1 Test per tube

Product catalog No: 25238-00

ReaPan B27

Manufactured by
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 ReaPan B27
1. 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).

2. SUMMARY AND EXPLANATION

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

B locus in the major histocompatibility complex (MHC) presents
microbial antigens to T-Lymphocytes. The onset of seronegative
spondylo-arthropathies that includes ankylosing spondylitis, Reiter’s
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 tests
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are 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 to
screen 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.

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 demonstrated
that 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 B7
antigens (1).
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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.

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

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tubes.

Precautions
1. Warning: The ReaPan B27 reagent contains Sodium azide.
Sodium azide is harmful if swallowed. Wear suitable protective
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 according
to the manufacturer’s 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.

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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.
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.

5. 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 manufacturer’s 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.

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6. SPECIMEN COLLECTION

Follow the collection tube manufacturer’s guidelines for the minimum

volume of blood to be collected.

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

25°C) and should be stained and analyzed ideally within 24 hours of
draw.

Important!

Refrigerated, hemolyzed, and previously fixed blood specimens can

yield erroneous results and should be rejected.

7. PROCEDURE
Reagent Provided
ReaPan B27 reagent
Reagents and materials required but not provided
1. Blood collection tube

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 2. Calibrated pipettes
3. Vortex mixer
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 25µL of blood into the single use HLA B27 reagent
tube.
2. Vortex each tube thoroughly for 30 seconds. Incubate for 30
minutes at room temperature. Protect the tube from direct
light.
3. Add 300µL of FACSLyse™ or equivalent fix/lyse solution to
each tube and vortex for 20 seconds. Return tubes to the dark
for 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 manufacturer’s instructions (For example, In the case of
FACScan™, 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:

• Set the compensation settings on all channels to

zero
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 • 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.
• 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.

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 • 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 to
acquire 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 to
600±10 channels (Histogram stats – M1 – Median
Fluorescence Intensity) on View
4. Similarly adjust FL1 and FL3 voltage gain to set the R3 gated
beads on FL1 at a MdFI of 600±10 and 400±10 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 record
the fluorescence intensity on histogram stats (of all events).
6. Enter the K value for the particular batch of reagent from the
label on the pouch and calculate RMX value as shown.

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 MdFIsample – K x MdFIbeads on FL2
RMX Value* =
MdFIbeads on FL2
*K is a constant unique to each reagent lot given on the Reagent Pouch Label
If RMX Value ≤0, then sample is HLA B27 negative
If RMX Value >0, then sample is HLA B27 positive

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

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8. 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 manufacturer’s 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 the
Phycoerythrin (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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9. 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. ReaMetrix’s 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.

10. REFERENCES

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

Hooijkaas, and Jan W. Gratama. Flow Cytometric HLA-B27
Screening: Cross-ReactivityPatterns of Commercially Available Anti–
HLA-B27 Monoclonal Antibodies With Other HLA-B Antigens.
Cytometry Part B (Clinical Cytometry) 54B:28–38 (2003)

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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
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:10–15 (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) 42:95–105
(2000)

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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
Rev No. 2.0, 21-Apr-09

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