1. Automation of Immunofluorescence Assays in a Clinical Laboratory- An Unmet Need Matthew J. McGinniss PhD FACMG Senior Director, Laboratory Operations Prometheus Laboratories Inc.

2. Matthew J. McGinniss PhD FACMG  Board Certified  Clinical Molecular Genetics ABMG  Molecular Diagnostics, ABCC  PhD Cell Biology, University of Vermont, 1988  Johns Hopkins University School of Medicine, Postdoctoral Fellow, 1988-1992  Children’s Hospital – San Diego, 1993-2000  University of California – San Diego, Adjunct Asst Prof Pediatrics 1994-2004; Voluntary Asst Clinical Prof 2004-present  Sequenom, Inc, 2000-2003  Quest Diagnostics, 2003-2007  Prometheus Laboratories Inc. 2007- present

3. Why Automate? Reduce variability and improve quality Reduce labor and test costs Improve workflow in the laboratory Avoid potential ergonomic issues

4. Outline Operational constraints of a clinical laboratory Current workflow of our two immuno- fluorescence assays (ANCA and EMA) Unmet needs, opportunities and potential solutions for automation

5. Quality Assurance- Clinical Laboratory 1.Use of Validated Tests (FDA-approved kits, analyte- specific reagents (ASRs) and laboratory developed tests) 2.Use of established Reference Materials (eg. DNA samples, cell lines ) as “positive controls” 3.Adequate Staff Training 4.Use of “Best Practice Guidelines” 5.Laboratory Accreditation 6.External Quality Assessment / Proficiency Testing

6. Some Regulatory Constraints CLIA and CMS College of American Pathologists (CAP) State of California New York State ISO 9001,15189,13485 FDA proposed regulations on in vitro diagnostic multivariate index assays (IVDMIAs) as “medical devices”

7. Prometheus Laboratories Inc Number of Employees : ~310 Annual net sales (2006) : $68 million CLIA Certified Laboratory : San Diego, CA Diagnostic Specialty : Gastrointestinal, Autoimmune, Inflammatory Number of Laboratory Tests: 12 DNA 4 Serology 3 Enzyme 1 Other 4

8. Established Leader in Gastroenterology Diagnostics IBD Serology 7, Celiac Serology, Celiac Genetics, TPMT Genetics, TPMT Enzyme,Thiopurine metabolites, FIBROSpect ® II Algorithm Development Publications

9. We are a busy lab ….

10. PROMETHEUS ® IBD Serology 7 ASCA IgA ASCA IgG Anti-OmpC IgA Anti-Cbir1 ANCA ELISA ANCA IFA perinuclear pattern ANCA IFA DNase sensitivity 5 immunoassays + 2 IFA assays + an algorithm

11. Anti-neutrophil Cytoplasmic Antibody (ANCA) Systemic vasculitis (Wegener’s granulomatosis, Churg-Strauss syndrome Microscopic polyangiitis ) Inflammatory Bowel Disease (Ulcerative colitis and Crohn’s disease) Rheumatoid Arthritis Systemic Lupus Erythematosis (SLE) Cystic Fibrosis Savige JA et al. 1998. J Clin Pathol 51:568-575

12. ANCA IFA – detection of anti-neutrophil cytoplasmic antibodies Source: www.bindingsite.co.uk/files/MKG265.pdf Our preparations are unique– we have optimized the detection of “ IBD- specific pANCA”

13. Endomysial Antibodies (EMA) Highly specific staining patterns in patients with Celiac disease and dermatitis herpetiformis Monkey esophagus is considered the tissue of choice for EMA assays Prepared slides are commercially available Bradwell et al. 1997. Atlas of Autoantibody patterns on tissues pp 56-59.

14. ANCA Labor-Intensive Slide Preparation Arrange for donor to collect whole blood sample Density centrifugation to isolate white cell component Count and dilute cell suspension Use Cytospin to adhere PMNs on glass slides Fix slides and then allow to air dry Store slides at -20 C Perform parallel testing to qualify new lot of slides before routine production use

15. Remove slides with adherent PMNs from freezer Add working DNase solution or buffer to appropriate wells Incubate at 37 C and wash slides Pipet diluted patient serum (and controls) to appropriate wells Incubate at 37 C and wash slides Pipet Antibody-FITC conjugate to each well Incubate at 37 C and wash slides Add Fluoromount G and cover slip to slide Examine each well with epifluorescence microscope Store used slides for up to one month ANCA Labor-Intensive Slide Analysis

16. Optimizing Fluorescence Microscopy  Characteristics of the fluorescence label  Use the appropriate objectives  Immersion oil is non-fluorescent  Use neutral density filters to reduce fading  Ensure the mercury lamp is centered and focused Lasslett A. 2005. Fluorescence in the pathology laboratory. The Biomedical Scientist pp. 548-550

17. Training and Quality Assurance Issues Variability noted with single donor PMNs We do not currently have an “Atlas” of images for training purposes Images are not currently digitized and archived Whole slide imaging has been explored for surgical pathology quality assurance Ho et al. 2006. Use of whole slide imaging in surgical pathology quality assurance: design and pilot validation studies. Hum Pathol 37(3): 322-331.

18. Proficiency Testing Proficiency testing programs are not available for IBD-specific pANCA, but are available for EMA IFA assays with CAP Immunology (Celiac Serology CES) May be cost-effective and advantageous to administer proficiency programs for IFA type assays with digital results Pilot programs in bright-field microscopy have explored the use of “virtual microscopy” and this may be effective for proficiency testing programs Marchevsky AM et al. 2006. The use of virtual microscopy for proficiency testing in gynecologic cytopathology. Arch Pathol Lab Med 130: 349-355

19. Commercially Available Solutions Source:www.bindingsite.co.uk/autoimmunediagnostics-8.asp

20. Commercially Available Solutions Source: www.biofilediagnostics.com/images/photos/ifa_processor.jpg

21. Commercially Available Solutions Source: www.htz.biz/beeline_220_ifa_main.htm

22. Opportunities  Automate some or all of the slide preparation steps  Real-time imaging on flat-screen monitor to improve training  Collect library / atlas of images for training and quality assurance purposes  Automate the image acquisition and result calling for both the ANCA and EMA IFA assays

23. Summary Currently examining options for digitizing the images from IFA assays for training and QA purposes We will be assessing the availability of fluorescence slide scanners to digitize and archive IFA images Automation of the slide preparation may also be possible with some currently available technologies Exploring ways to streamline production of donor neutrophils and the possibility of using a cell line in lieu of donor whole blood

24. Acknowledgements Mariko J. Matsutani Curtis A. McGuyer Robert M. Nakamura Henry Pan