pi-25124-00-rea t count (cd4cd3 & cd8cd3)

8/14/2019 Pi-25124-00-Rea t Count (Cd4cd3 & Cd8cd3)

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2 Tubes per test

Product Catalog No: 25124-00

Rea T-CountCD4/CD3

&CD8/CD3Reagent

Manufactured by



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Rea T-Count Reagent

1. INTENDED USE

The Rea T-Count reagent is a two color immunofluorescence reagentfor the labeling and identification of helper/inducer (CD3+CD4+) andcytotoxic/suppressor (CD3+CD8+) T lymphocytes combined with aprecise number of fluorescent counting beads for absolute CD4+,CD8+and Average CD3+ T-Cell counts. This reagent is intended for flowcytometry based analysis in unlysed human whole blood samples.

2. BACKGROUND

The Rea T-Count reagent contains fluorescently labeled antibodies that

bind to CD3, CD4, or CD8 antigens found on the surface of circulatingleukocytes.The CD3 antigen is a complex of at least six proteins known collectivelyas the T-cell receptor (TCR) complex. The antibody used in this reagent

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binds to the 20kDa ε chain of this complex.

The CD4 antigen is a 59kDa protein. It interacts with class II moleculesof the major histocompatibility complex and is the primary receptor for

the Human Immunodeficiency Virus (HIV).

The CD8 antigen is a complex consisting of two disulfide linkedsubunits. The antibody used in this reagent binds to the 32kDa α subunit of the complex. CD8 interacts with class I majorhistocompatibility complex molecules.

Cells that are both CD3+ and CD4+ are identified as helper/inducerlymphocytes. Decreased CD4+CD3+ cell counts have been associatedwith some forms of immunodeficiency. Suppressor/cytotoxiclymphocytes are the subset of cells that have both CD3 and CD8receptors. Increased CD8+CD3+ cell counts have been observed in

some cases of immunodeficiency.

3. Rea T-Count Reagent

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The Rea T-Count reagent consists of a single unit comprising of twotubes, with dried down reagents formulated in buffered saline, sodiumazide and stabilizers. One tube contains CD4 monoclonal antibody(clone RPA-T4) labeled with Phycoerythrin (PE) and CD3 monoclonal

antibody (clone UCHT1) labeled with PE-Dyomics649.The other tubecontains CD8 monoclonal antibody (Clone LT8) labeled withPhycoerythrin (PE) and CD3 monoclonal antibody (clone UCHT1)labeled with PE-Dyomics649. The monoclonal antibodies used in theRea T-Count were assigned these specificities at the 8th InternationalWorkshop on Human Leukocyte Differentiation Antigens. A precise

number of fluorescent counting beads are included in each tube to allowsingle-platform determination of absolute CD4+ and CD8+ T-cellcounts. The Rea T-Count reagent is provided in dried form anddispensed in FACSCount TM compatible sample tubes with each unitcontaining one ready-to-use test.

Precautions

1. Warning : The Rea T-Count reagent contains sodium azide. Sodiumazide is harmful if swallowed. Wear suitable protective clothing. If

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swallowed, seek medical advice immediately. Contact with acidsliberates toxic gas. Azides should be flushed with large amounts ofwater during disposal to avoid deposits in lead or copper plumbing.

2. Warning : All blood specimens are considered bio-hazardous.Handle them as if they are capable of transmitting infection anddispose off with proper precautions and in accordance withgovernmental regulations.

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

results. Use a calibrated pipette and operate according to themanufacturer’s instructions.

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 T-Count reagent.3. The Rea T-Count reagent is light sensitive. Do not expose to direct

light either during storage or when mixed with blood.6



4. INSTRUMENT

The Rea T-Count reagent is designed to be used on FACSCount TM

manufactured by Becton Dickinson equipped with a convection cooled540nm laser that is capable of detecting two fluorescent colors andmeasures relative cell size. The CD3 cells fluoresce red and CD4 andCD8 cells fluoresce yellow. This instrument can be used forenumerating absolute lymphocyte counts namely; helper/inducer(CD3+CD4+) and cytotoxic /suppressor (CD3+CD8+). Instrument

should be calibrated by setting photomultiplier tube voltages,fluorescence compensation, and checking instrument sensitivityaccording to the manufacturer’s guidelines.

5. SPECIMEN COLLECTION

The blood sample should be collected in a sterile blood collection tubecontaining K 3-EDTA. Follow the collection tube manufacturer’sguidelines for the minimum volume of blood to be collected.

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The anti-coagulated blood must be stored at room temperature (20 ° C -25 ° C) and should be stained and analyzed within 24 hours of draw.

Refrigerated, hemolyzed, and previously fixed blood specimens can

yield erroneous results and should be rejected.

6. PROCEDURE

Reagent Provided

1. Rea-T Count Reagent in a dried format (10 units/pack, each unitcontains two tubes)-Part No: 25124-00

2. ReaFix Fixative solution (10X concentrate)- Part No.:25325-003. ReaFix Diluent Solution- Part No.:25326-00

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Refer to the ReaFix Fixative solution (10X) package insert fordilution instructions and warnings.

Reagents and materials required but not provided

1. Blood collection tube containing K 3EDTA2. Calibrated pipettes3. Vortex mixer4. Becton Dickinson FACSCount TM Instrument

(Becton Dickinson Cat No: 337858)5. Sheath fluid (Becton Dickinson FACSFlow ™ Catalog No. 340398

or equivalent)6. BD FACSCount TM Control kit (Becton Dickinson Cat No: 340166)7. BD FACSCount TM Reagent Kit to set up controls (Becton

Dickinson Catalog No. 340167)8. BD FACSCount ™ CD4 Software and User’s Guide Assembly

(Becton Dickinson Catalog No. 339011)

Preparation of Control Samples

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Follow Manufacturer’s instructions for setting up the control in theFACSCount TM Instrument (Use BD FACSCount TM Reagent for settingthe control)

Preparation of the patient samples

1. Blood samples must be collected in a (K 3EDTA) vacutainer tube andbe stored no longer than 48 hours at room temperature.

2. Place the samples in the workstation.3. Take one reagent pair per sample from the foil bag containing the

Rea T-Count tubes. Reseal the foil bag and return unused reagentpairs to the appropriate place. DO NOT refrigerate the reagents.

4. Label the tab of each reagent pair with the sample number found onthe blood tube.

5. Mix blood sample by inverting the tube 5 times.6. Reverse Pipette 50 µL of whole blood into each of the 2 reagent

tubes. Use a new pipette tip for each tube.7. Cap the tubes and vortex vigorously for 30 seconds each.

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8. Incubate for 60 minutes to 120 minutes at room temperature. Placereagent pairs in the workstation and close the cover to protect thereagents from light.

9. Uncap tubes and pipette 450 µL of 1X Working ReaFix fixativesolution into each reagent tube. Use a new pipette tip for each tube.Refer to the ReaFix Fixative solution (10X) package insert for dilutioninstructions to prepare 1X working ReaFix Fixative Solution.

10.Recap the tubes and vortex for 15 seconds each.11. Run the tubes on the FACSCount TM within 24 hours of preparation.

Store samples at room temperature in the workstation until they arerun 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

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Running patient samples

1. Vortex the reagent pair for 15 seconds.

2. Uncap the CD4 tube and place the reagent pair in the sample holderso the CD4 tube is in the run position.

3. Press [run]4. Remove reagent pair and recap CD4 tube.5. Uncap CD8 tube and place so the CD8 tube is in the run position.

6. Press [run]7. After a successful run the results will print out. If the sample fails

review reported error and refer to BD FACSCount TM guide to rectifyproblem.

8. The patient results are displayed on the screen and are automatically

printed.9. The sample printout contains the following information:

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10. Reagent information-reagent lot code and reference beadcounts entered for the sample run. (This number should match thereagent lot code for the control run.)

11. Date (mm / dd / yy) and time the patient sample was run12. Control information-control run results, date controls were run,

reagent lot code entered for the control run, control lot code.13. Patient accession number14. Patient results15. Absolute Counts - cells/ µl of CD4 & CD8

16. Total CD3 Average17. CD4/CD8 ratio18. Remove the reagent pair, recap the CD8 tube and discard in the

biohazard waste container.19. Repeat above steps until all patient samples have been run.

20. Discard the reagent pair in an appropriate biohazard container.21. Shut down instrument as per manufacturer’s instructions.

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22. A sample report is generated after every sample run. The samplereport is attached below (Refer Figure 1)

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15Figure 1: Patient Sample



7. LIMITATIONS

1. The Rea T-Count reagent has only been validated with

K3EDTA or K 2EDTA treated whole blood.

8. WARRANTY

This product is warranted only to conform to the quantity and contentsstated on the label at the time of delivery to the customer. There are no

warranties, expressed or implied, that extend beyond the description onthe label of the product. ReaMetrix sole liability is limited to replacementof the product. Reametrix is not liable for property damage, personalinjury, or economic loss caused by the product.

Note: FACSCount TM is a registered trade name of Becton-Dickinson.

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Manufactured by ReaMetrix India Pvt. Ltd.

Manufacturing License Number: KTK/25/519/2006 50-B, II Phase, Peenya Industrial AreaPeenya, Bangalore 560058, IndiaPh: +91-80-28378693/5, Fax: +91-80-41172451E-mail: [email protected],

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www.reametrix.com

Rev No. 3.0, 29-Sep-09

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pi-25124-00-rea t count (cd4cd3 & cd8cd3)

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