Inflammatory bowel diseases in Indo-Canadians: Laboratory studies
Laboratory studies: For each patient, all blood samples were collected into vacutainer glass tubes (Becton Dickinson, Franklin, New Jersey) by a laboratory technologist without knowledge of the patient’s clinical history, investigation results or diagnosis. Blood was also obtained for hematological studies (hemoglobin, white blood cell count and platelet count), erythrocyte sedimentation rate, antinuclear antibodies, liver chemistry tests (aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase), serum iron studies and serum protein tests, including serum albumin.
Finally, blood samples were collected into vacutainer glass tubes, allowed to clot at room temperature and used for detection of ANCA via two methods: ANCA immunofluorescence and ELISA. Serological studies for ANCA were done by a single laboratory technologist blinded to the clinical details and diagnosis. If ANCA immunofluoresence was positive, ANCA ELISA was done for autoantibodies to myeloperoxidase (MPO) and serine protease-3 (PR-3). ANCA immunofluorescence: Indirect immunofluorescence for c-ANCA, p-ANCA and atypical p-ANCA was detected with a standardized fluorescent antibody detection method, using a proprietary kit purchased from a commercial supplier (Inova Diagnostics Inc, San Diego, California). Slides were supplied with an adherent layer of cultured human neutrophils. The culture conditions were designed to ensure stability and strong expression of the primary cyto- plasmic granules. The adherent neutrophils were fixed by the manufacturer with either ethanol or formalin. The primary screen for c-ANCA or p-ANCA involved incubation of serum at a one to 20 dilution in phosphate-buffered saline with ethanol-fixed slides for 25 mins at room temperature followed by a 5 min wash with phosphate-buffered saline. This was followed by another 25 min incubation using affinity-purified antihuman immunoglobulin (Ig) G with a fluorescent tag and a further 5 min wash with phosphate-buffered saline. Coverslips were then applied to the slides, and these were examined with a fluorescence microscope at x500 magnification.
Positive ANCA cellular fluorescence was recorded and the distribution patterns of fluorescence designated as c-ANCA, p-ANCA or atypical p-ANCA. Sera that were positive for either p-ANCA or atypical p-ANCA were further evaluated using formalin-fixed slides because formalin destroys nuclear antigens and atypical p-ANCA. In addition, formalin fixes both c-ANCA and p-ANCA antigens in the cytoplasm so that a false positive pattern is not observed. Antinuclear antibody-positive sera were excluded because these may mimic ANCA.
ELISA assays: ANCA ELISA assays were performed with a standardized method using commercial kits (Quanta-Lite MPO and PR-3 ELISA, Inova Diagnostics Inc). The test kits use microtitration strips containing wells coated with either PR-3, a primary antigen related to the c-ANCA pattern, or MPO, a primary antigen associated with the p-ANCA pattern. Diluted serum was applied and incubated. If specific antibodies are present, they bind to the wells. Unbound material is initially removed by washing, and bound antibody is detected by adding enzyme-labelled antihuman IgG, followed by a second washing, then incubated with a nitrophenol substrate. Wells with PR-3 or MPO antibodies are quantified by a colorimetric method. Your life is worth living. Buy sale cialis tablets online
