2/2014 23-7430-02 IVD BD Oncomark CD8/CD56/CD3 Catalog No. 333171 BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. 2014 BD Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA 95131 USA Benex Limited Pottery Road, Dun Laoghaire, Co. Dublin, Ireland Tel +353.1.202.5222 Fax +353.1.202.5388 BD Biosciences European Customer Support Tel +32.2.400.98.95 Fax +32.2.401.70.94 help.biosciences@europe.bd.com 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 bdbiosciences.com ClinicalApplications@bd.com 1. INTENDED USE BD Oncomark CD8 FITC/CD56 PE/ CD3 PerCP-Cy 5.5 * is intended for in vitro flow cytometric immunophenotyping of normal and abnormal populations of large granular lymphocytes. 1-4 These types of cells express the CD56 antigen and frequently are dimly stained with CD8. 5 Cells of natural killer (NK) origin are CD3 negative. 5 A subset of CD3-positive T cells express the CD56 antigen. 6 Tand non-t variants of NK lymphoproliferative disorder have been recognized, 1,4 including aggressive variants. 3,7 CD8/ CD56/CD3 assays are used in the diagnosis of hematologic disorders. 1-4 2. COMPOSITION CD8, clone SK1, is derived from the hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood T lymphocytes. CD56, clone NCAM16.2, 8 is derived from the hybridization of mouse P3-X63- Ag8.653 cells with spleen cells from BALB/c mice immunized with immunoaffinity-enriched adult human brain neural cell adhesion molecule (NCAM). * Cy is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and is made and sold under license from GE Healthcare. This product is licensed for sale only for in vitro diagnostics. It is not licensed for any other use. If you require any additional license to use this product and do not have one, return this material, unopened, to BD Biosciences, 2350 Qume Dr., San Jose, CA 95131, and any money paid for the material will be refunded. 1
CD3, clone SK7, 9-12 is derived from the hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human thymocytes. CD8 and CD3 are each composed of mouse IgG 1 heavy chains and kappa light chains. CD56 is composed of mouse IgG 2b heavy chains and kappa light chains. This reagent is supplied as a combination of CD8 FITC, CD56 PE, and CD3 PerCP-Cy5.5 in 1 ml of phosphatebuffered saline (PBS) containing bovine serum albumin (BSA) and 0.1% sodium azide. Antibody purity is as follows. FITC, PE, PerCP-Cy5.5: 20% free fluorophore at bottling, as measured by size-exclusion chromatography (SEC) 3. STORAGE AND HANDLING The antibody reagent is stable until the expiration date shown on the label when stored at 2 C 8 C. Do not use after the expiration date. Do not freeze the reagent or expose it to direct light during storage or incubation with cells. Keep the outside of the reagent vial dry. Do not use the reagent if you observe any change in appearance. Precipitation or discoloration indicates instability or deterioration. 4. REAGENTS OR MATERIALS REQUIRED BUT NOT PROVIDED Falcon disposable 12 x 75-mm polystyrene test tubes or equivalent Micropipettor with tips Falcon is a registered trademark of Corning Incorporated. Vortex mixer BD FACS lysing solution (10X) (Catalog No. 349202). For dilution instructions and warnings, refer to the instructions for use (IFU). Centrifuge BD CellWASH (Catalog No. 349524) or a wash buffer of PBS with 0.1% sodium azide BD CellFIX (Catalog No. 340181) or 1% paraformaldehyde solution in PBS with 0.1% sodium azide. Store at 2 C 8 C in amber glass for up to 1week. Properly equipped cytometer. Flow cytometers must have laser excitation set at 488 nm and must be equipped to detect light scatter and the appropriate fluorescence, and have the appropriate analysis software installed for data acquisition and analysis. Refer to your instrument user s guide for instructions. 5. SPECIMEN(S) BD Oncomark CD8 FITC/CD56 PE/CD3 PerCP-Cy5.5 can be used for immunophenotyping by flow cytometry with peripheral blood and bone marrow aspirates collected in BD Vacutainer EDTA tubes. Each type of specimen can have different storage conditions and limitations that should be considered prior to collection and analysis. 13,14 WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 15,16 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. 2
6. PROCEDURE Viability of samples should be assessed and a cutoff value established. A cutoff value of at least 80% viable cells has been suggested. 13 To avoid serum interference when using these reagents, it is necessary to pre-wash the sample using at least 25 volumes excess 1X PBS with 0.1% sodium azide (48 ml of 1X PBS with sodium azide per 2 ml of whole blood to be washed). Mix well. Pellet cells by centrifugation and resuspend in 1X PBS with 0.1% sodium azide to the original volume. 1. Add 20 µl of BD Oncomark CD8/ CD56/CD3 reagent to 100 µl of whole blood or prefiltered bone marrow in a 12 x 75-mm tube. 2. Vortex gently and incubate for 15 to 20 minutes in the dark at room temperature (20 C 25 C). 3. Add 2 ml of 1X BD FACS lysing solution. 4. Vortex gently and incubate for 10 minutes in the dark at room temperature. 5. Centrifuge at 300g for 5 minutes. Remove the supernatant. 6. Add 2 to 3 ml of BD CellWASH solution (or wash buffer) and centrifuge at 200g for 5 minutes. Remove the supernatant. 7. Add 0.5 ml of BD CellFIX solution or 1% paraformaldehyde solution and mix thoroughly. Store at 2 C 8 C until analyzed. Stained samples should be analyzed within 24 hours of staining. Flow Cytometric Analysis 1. Set up the instrument as recommended by the manufacturer. Run a control sample daily from a normal adult subject or a commercially available whole blood control to optimize instrument settings and as a quality control check of the system. 2. Vortex the cells thoroughly at low speed to reduce aggregation before running them on the flow cytometer. 17 3. Run the sample on the flow cytometer. Verify that all populations are on scale. Optimize the instrument settings, if needed. 4. Acquire and analyze list-mode data using appropriate software. 5. On the appropriate plots, use the required combination of gates, regions, or quadrants to isolate the population of interest (Figure 1). Figure 1 Dot plots displaying region R1 and quadrants 3
6. Determine antigen expression based on the sample negative population. 7. PERFORMANCE CHARACTERISTICS Specificity CD8 recognizes the 32-kilodalton (kda) α subunit of a disulfide-linked bimolecular complex. 18,19 The majority of peripheral blood CD8 + T lymphocytes expresses an α/β heterodimer (32, 30 kda), while CD8 + CD16 + natural killer (NK) lymphocytes and CD8 + T-cell receptor (TCR)-γ/δ + T lymphocytes express an α/α homodimer (30 kda). CD8 + TCR-α/β + T lymphocytes can express either an α/α homodimer or an α/β heterodimer. 18,19 CD56 (NCAM16.2) recognizes an extracellular immunoglobulin-like domain common to three molecular weight forms (120, 140, and 180 kda) of NCAM. 20-22 CD3 reacts with the epsilon chain of the CD3 antigen/tcrcomplex. 23 The antigen recognized by CD3 antibodies is noncovalently associated with either α/β or γ/δ TCR (70 to 90 kda). 24 Antigen Distribution The CD8 antigen is present on the human suppressor/cytotoxic T-lymphocyte subset, 9,25-29 as well as on a subset of NK lymphocytes. 30 The CD8 antigen is expressed on 19% to 48% of normal peripheral blood lymphocytes 31 and 60% to 85% of normal thymocytes. 9,25 The CD56 antigen is present on approximately 10% to 25% of peripheral blood lymphocytes. 32 It is present on essentially all resting and activated CD16 + NK lymphocytes and approximately 5% of CD3 + peripheral blood lymphocytes. 32 The CD3 antigen is present on 61% to 85% of normal peripheral blood lymphocytes. 31 8. LIMITATIONS Use of therapeutic monoclonal antibodies in patient treatment can interfere with recognition of target antigens by this reagent. This should be considered when analyzing samples from patients treated in this fashion. BD Biosciences has not characterized the effect of the presence of therapeutic antibodies on the performance of this reagent. Use of this reagent combination for diagnostic evaluation of hematologic disorders should be performed in the context of a thorough immunophenotypic analysis including other relevant markers. Procedures using BD Oncomark reagents must adhere to the instructions for use for the specific instrument, software, and quality control procedures used by your laboratory. Reagent performance data was collected typically with EDTA-treated specimens. Reagent performance can be affected by the use of other anticoagulants. 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. 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. 4
TROUBLESHOOTING Problem Possible Cause Solution Poor resolution between debris and lymphocytes Staining dim or fading REFERENCES Cell interaction with other cells and platelets Rough handling of cell preparation Inappropriate instrument settings Cell concentration too high at staining step Insufficient reagent Cells not analyzed within 24 hours of staining Few or no cells Cell concentration too low Cytometer malfunctioning Prepare and stain another sample. Check cell viability; centrifuge cells at lower speed. Follow proper instrument setup procedures; optimize instrument settings as required. Check and adjust cell concentration or sample volume; stain with fresh sample. Repeat staining with increased amount of antibody. Repeat staining with fresh sample; analyze promptly. Resuspend fresh sample at a higher concentration; repeat staining and analysis. Troubleshoot instrument. 1. Chan WC, Gu LB, Masih A, et al. Large granular lymphocyte proliferation with the natural killer-cell phenotype. Am J Clin Pathol. 1992;97:353-358. 2. Okuno SH, Tefferi A, Hanson C, Katzmann JA, Li CY, Witzig TE. Spectrum of diseases associated with increased proportions or absolute numbers of peripheral blood natural killer cells. Br J Haematol. 1996;93:810-812. 3. Macon WR, Williams ME, Greer JP, et al. Natural killer-like T-cell lymphomas: aggressive lymphomas of T-large granular lymphocytes. Blood. 1996;87:1474-1483. 4. Loughran TP, Jr. Clonal diseases of large granular lymphocytes. Blood. 1993;82:1-14. 5. Ritz J, Schmidt RE, Michon J, Hercend T, Schlossman SF. Characterization of functional surface structures on human natural killer cells. Adv Immunol. 1988;42:181-211. 6. Ortaldo JR, Winkler-Pickett RT, Yagita H, Young HA. Comparative studies of CD3 and CD3 + CD56 + cells: examination of morphology, functions, T cell receptor rearrangement, and pore-forming protein expression. Cell Immunol. 1991;136:486-495. 7. Gentile TC, Uner AH, Hutchison RE, et al. CD3 +, CD56 + aggressive variant of large granular lymphocyte leukemia. Blood. 1994;84:2315-2321. 8. Ritz J, Trinchieri G, Lanier LL. NK-cell antigens: section report. In: Schlossman SF, Boumsell L, Gilks W, et al, eds. Leucocyte Typing V: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1995;2:1367-1372. 9. Ledbetter JA, Evans RL, Lipinski M, Cunningham- Rundles C, Good RA, Herzenberg LA. Evolutionary conservation of surface molecules that distinguish T-lymphocyte helper/inducer and T cytotoxic/ suppressor subpopulations in mouse and man. J Exp Med. 1981;153:310-323. 10. Haynes BF. Summary of T-cell studies performed during the Second International Workshop and Conference on Human Leukocyte Differentiation Antigens. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human T Lymphocytes. New York, NY: Springer-Verlag; 1986;1:3-30. 11. Kan EAR, Wang CY, Wang LC, Evans RL. Noncovalently bonded subunits of 22 and 28 kd are rapidly internalized by T cells reacted with Anti Leu-4 antibody. J Immunol. 1983;131:536-539. 12. Knowles RW. Immunochemical analysis of the T-cell specific antigens. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human T Lymphocytes. New York, NY: Springer- Verlag; 1986;1:259-288. 13. Rothe G, Schmitz G. Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Leukemia. 1996;10:877-895. 14. Stelzer GT, Marti G, Hurley A, McCoy P Jr, 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;30:214-230. 15. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2005. CLSI document M29-A3. 5
16. 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;37:377-388. 17. Jackson AL, Warner NL. Preparation, staining, and analysis by flow cytometry of peripheral blood leukocytes. In: Rose NR, Friedman H, Fahey JL, eds. Manual of Clinical Laboratory Immunology. 3rd ed. Washington, DC: American Society for Microbiology; 1986:226-235. 18. Moebius U. Cluster report: CD8. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989:342-343. 19. Terry LA, Disanto JP, Small TN, Flomenberg N. Differential expression of the CD8 and Lyt-3 antigens on a subset of human T-cell receptor γ/δbearing lymphocytes. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989:345-346. 20. Lanier LL, Chang C, Azuma M, Ruitenberg JJ, Hemperly JJ, Phillips JH. Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56). J Immunol. 1991;146:4421-4426. 21. Schubert J, Lanier LL, Schmidt RE. Cluster report: CD56. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989:699-702. 22. Cunningham BA, Hemperly JJ, Murray BA, Prediger EA, Brackenbury R, Edelman GM. Neural cell adhesion molecule: structure, immunoglobulinlike domains, cell surface modulation, and alternative RNA splicing. Science. 1987;236:799-806. 23. van Dongen JJM, Krissansen GW, Wolvers-Tettero ILM, et al. Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies. Blood. 1988;71:603-612. 24. Clevers H, Alarcón B, Wileman T, Terhorst C. The T cell receptor/cd3 complex: a dynamic protein ensemble. Annu Rev Immunol. 1988;6:629-662. 25. Evans RL, Wall DW, Platsoucas CD, et al. Thymusdependent membrane antigens in man: inhibition of cell-mediated lympholysis by monoclonal antibodies to the T H2 antigen. Proc Natl Acad Sci USA. 1981;78:544-548. 26. Engleman EG, Benike CJ, Evans RL. Circulating antigen-specific suppressor T cells in a healthy woman: mechanism of action and isolation with a monoclonal antibody. Clin Res. 1981;29:365A. 27. Kotzin BK, Benike CJ, Engleman EG. Induction of immunoglobulin secreting cells in the allogeneic mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets in man. J Immunol. 1981;127:931-935. 28. Engleman EG, Benike CJ, Glickman E, Evans RL. Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocyte subpopulations block the mixed leukocyte reaction in man. J Exp Med. 1981;153:193-198. 29. Ledbetter JA, Frankel AE, Herzenberg LA. Human Leu T-cell differentiation antigens: quantitative expression on normal lymphoid cells and cell lines. In: Hämmerling G, Hämmerling U, Kearney J, eds. Monoclonal Antibodies and T Cell Hybridomas: Perspectives and Technical Notes. New York, NY: Elsevier/North Holland; 1981:16-22. 30. Lanier LL, Le AM, Phillips JH, Warner NL, Babcock GF. Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol. 1983;131:1789-1796. 31. Reichert T, DeBruyère M, Deneys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopath. 1991;60:190-208. 32. Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH. The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol. 1986;136:4480-4486. 6