company,
Philadelphia, Penn., 1998