Pi-25323-00-reapan S34

1 Test per tube

Product catalog No: 25323-00

ReaPan S34 Manufactured by

ReaPan S34 1. INTENDED USE The ReaPan S34 reagent uses principle of Immuno-fluorescence to enumerate Hematopoietic stem cells in cell preparations. It is available in a unitized, dried-down format and is ideal for enumerating viable CD34+ cells in peripheral blood, cord blood, and mobilized stem cell preparations on a flow cytometry platform. This reagent is intended for specific use on open system flowcytometer. (Eg.BDFACScan™, BDFACSCalibur™ ,FACSCanto™) 2. BACKGROUND The ReaPan S34 allow for flow cytometry based rapid and accurate assessment of live hematopoietic progenitor cells in accordance with the International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines. Success of Hematopoietic transplantations and hematopoietic progenitor cell storage is relative to the absolute live CD34+ hematopoietic progenitor cells (HPC’s).

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The ReaPan S34 reagent facilitates a robust, single platform method of enumerating viable human CD34+ cells. The CD34 antigen is an 110Kda single chain type1 trans-membrane glycoprotein, associated with human hematopoietic progenitor cells (1). Reagent contains workshop approved anti mouse anti-human monoclonal antibody specific to the CD45 antigen, CD34 specific Class III mouse anti-human monoclonal antibody, and 7-Amino Actinomycin-D (7-AAD) dye (for the detection of dead cells). Viability dye 7- Amino Actinomycin D is a DNA intercalating agent that stains only cells in the late phases of cell death when the integrity of both the cell and nuclear membranes is lost(6). It is important to exclude CD34 dead cells to assist in counting true live CD34+ cells (5). Investigators have demonstrated that graft containing 4-5X 106 CD34+ cells /Kg recipient ideal body weight(IBW) will result in rapid, durable, trilineage engraftment(4). 3. REAGENT The ReaPan S34 reagent is formulated in buffered saline with sodium azide and stabilizers. R-Phycoerythrin (PE) – is labeled to mouse anti-human

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monoclonal CD34 antibody (clone-581), Atto-488 labeled mouse Anti-human CD45 antibody (clone-H130) and optimized concentrations of 7-AAD (1,7). 7AAD is important to exclude CD34 dead cells to assist in counting true live CD34+ cells (5). Specificity of the reagent enhanced by the use of class III mouse monoclonal antibody to CD34. Class III epitopes have broader distribution on both normal HPC’s and blast cells (3, 4). A precise number of fluorescent beads are included to facilitate reference number for absolute cell count. Reagent is provided as a ready-to-test, in flow cytometer compatible sample tubes. 4. Precautions • Warning: The ReaPanS34 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. Azide should be flushed with large amounts of water during disposal to avoid deposits in lead or copper plumbing. • Warning: All blood specimens are considered biohazards. Handle them as if they are capable of transmitting infection and dispose off with proper

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precautions in accordance with governmental regulations. Warning: The ReaPan S34 reagent contains 7AAD- viability dye. Pure 7AAD is a potential carcinogen, although this compound is highly diluted in the reagent it is recommended to avoid contact with eyes and skin. 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. The ReaPanS34 reagent and the reference count beads are in dried form. It is critical to ensure vigorous vortexing after addition of blood sample to ensure solubilization of the reagent and the beads. (Note: Vigorous Vortexing will not affect the CD34+ population). The Total leukocyte count should Not exceed 30X103 cells/µL and CD34+ cell count should not exceed 2000 cells/ µL( Please perform Dilution if the Count exceeds above the mentioned value) Stained samples must be analyzed within Three hours to ensure precise viable cell counting.

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The use of temperatures condition, Incubations time and mixing (Vortex) other than specified, may give erroneous results. 5. Storage and Handling • Store the reagent at room temperature in a dry place. Do not use the reagent after the expiry date. (Refer the label) • Do not freeze the ReaPan S34™. • The ReaPanS34 reagent is light sensitive. Do not expose to direct light either during storage or sample processing. 6. Indication of Stability : ReaPan S34 reagent is a dry glassy coating at the bottom of the reaction tube. Do not use if the reagents appears to be moist or it has become dislodged from the bottom of •

the reaction tube. 7. Instrument The ReaPanS34 reagent has been tested on the FACScan™ and FACSCalibur™,FACSCanto™ systems manufactured by Becton-Dickinson and other open Flow Cytometers. ReaMetrix recommends running this reagent on these instruments. The instrument should be calibrated for setting

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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 cytometer other than those mentioned above. 8.

SPECIMEN COLLECTION Universal precautions and good laboratory practice (GLP) should be adopted while processing blood and human tissue samples. The peripheral Blood, Placental blood, Apheresis sample or bone marrow 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 anti-coagulated sample must be stored at room temperature (20°C 25°C) and should be analyzed ideally within 24 hours of collection and within three hours of staining. Cryopreserved blood and bone marrow should be processed preferably within 30 minutes after thawing (7).

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9. PROCEDURE Reagent Provided ReaPan S34 reagent Reagents and materials required but not provided Blood collection tube containing K3-EDTA Calibrated pipettes Vortex mixer ReaLyse Lysing Solution (RMX Catalog No. 25237-00) or similar blood fix/lyse solution. • Sheath fluid (BD FACSFlow™ Catalog No. 340398 or equivalent) • Calibrite Beads

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Assay Protocol • • •

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Mix blood sample (invert blood tube at least 10 times) and pipette 50µL of blood into the single use ReaPanS34 reagent tube. Vortex each tube thoroughly for 30 seconds. Incubate for 30 minutes at room temperature. Protect the tube from direct light. Add 450µL of ReaLyse™ 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. Vortex sample tube thoroughly (at low speed) and load onto cytometer for analysis. Analyze the sample within 3.0 hrs from staining and fixing the sample.

Flow Cytometer Acquisition and Analysis This protocol assumes that the flow cytometer has been setup according to the

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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 FACSCalibur™) or 650 nm LP(BD FACScan™) Instrument Setting: Before collecting the sample data it is necessary to optimize instrument electronics for acquiring good quality data. Instrument controls (detectors, compensation, threshold and amplifiers) are adjusted optimally to display cell population of interest (This step is critical and must not be skipped to ensure the accuracy of the test results). • Follow manufacturer’s guidelines to calibrate the system. • Change the threshold channel From FL3 to FL1 (300±50)

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ISHAGE recommended cell enumeration strategy: CD45+ events are selected precisely to include all the CD45+ cells to establish the minimum size range for lymph blast region (R1).The lower extremity of R1 is set low enough to include all CD45+ dim CD34+events. It has been demonstrated that the pre-selection of Lymphocytes increases the specificity of the test by eliminating nonspecifically stained debris and platelets (1). Region-2 is adjusted to select all the CD34+ dim and bright population to include all CD34+ cells. Region 3 is created as an amorphous region to best delineate live from dead cells. Region -4 is gated on the cumulative regions R1, R2&R3 which demonstrates CD34+ cells which CD45 dim and are live.CD34+ positive cells share the morphological characters of lymphocytes. True live CD34+ cells are selected on Region-6(R6) by Intersecting R1*R2*R3*R4 cell population with respect to lymphocyte population (View-5) and by eliminating platelet aggregates and monocytes (1) Draw dot-plot (View-1 View-2, View 3, View-4, View-5, View 6, View-7, and View-8

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View-1: CD45-A488 fluorescence (FL1) versus side scatter (SSC-Lin scale). View-2: CD34-PE Fluorescence (FL2) versus side scatter(SSC-Linscale) View-3: 7-AAD fluorescence (FL3) versus side scatter(SSC-Linear scale) View-4:CD45-A488 Fluorescence(FL1) versus side scatter(SSCLinear scale) View-5 Forward scatter(Linear) versus Side scatter (Linear)- for CD45 reference View-6 Forward scatter(Linear) versus Side scatter (Linear)- for Absolute CD34+ cell counting View-7 Forward scatter(Linear) versus Side scatter (Linear)- For selection of beads. View-8 CD45-A488 Fluorescence (FL1) versus 7-AAD Fluorescence (FL3) For selection of beads.

Set the number of events to capture at 3000 for R8 gate (View8)

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View-1

View-2

View-3

Dot plot views: • View-1 CD45-A488 fluorescence (FL1 - log scale) versus side-scatter (SSC – Lin scale) to establish lymph blast region. • View-2 CD34-PE fluorescence (FL2-log) versus –side scatter (SSC – Lin scale), to capture CD34+ cells (R2), • View-3 7-AAD fluorescence (FL3-log) versus –side scatter (SSC – Lin scale), to eliminate dead cells from live cells (R3)

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View-4

View-5

View-6

Dot plot views: • View-4 Intersection population of (R1*R2*R3) fluorescence (FL1 - log scale) versus side-scatter (SSC – Linear scale) to capture CD34+ cells (R4). • View-5 CD45-A488 (R5) population plotted on forward scatter (FSc-Lin) versus –side scatter (SSC – Lin) as a reference plot for scatter properties. • View-6 R4 gate applied on forward scatter (FSc-Lin) versus –side scatter (SSC – Lin). , to eliminate non CD34+ cells (R3).

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View-7

View-8

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Dot plot views: • View-7 Beads (R7) population is selected on forward scatter (FSC-Lin) versus –side scatter (SSC – Lin). • View-8 Beads (R7) population plotted on forward scatter (FSC-Lin) versus –side scatter (SSC – Lin)to eliminate any cell contamination and get absolute beads count. • View-9 population plotted on forward CD45-A488 (FL1-Log) CD34-PE (FL2-Log) to refer the threshold on FL1 Channel and avoid any loss of CD34+ cells.

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10. Calculations for CD34+ Counts: Use the following Equation to enumerate absolute live CD34+ cell events Acquire the regional stats for R6 on View -6 for CD34+ live Cells. Acquire Gate Stats for G8 for bead events (R8) on view8. Viable CD34+ cells/µl = CD34+ events in region R6 Beads per Test ---------------------------------X --------------------- X Dilution Bead events in ( R8) Sample Volume µl (50)

*The bead per test value is provided on the reagent pouch and varies form lot to lot.

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11. 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.

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12. REFERENCES 1. Sutherland DR., Marsh J.C. , David J. et al, Differential sensitivity of CD34 epitopes to cleavage by pasteurella hemolytica glycoproteases implication for purification of CD34 Positive progenitor cells. Exp Hematol 1992;20;590-599. 2. Michael Keeney,Ian Chin Yee, Karin Weir, Jan Popma, Rakash Nayar and D.Robert Sutherland.Single platform cytometric absolute CD34+ cell counts based on the ISAGE guidelines. Cytometry (Communications in clinical cytometry) 34:61-70(1998) 3. Mario Otto,Xiaohua Chen,William J.Martin,Wing Leung,James Knowles,Marti Holladay,Jim Houston, Rupert handgretinger and Raymond C. Barfield.-Selection of stem cells by using antibodies that target different CD34 epitopes yields different patterns of T cell differentiation Stemcells :2007:25:537-542. 4.

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5. SJ Noga,GB Vogelsang, SC Miller, S Meusel, K Loper, R Case, B Myers, L Rogers, I Flinn, M Borowitz and PO Donnell. Using point of care CD34 enumeration to optimize PBMC collection conditions. CytoTherapy(2001) vol 3, No.1, 11-18. 6. P.Pranke, J.Hendrikx, G.Alespeiti,N.Nardi, P.Rubinstein, J.VisserComparative quantification of Umbilical cord blood CD34+ and CD34+ bright cells using the Procount™ BD and ISHAGE protocols.-Brazilian journal of medical and biological research(2006)39:901-906. 7. Heeje Kim, Katharine A. Whartenby, Robert W. Georganatas III, John Wingard, Curt I. Civin. Human CD34+ Hematopietic stem / Progenitor cells express high levels of FLIP and are resistant to Fas mediated Apoptosis. Stem Cells 2002; 20; 174-182. 8. AFCG Guidelines 2006

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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: [email protected] www.reametrix.com Rev No. 2.0, 14-Apr-09

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