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1 Test per tube

Product catalog No: 25238-00

ReaPan B27

Manufactured by

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ReaPan B27

. INTENDED USE

The ReaPan B27 Reagent is a two-color immunofluorescence stain,suitable for determining HLA-B27 antigen expression by flow cytometry

in lysed human whole blood samples (lyse-no wash technique). This

reagent is intended for specific use on open system flow cytometers (Eg.

BD FACScan and BDFACSCalibur cytometers).

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. SUMMARY AND EXPLANATION

Human Leukocyte Antigen B27, a class I surface antigen encoded by the

B locus in the major histocompatibility complex (MHC) presentsmicrobial antigens to T-Lymphocytes. The onset of seronegative

spondylo-arthropathies that includes ankylosing spondylitis, Reiters

disease, psoriatic arthritis and inflammatory bowel disease (1,2,3,4) is

associated with the expression of HLA-B27 antigen on T-Lymphocytes.

Screening for HLA-B27 is thus of clinical relevance in conjunction with

the symptomatic presentation of the disorder. Micro-lymphotoxicity testsare conventionally used in HLA-typing but are typically time-consuming

and expensive. Flow cytometry has gradually evolved into a faster and

reliable method for screening samples for HLA-B27 antigen (5,6).

Commercially available reagents for flow cytometry based HLA-B27

typing either immunospecifically select T-Lymphocyte population toscreen for HLA-B27 antigen so as to increase the specificity or minimize

the cross-reactivity of the Anti-B27 antibody to B7 antigen in order to

reduce false positives.

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3. PRINCIPLES OF THE PROCEDURE

T-lymphocytes selected through gating of CD3 specific populations are

analyzed for staining by HLA-B27 conjugates. It has been demonstratedthat the preselection of T-Lymphocytes increases the specificity of the

test by eliminating the background caused from other leukocyte

populations (8). Another method to reduce false-positives is by the

addition of anti-B7 antibody to the reagent cocktail. The anti-B7 antibody

competes with the B27 antibody for the B7 antigen and in this manner

suppresses the incidence of cross-reactivity of anti-B27 antibodies to B7antigens (1).

The ReaMetrix reagent for HLA-B27 typing improves the specificity by

having two clones of the anti-HLA-B27 antibody and an antibody against

the B7 antigen. By having two clones, multiple epitopic regions of the

B27 antigen are targeted ensuring that more B27 antigens are identified.The anti-B7 antibody binds preferentially to the B7 antigen thereby

reducing cross-reactivity.

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The test result is a direct extrapolation of the fluorescent staining

intensity of the HLA-B27 conjugates with respect to a cut-off value.

4. REAGENT

The ReaPan B27 reagent is formulated in buffered saline with sodium

azide and stabilizers. It contains R-Phycoerythrin (PE) labeled

monoclonal Anti-HLA-B27 antibodies (clones FD705 and HLA-ABC-m3),

PE-Dyomics649 labeled Anti-CD3 monoclonal antibody (clone UCHT1)and unlabeled Anti-B7 antibody (clone BB7.1) (1,7). A precise number of

fluorescent beads are included to facilitate internal calibration of the

reagent and determine RMX value. The ReaPan B27 reagent is provided

as a ready-to-use test, dried down in flow cytometer compatible sample

tubes.

Precautions

1. Warning: The ReaPan B27 reagent contains Sodium azide.

Sodium azide is harmful if swallowed. Wear suitable protective

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clothing. If swallowed, seek medical advice immediately. Contact

with acids liberates toxic gas. Azides should be flushed with

large amounts of water during disposal to avoid deposits in lead

or copper plumbing.2. Warning: All blood specimens are considered biohazards.

Handle them as if they are capable of transmitting infection and

dispose off with proper precautions in accordance with

governmental regulations.

3. The addition of the precise volume of blood is critical to obtain

correct results. Use a calibrated pipette and operate accordingto the manufacturers instructions.

4. The ReaPan B27 reagent and the control beads are in dried

form. It is absolutely critical to ensure vigorous vortexing after

addition of blood sample to ensure solubilization of the reagent

and the beads.

Storage and Handling

1. Store the reagent at room temperature in a dry place. Do not

use the reagent after the expiry date on the label.

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2. Do not freeze the ReaPan B27.

3. The ReaPan B27 reagent is light sensitive. Do not expose to

direct light either during storage or when mixed with blood.

. INSTRUMENT

The ReaPan B27 reagent has been tested on the FACScan and

FACSCalibur systems manufactured by Becton-Dickinson. ReaMetrix

recommends running this reagent on these instruments. The instrument

should be calibrated for setting photomultiplier tube voltages,fluorescence compensation, and checking instrument sensitivity

according to the manufacturers guidelines.

It is the responsibility of the user to optimize the performance of the

reagent for use in flow cytometers other than those mentioned above.

. SPECIMEN COLLECTION

Follow the collection tube manufacturers guidelines for the minimum

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volume of blood to be collected.

The anti-coagulated blood must be stored at room temperature (20C -

25C) and should be stained and analyzed ideally within 24 hours ofdraw.

Important!

Refrigerated, hemolyzed, and previously fixed blood specimens can

yield erroneous results and should be rejected.

. PROCEDURE

Reagent Provided

ReaPan B27 reagent

Reagents and materials required but not provided

1. Blood collection tube

2. Calibrated pipettes

3. Vortex mixer

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4. ReaLyse Solution

5. Sheath fluid (BD FACSFlow Catalog No. 340398 or

equivalent)

6. Calibrite Beads

Assay Protocol

1. Mix blood sample (invert blood tube at least 10 times) and

pipette 25L of blood into the single use HLA B27 reagenttube.

2. Vortex each tube thoroughly for 30 seconds. Incubate for 30

minutes at room temperature. Protect the tube from direct

light.

3. Add 300L of FACSLyse or equivalent fix/lyse solution to

each tube and vortex for 20 seconds. Return tubes to the darkfor at least 15 minutes to ensure complete lysis.

4. Vortex sample tube thoroughly (at low speed) and load onto

cytometer for analysis.

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Flow Cytometer Acquisition and Analysis

This protocol assumes that the flow cytometer has been setup according

to the manufacturers instructions (For example, In the case ofFACScan, the instrument should be setup and calibrated using

CaliBrite beads and FACSComp software). Open the CellQuest

Pro software and connect to the cytometer.

The fluorescence filters referred to in the subsequent sections are given

below:

FL1 530 30 nm

FL2 585 42 nm

FL3 670 nm LP (BD FACS Calibur) or 650 nm LP (BD

FACScan)Open a new document (File>New document) and select the option to

view instrument settings:

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Set the compensation settings on all channels to

zero

Set the threshold on FL3

Set Intensity to Channel values (CellQuest Pro

toolbar>Plots>Log Data Units>Channel Values)

1. Draw two dot-plot (View-1 and View-2) and four histogram (View-3,

View-4, View-5, View-6) views View-1 (Dot Plot): CD3 PE-Dy649 fluorescence (FL3)

versus side scatter (SSC Linear scale).

View-2 (Dot Plot): FL2 versus FL1 of events gated as

beads from the first view to capture the fluorescent reference

beads. The beads have a high FL1 and FL2 Fluorescence

Intensity

View-3 (Histogram): HLA-B27-PE fluorescence (FL2)

versus counts of events gated as CD3 positive (R1) from

View-1.

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View-4, View-5, and View-6 (Histogram): Fluorescent

bead intensity (on FL1, FL2 & FL3) obtained from R3 of View

2 versus Counts.

Set the number of events to capture at 4000 for R-3

gate (View 2) before data acquisition. When 4000 beads are

acquired in the gate, acquisition stops.

Select the histogram statistics option for View-3, View-4

and View-5 & View-6

2. Adjust the Side-scatter and FL3 voltage settings on View 1 toacquire CD3 positive (R1) and bead populations (R2) as

shown. Ensure that both the beads and the lymphocyte

populations are acquired and can be visualized as separate

from the background.(Note: Multi-colored gating can be used

to prevent unintentional thresholding out beads)

3. Adjust the FL2 voltage settings to position R3 gated beads to60010 channels (Histogram stats M1 Median

Fluorescence Intensity) on View

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4. Similarly adjust FL1 and FL3 voltage gain to set the R3 gatedbeads on FL1 at a MdFI of 60010 and 40010 channels

respectively. (The above steps are critical for the accuracy of

the test results and must not be skipped)

5. Obtain the R1-gated HLA-B27 population on View 3 to recordthe fluorescence intensity on histogram stats (of all events).

6. Enter the K value for the particular batch of reagent from thelabel on the pouch and calculate RMX value as shown.

If RMX Value0, then sample is HLA B27 negative

If RMX Value >0, then sample is HLA B27 positive

RMX Value* =MdFI

sam le K x MdFI

beadson FL2

MdFIon FL2

*K is a constant unique to each reagent lot given on the Reagent Pouch Label

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View 1 View 2

Dot plot views:View-1:CD3-PE-Dy649 fluorescence (FL3 - log scale) versus side-scatter

(SSC Linear scale) to capture CD3+ (R1) and fluorescence beads (R2) View-2: FL1

versus FL2 (both on log scale) and R2 gate applied to obtain a clean reference beads

population (R3)

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View 3

View 4 View 5 View 6

Histogram views: View-3: HLA-B27-PE (gated from R1 of View 1) fluorescence (FL2)

versus CountsView-4View-5 View-6: MdFI of bead intensity on FL1, FL2 & FL3

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. LIMITATIONS

1. The ReaPan B27 reagent has only been validated with K3EDTA

treated whole blood.2. It is the responsibility of the user to ensure that the flow cytometer

instrument is calibrated according to manufacturers instructions.

3. It is important that the gain settings are adjusted to position the

fluorescent beads as described in Section 6.0. The R-1 gate must be

visually checked to ensure adequate T-lymphocyte gating.

4. The test result bears a direct relation to the intensity of thePhycoerythrin (PE) in the reagent. Exposure of reagent to direct light

or use of expired reagents may prove detrimental to the accuracy of

the test results.

5 The HLA-B27 test result is not indicative of the presence or absence

of a disease condition. HLA-B27 expression along with symptomatic

presentation and clinical history of the patient should be carefully

considered by the physician in making a diagnosis (7).

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. WARRANTY

This product is warranted only to conform to the quantity and contents

stated on the label at the time of delivery to the customer. There are no

warranties, expressed or implied, that extend beyond the description on

the label of the product. ReaMetrixs sole liability is limited to

replacement of the product. Reametrix is not liable for property damage,

personal injury, or economic loss caused by the product.Note: FACScan, CaliBRITE, FACSLyse, FACSComp, FACSCalibur are all

registered trade names of Becton-Dickinson.

. REFERENCES

1. Wilfried H. B. M. Levering, Henk Wind, Kees Sintnicolaas, Herbert

Hooijkaas, and Jan W. Gratama. Flow Cytometric HLA-B27Screening: Cross-ReactivityPatterns of Commercially Available Anti

HLA-B27 Monoclonal Antibodies With Other HLA-B Antigens.

Cytometry Part B (Clinical Cytometry) 54B:2838 (2003)

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2. Michael J. Warzynski. HLA-B27 TESTING: MEAN CHANNELS, QC,

AND SURVEYS. Cytometry (Communications in Clinical Cytometry)

30:208211 (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

Seronegative Spondyloarthritis 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:1015 (2005)

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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)

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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) 42:95105

(2000)

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Manufactured by ReaMetrix India Pvt. Ltd.

50-B, II Phase, Peenya Industrial Area

Peenya, Bangalore 560058, India

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Ph: +91-80-28378693/5, Fax: +91-80-41172451

E-mail: info@reametrix.com

www.reametrix.comRev No. 2.0, 21-Apr-09

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