reapan b27
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© 2008 Reametrix, Inc. All rights reserved.
ReaPan B27A Reagent for HLA-B27 Typing
September 2008
2© 2008 Reametrix, Inc. All rights reserved.
What is HLA-B27 ?
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”
HLA-B27 has been associated with seronegative spondyloarthropathies:
Condition Relative association with HLA-B27 antigen
Ankylosing Spondylitis >90%
Rheumatoid Arthritis 80%
Reiter’s Syndrome 50-80%
Inflammatory Bowel Disease
50%
Anterior Uveitis 10%
3© 2008 Reametrix, Inc. All rights reserved.
Accuracy of information – Considered a gold standard
Diagnostic Methods in HLA-B27 typing
PCR Platform Flow cytometry Platform
Ease of operationSpeed of work flow
Requires high level of expertise Laborious work flow (6-8 hours)High Cost
Reduced sensitivity and specificity in comparison to PCRHigh Cost
HLA-B27 typing
Limitations of PCR based testing
Advantages of PCR based testing
Advantages of Flow Cytometry based testing
Limitations of Existing Flow Cytometry based testing
4© 2008 Reametrix, Inc. All rights reserved.
Relative reagent performance for HLA-B27 typing
Typing by PCR platform is highly accurate
Flow cytometry platforms are less expensive and provide quicker turn around time
Accuracy of Test Ease of Work Flow Assay Time Cost of Assay
Parameter
Perf
orm
an
ce R
ati
ng
PCR
Flow Cytometry
5© 2008 Reametrix, Inc. All rights reserved.
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© 2008 Reametrix, Inc. All rights reserved.
Designing a reagent for B27 typing
– an India centric approachRequirements for a ‘new’ B27 Test
Accuracy offered by PCR
Ease of work flow offered by Flow Cytometry
India Centric FactorsAffordable reagent to make testing more accessibleProvide Reagent in a dry format for ease of storage
ReaMetrix Solution: A Flow-Cytometry based test that offers accuracy closest to that of
PCR
7© 2008 Reametrix, Inc. All rights reserved.
Rational design of B27 Reagent – The ReaMetrix approachAccuracy
Increase sensitivity by using 2 Anti-HLA B27 antibody clones (FD705 and HLA-ABC-M3)7
Increase specificity by minimizing cross reactivity with HLA-B7 antigen using Anti-HLA-B7 antibody 7
Reduce noise by selecting CD3+HLA-B27+ population3 and using bright fluorophores (R-PE vs. FITC)Use Internal Fluorescence Standards for every test to assure result quality
Ease of work flowA Lyse-No wash approach to reduce assay time (<1 h)
Design for the Indian diagnostic settingProvide reagents in a dried format to simplify storage conditionsReduce cost of test through innovation on process developmentReduce ambiguity in typing results
Reagent to meet the desired performance requirements – ReaPan B27
8© 2008 Reametrix, Inc. All rights reserved.
Simplified Work FlowIncubation of sample in unitized dried reagent tubeLysis of RBCs Fix - No wash Analysis by flow cytometry
Stain to Analysis in 45 min.
Dried Reagent Tube
Add 25µl of Sample
Vortex & Incubate for
30minsAdd 300µl of ReaLyse
Solution & Incubate for 15mins
Analyze by Flow
Cytometry
ReaPan B27 assay protocol
9© 2008 Reametrix, Inc. All rights reserved.
Beads
Beads
CD3+
Beads
CD3+ HLA-B27+
Acquisition strategy
Standardized TemplatesNo compensation adjustmentsInternal Fluorescence standards (beads) to “calibrate” instrument for each sample
Results are reported as RMX values
Sign of the RMX value indicates positive or negative typing
10© 2008 Reametrix, Inc. All rights reserved.
Design comparison of flow based reagents
Parameter ReaPan B27 BD’s HLA-B27 Reagent
Coulter’s HLA-B27 Reagent
Selection of Population
HLA-ABC-m3 and FD705 clones for HLA B-27 selectionLymphocyte population selection using CD3 HLA B7 Ab to remove cross reactivity
GS145.2 clone for HLA B-27 selectionLymphocyte population selection using CD3
HLA-ABC-m3 clone for HLA B-27 selectionHLA-B7 Ab to remove cross reactivity
Selection of Fluorophores
CD3 labeled with CD3-PE-Dyo649 and HLA B27 labeled with PE
CD3 labeled with PE and HLA B27 with FITC
HLA B27 labeled with FITC and B7 labeled with PE
Discrimination of Positive and Negative Populations
Internal standard per test
Calbrite Beads and specific fluorescence intensity beads to establish threshold
Requires control samples
ReaPan B27 has been designed for higher specificity and sensitivity
11© 2008 Reametrix, Inc. All rights reserved.
Relative reagent performance for HLA-B27 typing
ReaPan B27 Accuracy closest to PCROperability easier than existing flow based reagentsRobust performance guaranteed by Internal Standards
Accuracy of Test Ease of WorkFlow
Assay Time Cost of Assay
Parameter
Perf
orm
an
ce R
ati
ng
PCR
Flow Cytometry
ReaPan B27
12© 2008 Reametrix, Inc. All rights reserved.
Decision MatrixBd’s HLA-B27
Reagent TOTALPositive Negative
ReaPan B27
Positive 27 2 29
Negative 1 41 42
TOTAL 28 43 71
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
13© 2008 Reametrix, Inc. All rights reserved.
Reference articles - Ankylosing Spondylitis
1. 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)
2. Michael J. Warzynski. HLA-B27 TESTING: MEAN CHANNELS, QC, AND SURVEYS. Cytometry (Communications in Clinical Cytometry) 30:208–211 (1997)
3. 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)
4. P. S. Dhurandhar and U. Shankarkumar. HLA Association in SeronegativeSpondyloarthritis Patients From Mumbai, India. Int J Hum Genet, 7(3): 235-239 (2007)
14© 2008 Reametrix, Inc. All rights reserved.
5. 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)
6. 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
7. 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
8. 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)
Reference articles - Ankylosing Spondylitis
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9. 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)
10. Wilfried Levering External quality assessment in flow cytometry. Educational aspects and trends toward improvement. Doctoral Thesis
11. 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)
12. 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
Reference articles - Ankylosing Spondylitis
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Reference articles - Uveitis
1. 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
2. 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
3. Acute Anterior Uveitis and HLA-B27 J . Chang , P . McCluskey , D . Wakefield Survey of Ophthalmology ,2005 June; Volume 50 Issue 4, Pages 364 – 388
4. HLA-B27+ anterior uveitis with or without associated spondyloarthritis: Clinical and immunological features RIVERA-CÍVICO et al. Ann Rheum Dis.1999; 58: 721-722
17© 2008 Reametrix, Inc. All rights reserved.
Manufactured by ReaMetrix India Pvt. Ltd.50-B, II Phase, Peenya Industrial Area
Peenya, Bangalore 560058, IndiaPh: +91-80-28378693/5, Fax: +91-80-
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