BD Oncomark IVD. CD2 FITC/CD7 PE/CD3 PerCP-Cy5.5

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1 BD Oncomark CD2 FITC/CD7 PE/CD3 PerCP-Cy5.5 IVD Catalog No / BD Biosciences Becton, Dickinson and Company 2350 Qume Drive San Jose, CA USA Tel (877) Fax (408) BENEX Limited Bay K 1a/d Shannon Industrial Estate Shannon, County Clare Ireland Tel (353) Fax (353) BD Biosciences Centralized European Office Erembodegem Dorp-86 B-9320 Erembodegem-Aalst, Belgium Tel (32) Fax (32) Becton, Dickinson and Company 1 Becton Drive Franklin Lakes, NJ USA Tel (201) INTENDED USE BD Oncomark CD2 FITC/CD7 PE*/ CD3 PerCP-Cy5.5* is intended for in vitro flow cytometric immunophenotyping of normal T-cell development 1 and aberrant expression 2 of the T-cell surface markers in various T-cell neoplasias. While the antigens recognized by these reagents are present on normal T cells, one or more of them is frequently expressed at different levels, or possibly absent, in abnormal T cells. 3,4 One or more of the antigens detected by these antibodies might be aberrantly expressed in myeloid leukemia. 5-7 CD2/CD7/CD3 assays are used in the diagnosis of hematologic disorders COMPOSITION CD2, clone S5.2, is derived from the hybridization of mouse Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with T lymphocytes activated by mixed lymphocyte culture. CD7, clone M-T701, 8 is derived from the hybridization of mouse P3-X63-Ag8.653 cells with spleen cells from BALB/c mice immunized with P-CLL and Jurkat cells. 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. CD2 is composed of mouse IgG 2a heavy chains and kappa light chains. CD7 amd CD3 are each composed of mouse IgG 1 heavy chains and kappa light chains. * Patents PE: US 4,520,110; 4,859,582; 5,055,556; Europe 76,695; Canada 1,179,942 PerCP: US 4,876,190 Cy5.5: US 5,268,486; 5,486,616; 5,569,587; 5,569,766; 5,627,027 1

2 This reagent is supplied as a combination of CD2 FITC, CD7 PE, and CD3 PerCP-Cy5.5 in 1 ml of phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) and 0.1% sodium azide. WARNING Sodium azide is harmful if swallowed (R22). Keep out of reach of children (S2). Keep away from food, drink, and animal feedingstuff (S13). Wear suitable protective clothing (S36). If swallowed, seek medical advice immediately and show this container or label (S46). Contact with acids liberates very toxic gas (R32). Azide compounds should be flushed with large volumes of water during disposal to avoid deposits in lead or copper plumbing where explosive conditions can develop. 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 to 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 BD Falcon disposable 12 x 75-mm capped polystyrene test tubes (BD Catalog No ) or equivalent Micropipettor with tips (BD Electronic Pipette, BD Catalog No [US], [Europe]) Vortex mixer BD FACS lysing solution* (10X) (BD Catalog No ) For dilution instructions and warnings, refer to the product insert. Centrifuge BD CellWASH solution (BD Catalog No ) or a wash buffer of phosphate-buffered saline (PBS) with 0.1% sodium azide BD CellFIX solution (BD Catalog No ) or 1% paraformaldehyde solution in PBS with 0.1% sodium azide Store at 2 to 8 C in amber glass for up to 1 week. WARNING Formaldehyde is harmful by inhalation, in contact with skin, and toxic if swallowed (R20/21, 25). It is irritating to eyes and skin (R36/38). In case of contact with eyes, rinse immediately with plenty of water and seek medical advice (S26). Exposure can cause cancer. Contact with acids liberates very toxic gas (R32). Possible risks of irreversible effects (R68). Can cause sensitization by skin contact (R43). When using do not eat or drink (S20). 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. 2 * US Patent Nos. 4,654,312; 4,902,613; 5,098,849

3 Refer to your instrument user s guide for instructions. 5. SPECIMEN(S) BD Oncomark CD2 FITC/CD7 PE/CD3 PerCP-Cy5.5 can be used for immunophenotyping by flow cytometry with peripheral blood and bone marrow aspirates collected in EDTA (eg, BD Vacutainer 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 and gloves. 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% azide (ie, 48 ml of 1X PBS with azide per 2 ml whole blood to be washed). Mix well. Pellet cells by centrifugation and resuspend in 1X PBS with 0.1% azide to the original volume. 1. Add 20 µl of BD Oncomark CD2/ CD7/CD3 reagent to 100 µl of whole blood or prefiltered bone marrow in a 12 x 75-mm tube. 2. Vortex gently and incubate 15 to 20 minutes in the dark at room temperature (20 to 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 300 x g for 5 minutes. Remove the supernatant. 6. Add 2 to 3 ml of BD CellWASH solution (or wash buffer) and centrifuge at 200 x g 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 to 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 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. 3

4 5. On the appropriate plots, use the required combination of gates, regions or quadrants to isolate the population of interest (Figure 1). 0 SSC CD7 PE 10 4 Figure 1 Dot plots displaying region R1 and quadrants 6. Determine antigen expression based on the sample negative population. 7. PERFORMANCE CHARACTERISTICS Specificity CD2 recognizes a human lymphocyte antigen, Mr 45 to 50 kilodaltons (kd). 18 CD7 recognizes a human T- and natural killer (NK)-lymphocyte antigen, Mr 40 kd. 19,20 CD3 reacts with the epsilon chain of the CD3 antigen/t-cell antigen receptor (TCR) complex. 21 The antigen recognized by CD3 antibodies is noncovalently associated with either α/β or γ/δ TCR (70 to 90 kd) CD2 FITC 10 4 R1 0 FSC CD3 PerCP-Cy CD3 PerCP-Cy CD7 PE CD2 FITC 10 4 Antigen Distribution The CD2 antigen is present on approximately 75% of normal peripheral blood lymphocytes and 95% to 99% of thymocytes. 23 The CD2 antibody reacts with essentially all T lymphocytes and with a subset of NK lymphocytes. 24 The CD7 antigen is expressed throughout T-lymphocyte differentiation. 20 It is present on 85% to 90% of peripheral blood T lymphocytes. 20 In normal individuals, CD7 reacts with all CD8 + lymphocytes, approximately 90% of CD4 + lymphocytes, and most NK lymphocytes. 20 CD7 is weakly reactive with monocytes and does not react with granulocytes 19 or B lymphocytes. 20 In leukemias, the CD7 antigen is present on the majority of T-lymphoid lineages. 25 The CD3 antigen is present on 61% to 85% of normal peripheral blood lymphocytes 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 the BD Oncomark reagents must adhere to the instructions for use for the specific instrument, soft- 4

5 ware, and quality control procedures used by your laboratory. Reagent data performance 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 The product sold hereunder 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. 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, personal injury, or economic loss caused by the product. TROUBLESHOOTING Problem Possible Cause Solution Poor resolution between debris and lymphocytes Staining dim or fading 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. 5

6 REFERENCES 1. Terstappen LWMM, Huang S, Picker LJ. Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow. Blood. 1992;79: Porwit-MacDonald A. Multiparameter flow cytometry detection of minimal residual disease in T-cell acute lymphoblastic leukemia. Presented at the International Symposium on Minimal Residual Disease: From methodological problems to clinical goals; October 31-November 1, 1997; Salamanca, Spain. 3. Knowles DM. Immunophenotypic and antigen receptor gene rearrangement analysis in T cell neoplasia. Am J Pathol. 1989;134: Ginaldi L, Matutes E, Farahat N, De Martinis M, Morilla R, Catovsky D. Differential expression of CD3 and CD7 in T-cell malignancies: a quantitative study by flow cytometry. Br J Haematol. 1996;93: Khalidi HS, Medeiros LJ, Chang KL, Brynes RK, Slovak ML, Arber DA. The immunophenotype of adult acute myeloid leukemia: high frequency of lymphoid antigen expression and comparison of immunophenotype, French-American-British classification, and karyotypic abnormalities. Am J Clin Pathol. 1998;109: Launder TM, Bray RA, Stempora L, Chenggis ML, Farhi DC. Lymphoid-associated antigen expression by acute myeloid leukemia. Am J Clin Pathol. 1996;106: Macedo A, Orfao A, Vidriales MB, et al. Characterization of aberrant phenotypes in acute myeloblastic leukemia. Ann Hematol. 1995;70(4): Reiter C. Cluster report: CD7. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: 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: 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: 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: 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: Rothe G, Schmitz G. Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Leukemia. 1996;10: 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: Protection of Laboratory Workers from Occupationally Acquired Infections Second Edition; Approved Guideline. Villanova, PA: National Committee for Clinical Laboratory Standards; NCCLS document M29-A Centers for Disease Control. 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: 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: Bieber CP, Howard FD, Pennock J, Wong J, Shorthouse R, Stinson EB. Preparation, characterization, and primate testing of monoclonal antithymocyte globulin. Transplantation. 1981;31: Link M, Warnke R, Finlay J, et al. A single monoclonal antibody identifies T-cell lineage of childhood lymphoid malignancies. Blood. 1983;62: Palker TJ, Scearce RM, Hensley LL, Ho W, Haynes BF. Comparison of the CD7 (3A1) group of T cell workshop antibodies. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human T Lymphocytes. New York, NY: Springer-Verlag; 1986;1: 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:

7 22. Clevers H, Alarcón B, Wileman T, Terhorst C. The T cell receptor/cd3 complex: a dynamic protein ensemble. Annu Rev Immunol. 1988;6: Howard FD, Ledbetter JA, Wong J, Bieber CP, Stinson EB, Herzenberg LA. A human T lymphocyte differentiation marker defined by monoclonal antibodies that block E-rosette formation. J Immunol. 1981;126: Lanier LL, Phillips JH. A map of the cell surface antigens expressed on resting and activated human natural killer cells. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human Myeloid and Hematopoietic Cells. New York, NY: Springer-Verlag; 1986;3: Weiss LM, Crabtree GS, Rouse RV, Warnke RA. Morphologic and immunologic characterization of 50 peripheral T-cell lymphomas. Am J Pathol. 1985;118: Reichert T, DeBruyère M, Deneys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopath. 1991;60: