Automation of Immunofluorescence Assays in a Clinical Laboratory- An

Unmet Need

Matthew J. McGinniss PhD FACMG

Senior Director, Laboratory Operations

Prometheus Laboratories Inc.

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

Why Automate?

• Reduce variability and improve quality

• Reduce labor and test costs

• Improve workflow in the laboratory

• Avoid potential ergonomic issues

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

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

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”

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 4Serology 3Enzyme 1Other 4

Established Leader in Gastroenterology Diagnostics

• IBD Serology 7, Celiac Serology, Celiac Genetics, TPMT Genetics, TPMT Enzyme,Thiopurine metabolites, FIBROSpect®II

• Algorithm Development

• Publications

We are a busy lab….

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

Anti-neutrophil Cytoplasmic Antibody (ANCA)

• Systemic vasculitis (Wegener’s granulomatosis, Churg-Strauss syndromeMicroscopic 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

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”

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.

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

• 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

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

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.

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

Commercially Available Solutions

Source:www.bindingsite.co.uk/autoimmunediagnostics-8.asp

Commercially Available Solutions

Source: www.biofilediagnostics.com/images/photos/ifa_processor.jpg

Commercially Available Solutions

Source: www.htz.biz/beeline_220_ifa_main.htm

OpportunitiesAutomate 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

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

Acknowledgements

• Mariko J. Matsutani

• Curtis A. McGuyer

• Robert M. Nakamura

• Henry Pan