ReaPan B27

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Information about ReaPan B27
Health & Medicine

Published on February 18, 2009

Author: zmanian

Source: slideshare.net

ReaPan B27 A Reagent for HLA-B27 Typing September 2008

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: 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

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:

HLA-B27 typing Accuracy of information – Considered a gold standard Diagnostic Methods in HLA-B27 typing PCR Platform Flow cytometry Platform Ease of operation Speed of work flow Requires high level of expertise Laborious work flow (6-8 hours) High Cost Reduced sensitivity and specificity in comparison to PCR High Cost Limitations of PCR based testing Advantages of PCR based testing Advantages of Flow Cytometry based testing Limitations of Existing Flow Cytometry based testing

Accuracy of information – Considered a gold standard

Ease of operation

Speed of work flow

Requires high level of expertise

Laborious work flow (6-8 hours)

High Cost

Reduced sensitivity and specificity in comparison to PCR

High Cost

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 Performance Rating PCR Flow Cytometry

Typing by PCR platform is highly accurate

Flow cytometry platforms are less expensive and provide quicker turn around time

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

Designing a reagent for B27 typing – an India centric approach Requirements for a ‘new’ B27 Test Accuracy offered by PCR Ease of work flow offered by Flow Cytometry India Centric Factors Affordable reagent to make testing more accessible Provide Reagent in a dry format for ease of storage ReaMetrix Solution: A Flow-Cytometry based test that offers accuracy closest to that of PCR

Requirements for a ‘new’ B27 Test

Accuracy offered by PCR

Ease of work flow offered by Flow Cytometry

India Centric Factors

Affordable reagent to make testing more accessible

Provide Reagent in a dry format for ease of storage

Rational design of B27 Reagent – The ReaMetrix approach Accuracy 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+ population 3 and using bright fluorophores (R-PE vs. FITC) Use Internal Fluorescence Standards for every test to assure result quality Ease of work flow A Lyse-No wash approach to reduce assay time (<1 h) Design for the Indian diagnostic setting Provide reagents in a dried format to simplify storage conditions Reduce cost of test through innovation on process development Reduce ambiguity in typing results Reagent to meet the desired performance requirements – ReaPan B27

Accuracy

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+ population 3 and using bright fluorophores (R-PE vs. FITC)

Use Internal Fluorescence Standards for every test to assure result quality

Ease of work flow

A Lyse-No wash approach to reduce assay time (<1 h)

Design for the Indian diagnostic setting

Provide reagents in a dried format to simplify storage conditions

Reduce cost of test through innovation on process development

Reduce ambiguity in typing results

ReaPan B27 assay protocol Simplified Work Flow Incubation of sample in unitized dried reagent tube Lysis 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 30mins Add 300µl of ReaLyse Solution & Incubate for 15mins Analyze by Flow Cytometry

Simplified Work Flow

Incubation of sample in unitized dried reagent tube

Lysis of RBCs

Fix - No wash

Analysis by flow cytometry

Stain to Analysis in 45 min.

 Beads  Beads CD3+   Beads CD3+ HLA-B27+ Acquisition strategy Standardized Templates No compensation adjustments Internal 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

Standardized Templates

No compensation adjustments

Internal 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

Design comparison of flow based reagents ReaPan B27 has been designed for higher specificity and sensitivity Internal standard per test CD3 labeled with CD3-PE-Dyo649 and HLA B27 labeled with PE HLA-ABC-m3 and FD705 clones for HLA B-27 selection Lymphocyte population selection using CD3 HLA B7 Ab to remove cross reactivity ReaPan B27 Requires control samples Calbrite Beads and specific fluorescence intensity beads to establish threshold Discrimination of Positive and Negative Populations HLA B27 labeled with FITC and B7 labeled with PE CD3 labeled with PE and HLA B27 with FITC Selection of Fluorophores HLA-ABC-m3 clone for HLA B-27 selection HLA-B7 Ab to remove cross reactivity Coulter’s HLA-B27 Reagent GS145.2 clone for HLA B-27 selection Lymphocyte population selection using CD3 Selection of Population BD’s HLA-B27 Reagent Parameter

Internal standard per test

CD3 labeled with CD3-PE-Dyo649 and HLA B27 labeled with PE

HLA-ABC-m3 and FD705 clones for HLA B-27 selection

Lymphocyte population selection using CD3

HLA B7 Ab to remove cross reactivity

Requires control samples

Calbrite Beads and specific fluorescence intensity beads to establish threshold

HLA B27 labeled with FITC and B7 labeled with PE

CD3 labeled with PE and HLA B27 with FITC

HLA-ABC-m3 clone for HLA B-27 selection

HLA-B7 Ab to remove cross reactivity

GS145.2 clone for HLA B-27 selection

Lymphocyte population selection using CD3

Relative reagent performance for HLA-B27 typing ReaPan B27 Accuracy closest to PCR Operability easier than existing flow based reagents Robust performance guaranteed by Internal Standards Accuracy of Test Ease of Work Flow Assay Time Cost of Assay Parameter Performance Rating PCR Flow Cytometry ReaPan B27

ReaPan B27

Accuracy closest to PCR

Operability easier than existing flow based reagents

Robust performance guaranteed by Internal Standards

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

Reference articles - Ankylosing Spondylitis 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) Michael J. Warzynski. HLA-B27 TESTING: MEAN CHANNELS, QC, AND SURVEYS. Cytometry (Communications in Clinical Cytometry) 30:208–211 (1997) 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) P. S. Dhurandhar and U. Shankarkumar. HLA Association in SeronegativeSpondyloarthritis Patients From Mumbai, India. Int J Hum Genet, 7(3): 235-239 (2007)

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)

Michael J. Warzynski. HLA-B27 TESTING: MEAN CHANNELS, QC, AND SURVEYS. Cytometry (Communications in Clinical Cytometry) 30:208–211 (1997)

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)

P. S. Dhurandhar and U. Shankarkumar. HLA Association in SeronegativeSpondyloarthritis Patients From Mumbai, India. Int J Hum Genet, 7(3): 235-239 (2007)

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) 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 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 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

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)

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

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

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)

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) Wilfried Levering External quality assessment in flow cytometry. Educational aspects and trends toward improvement. Doctoral Thesis 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) 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

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)

Wilfried Levering External quality assessment in flow cytometry. Educational aspects and trends toward improvement. Doctoral Thesis

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)

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 - Uveitis 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 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 Acute Anterior Uveitis and HLA-B27 J . Chang , P . McCluskey , D . Wakefield Survey of Ophthalmology ,2005 June; Volume 50 Issue 4, Pages 364 – 388 HLA-B27+ anterior uveitis with or without associated spondyloarthritis: Clinical and immunological features RIVERA-CÍVICO et al. Ann Rheum Dis.1999; 58: 721-722

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

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

Acute Anterior Uveitis and HLA-B27 J . Chang , P . McCluskey , D . Wakefield Survey of Ophthalmology ,2005 June; Volume 50 Issue 4, Pages 364 – 388

HLA-B27+ anterior uveitis with or without associated spondyloarthritis: Clinical and immunological features RIVERA-CÍVICO et al. Ann Rheum Dis.1999; 58: 721-722

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: info@reametrix.com www.reametrix.com Further details:

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