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Product catalog No: 2524200
Rea TCount
CD4/CD3
Reagent
Manufactured by
Rea TCount Reagent CD4/CD3
1. INTENDED USE
The Rea TCount reagent is a two color immunofluorescence reagent
for the labeling and identification of helper/inducer (CD3+CD4+) T
lymphocytes combined with a precise number of fluorescent counting
beads for absolute CD4+ and CD3+ TCell counts. This reagent is
intended for flow cytometry based analysis in unlysed human whole
blood samples.
2. BACKGROUND
The Rea TCount reagent contains fluorescently labeled antibodies that
bind to CD3 and CD4 antigens found on the surface of circulating
leukocytes. The CD3 antigen is a complex of at least six proteins known
collectively as the Tcell receptor (TCR) complex. The antibody used in
this reagent binds to the 20kDa ε chain of this complex. The CD4
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antigen is a 59kDa protein. It interacts with class II molecules of the
major histocompatibility complex and is the primary receptor for the
Human Immunodeficiency Virus (HIV).
Cells that are both CD3+ and CD4+ are identified as helper/inducer
lymphocytes. Decreased CD4+CD3+ cell counts have been associated
with some forms of immunodeficiency.
3. Rea TCount Reagent
The Rea TCount reagent contains dried down reagents formulated in
buffered saline, sodium azide and stabilizers. The reagent has CD4
monoclonal antibody (clone RPAT4) labeled with Phycoerythrin (PE)
and CD3 (clone UCHT1) monoclonal antibody labeled with PE
Dyomics649.
1. Warning: The Rea TCount 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.
3. The addition of precise volume of blood is critical to obtain correct
results. Use a calibrated pipette and operate according to the
manufacturer’s instructions.
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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 Rea TCount reagent.
3. The Rea TCount reagent is light sensitive. Do not expose to direct
light either during storage or when mixed with blood.
4. INSTRUMENT
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5. SPECIMEN COLLECTION
The blood sample should be collected in a sterile blood collection tube
containing K3EDTA. Follow the collection tube manufacturer’s
guidelines for the minimum volume of blood to be collected.
The anticoagulated blood must be stored at room temperature (20°C
25°C) and should be stained and analyzed within 24 hours of draw.
6. PROCEDURE
Reagent Provided
1. ReaT Count Reagent Part No: 2524200
2. ReaFix Fixative solution (10X concentrate) Part No.:2532500
3. ReaFix Diluent Solution Part No.:2532600
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Refer to the ReaFix Fixative solution (10X) package insert for
dilution instructions and warnings.
Reagents and materials required but not provided
1. Blood collection tube containing K3EDTA
2. Calibrated pipettes
3. Vortex mixer
4. Becton Dickinson FACSCountTM Instrument (Becton Dickinson Cat
No: 337858)
5. Sheath fluid (Becton Dickinson FACSFlow™ Catalog No. 340398 or
equivalent)
6. BD FACSCountTM Control kit (Becton Dickinson Cat No: 340166)
7. BD FACSCountTM Reagent CD4/CD3 Kit to set up controls (Becton
Dickinson Catalog No. 342512)
Preparation of Control Samples
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Follow Manufacturer instructions for setting up the control in the
FACSCountTM Instrument (Use BD FACSCountTM Reagent for setting
the control)
Preparation of the patient samples
1. Blood samples must be collected in a (K3EDTA) vacutainer tube and
be stored no longer than 48 hours at room temperature.
2. Take one reagent tube per patient from the foil bag containing the
Rea Tcount tubes. Reseal the foil bag and return unused reagent
pairs to the appropriate place. DO NOT refrigerate the reagents.
3. Label the reagent with the sample number found on the blood tube.
4. Mix patient blood by inverting the tube 5 times.
5. Reverse Pipette 50 µL of whole blood into the reagent tubes. Use a
new pipette tip for each tube.
6. Cap the tubes and vortex vigorously for 30 seconds.
7. Incubate for 60 minutes at room temperature. Place the reagent
tubes in the workstation and close the cover to protect the reagents
from light.
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8. Uncap tubes and pipette 450 µL of ReaFix fixative solution into the
reagent tube. Use a new pipette tip for each tube. Refer to the
ReaFix Fixative solution (10X) package insert for dilution instructions
to prepare 1X working ReaFix Fixative Solution
9. Recap the tubes and vortex for 15 seconds.
10. Run the tubes on the FACSCountTM within 24 hours of preparation.
Store samples at room temperature in the workstation until they are
run on the instrument. Vortex upright for 5 seconds before running.
Entering patient information
1. Press [sample].
2. Enter or verify reagent lot code and bead counts as provided on Rea T
Count pouch.
3. Press [confirm]
4. Enter the patient accession number
Running patient samples
1. Vortex the reagent pairs for 15 seconds.
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2. Uncap the reagent tube and place the reagent tube in the sample
holder so the CD4 tube is in the run position.
3. Press [run]
4. After a successful run the results will print out. If the sample fails
review reported error and refer to BD FACSCountTM guide to rectify
problem.
5. The patient results are displayed on the screen and automatically
printed.
6. The sample printout contains the following information:
7. Reagent informationreagent lot code and reference bead counts entered
for the sample run. (This number should match the reagent lot code for
the control run.)
8. Date (mm / dd / yy) and time the patient sample was run
9. Control informationcontrol run results, date controls were run, reagent lot
code entered for the control run, control lot code.
10. Patient accession number
11. Patient results
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12. Absolute Counts cells/µl of CD4 & CD3
13. A sample report is attached below (Figure 1)
14. Remove the reagent tube, recap the reagent tube and discard in the
biohazard waste container.
15. Repeat above steps until all patient samples have been run.
16. Discard the reagent pair in an appropriate biohazard container.
17. Shut down instrument as per manufacturer’s instructions.
18. A sample report is generated after every sample run. The sample
report is attached below (Refer Figure 1).
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Figure 1: Patient Sample Report
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7. LIMITATIONS
1. The Rea TCount reagent has only been validated with K3EDTA or
K2EDTA treated whole blood.
8. 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 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: FACSCountTM is a registered trade name of BectonDickinson.
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Manufactured by ReaMetrix India Pvt. Ltd.
Manufacturing License Number: KTK/25/519/2006
50B, II Phase, Peenya Industrial Area
Peenya, Bangalore 560058, India
Ph: +918028378693/5, Fax: +918041172451
Email: info@reametrix.com,
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www.reametrix.com
Rev No. 3.0, 14Oct09