pi-25238-00-reapan b27
TRANSCRIPT
-
8/14/2019 PI-25238-00-ReaPan B27
1/23
1 Test per tube
Product catalog No: 25238-00
ReaPan B27
Manufactured by
-
8/14/2019 PI-25238-00-ReaPan B27
2/23
2
-
8/14/2019 PI-25238-00-ReaPan B27
3/23
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).
3
-
8/14/2019 PI-25238-00-ReaPan B27
4/23
. 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.
4
-
8/14/2019 PI-25238-00-ReaPan B27
5/23
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.
5
-
8/14/2019 PI-25238-00-ReaPan B27
6/23
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
6
-
8/14/2019 PI-25238-00-ReaPan B27
7/23
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.
7
-
8/14/2019 PI-25238-00-ReaPan B27
8/23
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
8
-
8/14/2019 PI-25238-00-ReaPan B27
9/23
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
9
-
8/14/2019 PI-25238-00-ReaPan B27
10/23
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.
10
-
8/14/2019 PI-25238-00-ReaPan B27
11/23
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:
11
-
8/14/2019 PI-25238-00-ReaPan B27
12/23
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.
12
-
8/14/2019 PI-25238-00-ReaPan B27
13/23
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
13
-
8/14/2019 PI-25238-00-ReaPan B27
14/23
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
14
-
8/14/2019 PI-25238-00-ReaPan B27
15/23
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)
15
-
8/14/2019 PI-25238-00-ReaPan B27
16/23
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
16
-
8/14/2019 PI-25238-00-ReaPan B27
17/23
. 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).
17
-
8/14/2019 PI-25238-00-ReaPan B27
18/23
. 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)
18
-
8/14/2019 PI-25238-00-ReaPan B27
19/23
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)
19
-
8/14/2019 PI-25238-00-ReaPan B27
20/23
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)
20
-
8/14/2019 PI-25238-00-ReaPan B27
21/23
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)
21
-
8/14/2019 PI-25238-00-ReaPan B27
22/23
Manufactured by ReaMetrix India Pvt. Ltd.
50-B, II Phase, Peenya Industrial Area
Peenya, Bangalore 560058, India
22
-
8/14/2019 PI-25238-00-ReaPan B27
23/23
Ph: +91-80-28378693/5, Fax: +91-80-41172451
E-mail: [email protected]
www.reametrix.comRev No. 2.0, 21-Apr-09
23