| | Background
From January 2004 to the end of January 2006 Alberta Agriculture, Food & Rural Development’s (AAFRD) Transmissible Spongiform Encephalopathy (TSE) Laboratory screened a total of over 41, 000 Bovine, Cervid and Ovine tissue samples for the abnormal prion protein (PrPres) associated with Bovine Spongiform Encephalopathy (BSE), Chronic Wasting Disease (CWD) and Scrapie, using the Bio-Rad TeSeE® ELISA test. The tissue samples required for this rapid screening test are either the obex region of the brainstem for cattle, elk, and sheep or the retropharyngeal lymph node (RPLN) for whitetail and mule deer. A tonsil is tested for sheep and deer if the obex and RPLN are not present. Practicing veterinarians, AAFRD post mortem room technicians, meat inspectors and Canadian Food Inspection Agency (CFIA) inspectors collect the appropriate samples(s) from the animal head and submit them to the TSE laboratory where trained technical staff weigh and collect the specific target tissues needed for testing (310-390 mg for obex and 180-220 mg for RPLN or tonsil). The sampled tissues are then placed into Bio-Rad screw top “grinding tubes” which are pre-filled with buffer and bar-coded. Testing then proceeds according to the manufacturer’s protocol that includes sample preparation stage, digestion stage and ELISA detection stage. The detection stage yields a sample Positive or Negative test result based on an Optical Density (O.D.) value that is measured and interpreted relative to positive and negative O.D. cut-off values established for each test run.
Quality Assurance Program for AAFRD’s TSE Surveillance Testing
Quality Assurance (QA) is important for all laboratory testing in order to maintain high standards, accuracy and consistency. The AAFRD TSE Laboratory has an ongoing QA program with the CFIA National BSE and CWD/Scrapie Reference Laboratories located in Winnipeg and Ottawa respectively. This QA program is required by the CFIA for all laboratories that are members of the National TSE Veterinary Diagnostic Laboratory Network. In addition, AAFRD’s TSE Laboratory carries out an in-house Bio-Rad/Immunohistochemistry QA program with AAFRD’s Histology Laboratory. The Standard Council of Canada (SCC) certified this QA program in 2005 under ISO 17025. All equipment used in the TSE laboratory is maintained, calibrated and validated according to ISO17025 requirements and Standard Operation Manuals, either by the TSE laboratory staff or Bio-Rad engineers.
To establish the test in AAFRD laboratory, inter-laboratory Bio-Rad TeSeE® validation studies were conducted between AAFRD and CFIA for BSE and CWD and Scrapie. Consequently, AAFRD lab received the certification from CFIA to use Bio-Rad TeSeE® for TSE. The results of the validation studies are tabled below.
Results of AAFRD - CFIA Inter-laboratory Bio-Rad TeSeE Validation Studies for BSE, CWD & Scrapie (2003 and 2004) |
TSE | # of Samples Tested | Sensitivity % | Specificity % |
BSE | 104 | 100 | 100 |
CWD | 389 | 100 | 99.4 |
Scrapie | 320 | 95 | 100 |
For every microplate of 96 tests, 6 commercial synthetic control samples are included (2 positive and 4 negative controls). All control data is recorded and plotted on monthly control charts for tracking. At the end of each month, the control data with averages is sent to the appropriate CFIA Reference Laboratory (Winnipeg for BSE and Ottawa for CWD and Scrapie) for test monitoring. Negative control data is of particular importance, because the cut-off limit for Positive Reactor is calculated based on the Negative control values. From March through August of 2005, of 840 negative control data, the values have been consistent with a mean value of 0.010 and Standard Deviation (SD) of 0.005.
Although the number of samples tested may vary with the seasons, typically 4 BSE and 4 CWD surveillance samples are randomly collected for QA purposes weekly. QA samples for Scrapie are collected approximately twice per month due to lower sample volume. Each target tissue is sampled such that duplicate homogenates are prepared. One of the homogenates is tested in AAFRD’s TSE laboratory using the Bio-Rad TeSeE® test. The duplicate homogenate is frozen. In addition, Immunohistochemistry (IHC) staining of the formalin-fixed QA target tissues is performed at AAFRD’s Histology Laboratory. Following interpretation of the IHC stained QA tissues by a pathologist, the formalin-fixed paraffin tissue blocks are sent along with the frozen homogenates for Bio-Rad TeSeE® and IHC QA testing at the CFIA Reference Laboratories. The Bio-Rad and IHC QA samples are batched and shipped to the CFIA laboratories approximately every six months. The parent tissue samples used for QA are stored at the AAFRD laboratory at –80°C until the IHC and Bio-Rad results are reported by the CFIA Reference Laboratories, at which time they may be disposed of.
To date, 142 bovine and 124 cervid samples from AAFRD have been QA tested by the CFIA Reference Laboratories. Sample O.D. values for the Bio-Rad ELISA have correlated well between the CFIA and AAFRD TSE Laboratories (Figure 1), plus a 100 percent inter-laboratory agreement between the Bio-Rad results and IHC interpretations.
In addition, annual Bio-Rad and IHC TSE proficiency test panels are supplied from CFIA to all the National TSE Diagnostic Network Laboratories. The results from all the TSE Network Laboratories are compared and the standard deviations calculated. The results of Bio-Rad TeSeE® ELISA and IHC BSE proficiency test panels analyzed at AAFRD’s laboratory in 2004 and 2005 and mixed CWD and Scrapie test panels analyzed in 2005 agreed 100% with CFIA test results.
To date AAFRD's TSE Laboratory has detected 4 BSE reactors and 14 CWD reactors using the Bio-Rad TeSeE® ELISA. CFIA, using IHC staining and Western Blot rapid test methods, have confirmed all 18 of these positive reactors
Note: CFIA's TSE Laboratory uses a manual method for sample purification while AAFRD's TSE Laboratory uses a robotic system (NSP, Bio-Rad). The high O.D. value of 0.127 for sample #52 is an anomaly that falls well below the positive cut-off value of 0.220 (varies slightly with each test based on control data). The CFIA mean O.D. value of 0.016 correlates highly with the AAFRD mean O.D. value of 0.015.
Chris Onderka, Eva Chow, Brian Miller, Michelle Longson, Cynthia Wittmeier, Marilyn Johnson, Celia Lo |
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