ReaPan B27

© 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%


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


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


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 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


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


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


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


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


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


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


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)


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)



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 - 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


Manufactured by ReaMetrix India Pvt. Ltd.50-B, II Phase, Peenya Industrial Area

Peenya, Bangalore 560058, IndiaPh: +91-80-28378693/5, Fax: +91-80-

41172451E-mail: [email protected]

www.reametrix.com

Further details:

Health & Medicine

ReaPan B27