BD IntraSure Kit IVD. Cell fixation and permeabilization reagents. 50 tests per kit Catalog No

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1 03/ IVD BD IntraSure Kit Cell fixation and permeabilization reagents 50 tests per kit Catalog No BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company BD Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA USA Benex Limited Pottery Road, Dun Laoghaire, Co. Dublin, Ireland Tel Fax BD Biosciences European Customer Support Tel Fax Becton Dickinson Pty Ltd, 4 Research Park Drive, Macquarie University Research Park, North Ryde NSW 2113, Australia Becton Dickinson Limited, 8 Pacific Rise, Mt. Wellington, Auckland, New Zealand 1. INTENDED USE The BD IntraSure kit is intended to fix and permeabilize cells to enable the staining of intracellular targets with minimal effect on the prior staining of cell surface antigens. Samples are acquired using a BD FACS flow cytometer equipped with the appropriate analysis software for data acquisition and analysis. 2. SUMMARY AND EXPLANATION Measurement of intracellular targets by flow cytometry is an essential analytical step in basic leukemia and lymphoma (L&L) diagnostics. 1 5 The BD IntraSure * kit assists in the process of staining intracellular markers of leukemia and lymphoma by fixing the cells of interest and permeabilizing the membrane to fluorescent antibody reagents, permitting analysis by flow cytometry. 3. PRINCIPLES OF THE PROCEDURE When whole blood is incubated with fluorochrome-conjugated monoclonal antibody reagents, the antibodies bind specifically to the appropriate leucocyte antigens on the cell surface. The BD IntraSure kit enables the user to fix and permeabilize stained cells so that fluorochrome-conjugated antibodies directed against intracellular targets can enter the cells and bind specifically to their targets. The fixation and permeabilization steps minimally affect the prior staining of cell surface markers. Thus, both cell surface and intracellular antigens can be simultaneously analyzed using flow cytometry. bdbiosciences.com ClinicalApplications@bd.com * This product is produced under license from Leeds Teaching Hospitals NHS Trust. 1

2 4. REAGENTS Reagents provided The BD IntraSure kit is provided as 5 ml of Reagent A, a proprietary buffered fixative containing 1% formaldehyde, and 2.5 ml of Reagent B, a proprietary buffered permeabilization solution containing a detergent. Precautions Do not use the reagents if you observe any change in their appearance. Precipitation or discoloration indicates instability or deterioration. Reagent A contains 1.0% formaldehyde, CAS number , and 0.35% methanol, CAS number Danger H341 Suspected of causing genetic defects. H350 May cause cancer. Route of exposure: Inhalative. H317 May cause an allergic skin reaction. Wear protective gloves / eye protection.wear protective clothing. Avoid breathing mist/vapours/spray. IF exposed or concerned: Get medical advice/attention. If skin irritation or rash occurs: Get medical advice/attention. Dispose of contents/container in accordance with local/regional/national/ international regulations. Reagent B contains sodium azide as a preservative. Storage and handling Store bottles at room temperature (20 C 25 C) and protect from light. Do not freeze. Each reagent is stable until the expiration date shown on the bottle label when stored as directed. 5. REAGENTS AND MATERIALS REQUIRED BUT NOT PROVIDED Disposable 12 x 75-mm capped polystyrene test tubes Micropipettor with tips Vortex mixer Centrifuge Fluorochrome-conjugated antibody appropriate for cell surface staining, for example, CD19 APC (Catalog No ) Fluorochrome-conjugated antibody reagent(s) for intracellular staining, eg, BD Oncomark CD3/Anti- Myeloperoxidase (MPO)/ CD79a(Catalog No ) BD FACS lysing solution, 10X concentrate (Catalog No ) For the dilution instructions and warnings, see the reagent instructions for use (IFU). BD CellWASH (Catalog No ) or a wash buffer of PBS with 0.1% sodium azide BD CellFIX (10X concentrate) (Catalog No )or 1% paraformaldehyde solution in PBS with 0.1 % sodium azide. Store at 2 C-8 C in amber glass for up to 1 week. For the dilution instructions and warnings, see the reagent IFU. 6. INSTRUMENTS Samples are acquired using the BD FACSCanto or BD FACSCanto II flow cytometers running BD FACSDiva software, or a BD FACSCalibur flow cytometer running BD CellQuest Pro software. 2

3 Other reagents required to set up and run the flow cytometer include: BD FACSFlow sheath fluid (Catalog No ) or equivalent. CAUTION Use only BD FACSFlow sheath fluid diluent to dilute BD Calibrite beads. Instrument setup beads, for example, BD FACS 7-color setup beads (Catalog No ), BD Calibrite 3 three-color kit and BD Calibrite APC beads (Catalog No and , respectively), or BD FACSDiva CS&T IVD beads (Catalog No and ). 7. SPECIMEN(S) The BD IntraSure reagents can be used with peripheral blood or bone marrow samples. Each type of specimen can have different storage conditions and limitations that should be considered prior to collection and analysis. 6,7 Samples with large numbers of nonviable cells can give erroneous results due to selective loss of populations and to increased nonspecific binding of antibodies to nonviable cells. Viability of samples should be assessed, and greater than 80% viable cells should be present. 6 WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 8,9 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves. 8. PROCEDURE Before you begin 1. Prepare at least 75 µl of a cell suspension with a white blood cell (WBC) count of up to 2.0 x 10 7 cells/ ml. 2. Check the cell viability to ensure that there are greater than 80% viable cells. 3. Prepare 1X BD FACS lysing solution. For the dilution instructions, see the reagent IFU. Staining and fixing of cell surface markers 1. Pipette 50 µl of the cell suspension into a test tube. 2. (Optional) If cell surface staining is to be performed, add the appropriate fluorochrome-labeled antibody reagent(s) and incubate according to the reagent IFU. 3. Add 100 µl of Reagent A to the tube. 4. Vortex to mix. 5. Incubate for 5 minutes in the dark at room temperature (20 C 25 C). Lysing and decanting 1. Add 2 ml of the 1X BD FACS lysing solution to the tube. 2. Vortex to mix. 3. Incubate for 10 minutes in the dark at room temperature (20 C 25 C). 4. Centrifuge at 800g 850g for 5 minutes. 5. Decant the supernatant. NOTE Decanting is preferable to aspirating. If aspirating, be careful not to disturb the cell pellet. 3

4 Permeabilizing and staining for intracellular markers 1. Pipette 50 µl of Reagent B into the tube. 2. Add the fluorochrome-conjugated antibody reagent(s) specific to the intracellular target(s) of interest, according to the IFU for each antibody reagent. 3. Vortex to mix. 4. Incubate for at least 15 minutes in the dark at room temperature (20 C 25 C). NOTE The duration of the incubation step may have to be optimized for different antibody reagents. 5. Add 2 to 3 ml of BD CellWASH solution or PBS with 0.1% sodium azide to the tube. 6. Vortex to mix. 7. Centrifuge at 800g 850g for 5 minutes. 8. Decant the supernatant. NOTE Decanting is preferable to aspirating. If aspirating, be careful not to disturb the cell pellet. 9. Resuspend the cell pellet in 0.5 ml of BD CellFIX solution or 1% paraformaldehyde. Vortex to mix. 10. Fix the cells for 30 minutes in the dark at 2 C 8 C. 11. Vortex before acquiring the sample tube. NOTE Refer to the reagent IFU for storage conditions of stained cells prior to acquisition. Flow cytometry Figure 1 shows acquisition and analysis performed on whole blood stained with CD3/Anti-MPO/CD79a and CD19 APC as a drop-in reagent. Cells were fixed and permeabilized using the BD IntraSure kit. Laser excitation is at 488 nm and 635 nm. Figure 1 Data acquired on a BD FACSCanto II flow cytometer and analyzed using BD FACSDiva software 9. PERFORMANCE CHARACTERISTICS Reproducibility Two operators performed two separate runs per day over a period of eight days on two BD FACSCanto II flow cytometers. For each run, duplicate samples of normal peripheral blood were stained using the CD19 PE-Cy 7 and CD3 FITC/anti-MPO/CD79a reagents, fixed and permeabilized with two lots of the BD IntraSure kit by each operator, acquired, and analyzed using BD FACSDiva software. Samples were gated on the appropriate cell population (for example, granulocytes for anti-mpo or lymphocytes for CD3, CD19 and CD79a). Cy is a trademark of GE Healthcare. 4

5 The overall reproducibility was calculated for Subset %P (percent positive) of the cell populations. The overall reproducibility comprises four components: operator/ instrument-to-operator/instrument, lot-tolot, run-to-run, and tube-to-tube reproducibility. See Table 1. Table 1 Reproducibility of Subset %P (overall) Subset DF a a. DF = degrees of Freedom c. CV = coefficient of variation SD b %CV c %MPO %CD %CD79a %CD Repeatability Two operators performed two separate runs per day over a period of eight days on two BD FACSCanto II flow cytometers. For each run, duplicate samples of normal peripheral blood were stained using the CD19 PE-Cy7 and CD3 /anti-mpo/cd79a reagents, fixed and permeabilized with two lots of the BD IntraSure kit by each operator, acquired, and analyzed using BD FACSDiva software. The intra-assay precision (tube-to-tube repeatability) was calculated for Subset %P of the cell populations. See Table 2. Table 2 Repeatability of Subset %P (tube-totube) Subset DF a a. DF = degrees of Freedom c. CV = coefficient of variation SD b %CV c %MPO %CD %CD79a %CD Accuracy A total of 116 fresh peripheral blood and 104 bone marrow specimens left over from diagnostic L&L testing at two external clinical sites were evaluated for equivalence in using the BD IntraSure kit or the FIX & PERM kit to fix and permeabilize the cells. Duplicate samples were stained with CD19 PE-Cy7, fixed and permeabilized using either the BD IntraSure kit or the FIX & PERM kit as a reference, and then stained for intracellular markers using BD Oncomark CD3/Anti-MPO/CD79a. Samples were acquired on a BD FACSCanto II flow cytometer using BD FACSDiva software. Each sample was analyzed separately for the difference in Mean Fluorescence Intensity (Delta MFI) between the positive cell population and the negative cell population for MPO +, CD3 +, CD79a +, and CD19 +, and Subset %P of cell populations for the same markers. The mean bias for Delta MFI between the BD IntraSure and FIX & PERM treated samples was calculated. See Table 3. The absolute difference in Subset %P values FIX & PERM is a shared registered trademark of CALTAG and AN-DER-GRUB. 5

6 for low bin specimens, and the relative difference in Subset %P values for high bin specimens between the BD IntraSure and FIX & PERM treated samples were calculated. See Table 4 and Table 5. Table 3 Mean bias of Delta MFI values between BD IntraSure and FIX & PERM Variable (MFI) N a a. N = number of samples Relative difference Mean bias SD b of mean bias CD MPO CD79a CD Table 4 Absolute difference in Subset %P values between BD IntraSure and FIX & PERM for low bin specimens Absolute difference Variable (% Subset) N a Mean Bias SD b CD MPO CD79a CD a. N = number of samples Table 5 Relative difference in Subset %P values between BD IntraSure and FIX & PERM for high bin specimens Variable (% Subset) N a a. N = number of samples Relative difference to reference Mean % Bias SD b CD MPO CD79a CD LIMITATIONS For In Vitro Diagnostic Use. The BD IntraSure kit is designed to be used in conjunction with BD FACS lysing solution. The BD IntraSure kit permeabilizes different cellular membranes to varying extents. Therefore the effectivity of detection depends on where a particular antigen is located in the cell. WARRANTY Unless otherwise indicated in any applicable BD general conditions of sale for non-us customers, the following warranty applies to the purchase of these products. THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT. 6

7 REFERENCES 1. Bennett F, Rawstron A, Plummer M, et al. B-cell chronic lymphocytic leukaemia cells show specific changes in membrane protein expression during different stages of cell cycle. Br J Haematol. 2007;139: de Tute RM, Jack AS, Child JA, Morgan GJ, Owen RG, Rawstron AC. A single-tube six-colour flow cytometry screening assay for the detection of minimal residual disease in myeloma. Leukemia. 2007;21: Dworzak MN, Fritsch G, Fröschl G, Printz D, Gadner H. Four-color flow cytometric investigation of terminal deoxynucleotidyl transferase positive lymphoid precursors in pediatric bone marrow: CD79a expression precedes CD19 in early B-cell ontogeny. Blood. 1998;92: Letestu R, Rawstron A, Ghia P, et al. Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process. Cytometry B Clin Cytom. 2006;70: Nakase K, Sartor M, Bradstock K. Detection of myeloperoxidase by flow cytometry in acute leukemia. Cytometry. 1998;34: Rothe G, Schmitz G. Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Leukemia. 1996;10: Stelzer GT, Marti G, Hurley A, McCoy PJ, Lovett EJ, Schwartz A. US-Canadian consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: standardization and validation of laboratory procedures. Cytometry. 1997;30: Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition.. Wayne, PA: Clinical and Laboratory Standards Institute; M29-A3. 9. Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37: