ReaPan B27


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ReaPan B27

  1. 1. ReaPan B27 A Reagent for HLA-B27 Typing September 2008
  2. 2. What is HLA-B27 ? <ul><li>Human Leukocyte antigen (HLA) are a group of proteins that help the body’s immune system to identify its own cells and to distinguish b/w “self” and “non self” </li></ul><ul><li>HLA-B27 has been associated with seronegative spondyloarthropathies: </li></ul>10% Anterior Uveitis 50% Inflammatory Bowel Disease 50-80% Reiter’s Syndrome 80% Rheumatoid Arthritis >90% Ankylosing Spondylitis Relative association with HLA-B27 antigen Condition
  3. 3. HLA-B27 typing <ul><li>Accuracy of information – Considered a gold standard </li></ul>Diagnostic Methods in HLA-B27 typing PCR Platform Flow cytometry Platform <ul><li>Ease of operation </li></ul><ul><li>Speed of work flow </li></ul><ul><li>Requires high level of expertise </li></ul><ul><li>Laborious work flow (6-8 hours) </li></ul><ul><li>High Cost </li></ul><ul><li>Reduced sensitivity and specificity in comparison to PCR </li></ul><ul><li>High Cost </li></ul>Limitations of PCR based testing Advantages of PCR based testing Advantages of Flow Cytometry based testing Limitations of Existing Flow Cytometry based testing
  4. 4. Relative reagent performance for HLA-B27 typing <ul><li>Typing by PCR platform is highly accurate </li></ul><ul><li>Flow cytometry platforms are less expensive and provide quicker turn around time </li></ul>Accuracy of Test Ease of Work Flow Assay Time Cost of Assay Parameter Performance Rating PCR Flow Cytometry
  5. 5. HLA-B27 typing in the Indian context Ankylosing Spondylitis (AS) is associated with HLA-B27 expression(>90%) Many cases suspected of AS are not referred for HLA-B27 typing High cost of Diagnosis BUT… WHY? High cost of HLA-B27 typing inhibits widespread prescription of the test
  6. 6. Designing a reagent for B27 typing – an India centric approach <ul><li>Requirements for a ‘new’ B27 Test </li></ul><ul><ul><li>Accuracy offered by PCR </li></ul></ul><ul><ul><li>Ease of work flow offered by Flow Cytometry </li></ul></ul><ul><ul><li>India Centric Factors </li></ul></ul><ul><ul><ul><li>Affordable reagent to make testing more accessible </li></ul></ul></ul><ul><ul><ul><li>Provide Reagent in a dry format for ease of storage </li></ul></ul></ul>ReaMetrix Solution: A Flow-Cytometry based test that offers accuracy closest to that of PCR
  7. 7. Rational design of B27 Reagent – The ReaMetrix approach <ul><li>Accuracy </li></ul><ul><ul><li>Increase sensitivity by using 2 Anti-HLA B27 antibody clones (FD705 and HLA-ABC-M3) 7 </li></ul></ul><ul><ul><li>Increase specificity by minimizing cross reactivity with HLA-B7 antigen using Anti-HLA-B7 antibody 7 </li></ul></ul><ul><ul><li>Reduce noise by selecting CD3+HLA-B27+ population 3 and using bright fluorophores (R-PE vs. FITC) </li></ul></ul><ul><ul><li>Use Internal Fluorescence Standards for every test to assure result quality </li></ul></ul><ul><li>Ease of work flow </li></ul><ul><ul><li>A Lyse-No wash approach to reduce assay time (<1 h) </li></ul></ul><ul><li>Design for the Indian diagnostic setting </li></ul><ul><ul><li>Provide reagents in a dried format to simplify storage conditions </li></ul></ul><ul><ul><li>Reduce cost of test through innovation on process development </li></ul></ul><ul><ul><li>Reduce ambiguity in typing results </li></ul></ul>Reagent to meet the desired performance requirements – ReaPan B27
  8. 8. ReaPan B27 assay protocol <ul><li>Simplified Work Flow </li></ul><ul><ul><li>Incubation of sample in unitized dried reagent tube </li></ul></ul><ul><ul><li>Lysis of RBCs </li></ul></ul><ul><ul><li>Fix - No wash </li></ul></ul><ul><ul><li>Analysis by flow cytometry </li></ul></ul><ul><ul><li>Stain to Analysis in 45 min. </li></ul></ul>Dried Reagent Tube Add 25µl of Sample Vortex & Incubate for 30mins Add 300µl of ReaLyse Solution & Incubate for 15mins Analyze by Flow Cytometry
  9. 9.  Beads  Beads CD3+   Beads CD3+ HLA-B27+ Acquisition strategy <ul><li>Standardized Templates </li></ul><ul><ul><li>No compensation adjustments </li></ul></ul><ul><ul><li>Internal Fluorescence standards (beads) to “calibrate” instrument for each sample </li></ul></ul><ul><li>Results are reported as RMX values </li></ul><ul><li>Sign of the RMX value indicates positive or negative typing </li></ul>
  10. 10. Design comparison of flow based reagents ReaPan B27 has been designed for higher specificity and sensitivity <ul><li>Internal standard per test </li></ul><ul><li>CD3 labeled with CD3-PE-Dyo649 and HLA B27 labeled with PE </li></ul><ul><li>HLA-ABC-m3 and FD705 clones for HLA B-27 selection </li></ul><ul><li>Lymphocyte population selection using CD3 </li></ul><ul><li>HLA B7 Ab to remove cross reactivity </li></ul>ReaPan B27 <ul><li>Requires control samples </li></ul><ul><li>Calbrite Beads and specific fluorescence intensity beads to establish threshold </li></ul>Discrimination of Positive and Negative Populations <ul><li>HLA B27 labeled with FITC and B7 labeled with PE </li></ul><ul><li>CD3 labeled with PE and HLA B27 with FITC </li></ul>Selection of Fluorophores <ul><li>HLA-ABC-m3 clone for HLA B-27 selection </li></ul><ul><li>HLA-B7 Ab to remove cross reactivity </li></ul>Coulter’s HLA-B27 Reagent <ul><li>GS145.2 clone for HLA B-27 selection </li></ul><ul><li>Lymphocyte population selection using CD3 </li></ul>Selection of Population BD’s HLA-B27 Reagent Parameter
  11. 11. Relative reagent performance for HLA-B27 typing <ul><li>ReaPan B27 </li></ul><ul><li>Accuracy closest to PCR </li></ul><ul><li>Operability easier than existing flow based reagents </li></ul><ul><li>Robust performance guaranteed by Internal Standards </li></ul>Accuracy of Test Ease of Work Flow Assay Time Cost of Assay Parameter Performance Rating PCR Flow Cytometry ReaPan B27
  12. 12. Sensitivity of ReaPan B27 Reagent = 96% Specificity of ReaPan B27 Reagent = 95% Accuracy of ReaPan B27 Reagent = 96% Comparison of assay performance with other reagents 71 43 28 TOTAL 42 41 1 Negative 29 2 27 Positive ReaPan B27 Negative Positive TOTAL Bd’s HLA-B27 Reagent Decision Matrix
  13. 13. Reference articles - Ankylosing Spondylitis <ul><li>Wilfried H. B. M. Levering, Henk Wind, Kees Sintnicolaas, Herbert Hooijkaas, and Jan W. Gratama. Flow Cytometric HLA-B27 Screening: Cross-Reactivity Patterns of Commercially Available Anti–HLA-B27 Monoclonal Antibodies With Other HLA-B Antigens. Cytometry Part B (Clinical Cytometry) 54B:28–38 (2003) </li></ul><ul><li>Michael J. Warzynski. HLA-B27 TESTING: MEAN CHANNELS, QC, AND SURVEYS. Cytometry (Communications in Clinical Cytometry) 30:208–211 (1997) </li></ul><ul><li>Jenn C. Chen, Bruce H. Davis, Nancy C. Bigelow, Carole Ceckowski, JoAnne Robinson,Cynthia Sounart-Miscovich, and Kame A. Steel. Flow Cytometric HLA-B27 Typing Using CD3 Gating and Molecules of Equivalent Soluble Fluorochrome (MESF) Quantitation. Cytometry (Communications in Clinical Cytornetry) 26:286-292 (1996) </li></ul><ul><li>P. S. Dhurandhar and U. Shankarkumar. HLA Association in SeronegativeSpondyloarthritis Patients From Mumbai, India. Int J Hum Genet, 7(3): 235-239 (2007) </li></ul>
  14. 14. <ul><li>Michael T. Seipp, Maria Erali, Rae Lynn Wies, and Carl Wittwer. HLA-B27 Typing: Evaluation of an Allele-Specific PCR Melting Assay and Two Flow Cytometric Antigen Assays. Cytometry Part B (Clinical Cytometry) 63B:10–15 (2005) </li></ul><ul><li>W.H.B.M. Levering, H. Wind, H. Hooijkaas, K. Sintnicolaas, B. Brando, J.W. Gratama. Flow cytometric screening for HLA-B27 on peripheral blood lymphocytes. J Biol Regul Homeost Agents 2003; 17: 241-6 </li></ul><ul><li>Johannes J.M.L. Hoffmann and Willy C.M. Janssen HLA-B27 phenotyping with flow cytometry:further improvement by multiple monoclonal antibodies. Clinical Chemistry 43:10, 1997 </li></ul><ul><li>Anne M. Ward and Afzal Nikaein. Comparison of Monoclonal Antibodies for Flow Cytometric Analysis of HLA-B27 Antigen. Cytometry (Communications in Clinical Cytometry) 22:65-69 (1995) </li></ul>Reference articles - Ankylosing Spondylitis
  15. 15. <ul><li>Wendy M. Reynolds, Philip R. Evans, Andrew C. Lane, W. Martin Howell, Peter J. Wilson, Raymond Wong, and John L. Smith Automated HLA-B27 Testing Using the FACSPrep/FACScan System. Cytometry (Communications in Clinical Cytometry) 18: 109-1 15 (1994) </li></ul><ul><li>Wilfried Levering External quality assessment in flow cytometry. Educational aspects and trends toward improvement. Doctoral Thesis </li></ul><ul><li>Wilfried H.B.M. Levering, Rene van den Beemd, Jeroen G. te Marvelde, Wil A.M. van Beers, Herbert Hooijkaas, Kees Sintnicolaas, and Jan W. Gratama. External Quality Assessment of Flow Cytometric HLA-B27 Typing. Cytometry (Communications in Clinical Cytometry) 42:95–105 (2000) </li></ul><ul><li>M. H. Nicknam, A.R.Jamshidi, M.D., M.Anjalikhani Hakemi, M.Sc., F. Khosravi, M.Sc., A. Amirkhani,m.Narouinejad, And B. Nikbin, Comparison of validity of microlymphocytotoxicity and flowcytometry methods with PCR for HLA-B27 antigen typing; MJIRI, Vol. 17, No. 1, 75-79, 2003 </li></ul>Reference articles - Ankylosing Spondylitis
  16. 16. Reference articles - Uveitis <ul><li>M. Huhtinen and A. Karma, Br HLA-B27 typing in the categorisation of uveitis in a HLA-B27 rich population - J Ophthalmol. 2000 April; 84(4): 413–416. doi: 10.1136/bjo.84.4.413 </li></ul><ul><li>PJ Derhaag, LP de Waal, A Linssen and TE Feltkamp, Acute anterior uveitis and HLA-B27 subtypes - Investigative Ophthalmology & Visual Science, 1998 July; Vol 29, 1137-1140 </li></ul><ul><li>Acute Anterior Uveitis and HLA-B27 J . Chang , P . McCluskey , D . Wakefield Survey of Ophthalmology ,2005 June; Volume 50 Issue 4, Pages 364 – 388 </li></ul><ul><li>HLA-B27+ anterior uveitis with or without associated spondyloarthritis: Clinical and immunological features RIVERA-CÍVICO et al. Ann Rheum Dis.1999; 58: 721-722 </li></ul>
  17. 17. Manufactured by ReaMetrix India Pvt. Ltd. 50-B, II Phase, Peenya Industrial Area Peenya, Bangalore 560058, India Ph: +91-80-28378693/5, Fax: +91-80-41172451 E-mail: Further details: