BD Oncomark CD103/CD22/CD20

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1 2/ IVD BD Oncomark CD103/CD22/CD20 50 Tests 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 bdbiosciences.com ClinicalApplications@bd.com 1. INTENDED USE BD Oncomark CD103 FITC/CD22 PE/ CD20 PerCP-Cy 5.5 * is intended for in vitro flow cytometric immunophenotyping of normal and abnormal subsets of B lymphocytes. The reagents detect expression of the CD103, CD22, and CD20 antigens. These antigens are highly expressed in the abnormal cells of hairy-cell leukemia. A subset of normal B cells expressing CD103 has been identified in peripheral blood. 1 CD103/CD22/CD20 assays are used in the diagnosis of hematologic disorders COMPOSITION CD103, clone Ber-ACT 8, 5 is derived from the hybridization of spleen cells from BALB/c mice with HTLV-1 positive cell line (MAPS16). 6 CD22, clone S-HCL-1, 7 is derived from the hybridization of mouse NS-1 myeloma cells with spleen cells from CD-1 mice immunized with whole cells and membrane preparations of hairy-cell leukemia cells. CD20, clone L27, is derived from the hybridization of mouse Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with the LB lymphoblastoid cell line. * 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 Drive, San Jose, CA 95131, and any money paid for the material will be refunded. 1

2 CD103 is composed of mouse IgG 1 heavy chains and lambda light chains. CD22 and CD20 are each composed of mouse IgG 1 heavy chains and kappa light chains. This reagent is supplied as a combination of CD103 FITC, CD22 PE, and CD20 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 Vortex mixer Falcon is a registered trademark of Corning Incorporated. BD FACS lysing solution (10X) (Catalog No ). For dilution instructions and warnings, refer to the instructions for use (IFU). Centrifuge BD CellWASH (Catalog No ) or a wash buffer of PBS with 0.1% sodium azide BD CellFIX (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. 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 CD103 FITC/CD22 PE/ CD20 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. 8,9 WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 10,11 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

3 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. 8 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 CD103/ CD22/CD20 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 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

4 6. Determine antigen expression based on the sample negative population. 7. PERFORMANCE CHARACTERISTICS Specificity CD103 recognizes the α E subunit of integrin α E β 7, an integrin also known as the human mucosa lymphocyte (HML) antigen 13,14 but not α 4, another member of the β 7 integrin subfamily. 14 Integrin α E β 7 is a trimeric protein complex of three components, 105 kilodaltons (kda) (β 7 ), 150 kda, and 25 kda. The CD103 epitope is localized on the 150-kDa chain. 6 CD22 recognizes a human B-lymphocyte antigen with a molecular weight of 135 kda. 15 The CD20 antigen is a phosphoprotein with a molecular weight of 35 or 37 kda, depending on the degree of phosphorylation. 16 The antigen is not glycosylated. 16 Antigen Distribution CD103 antigen is expressed on the surface of >90% of intestinal intraepithelial lymphocytes (iiels), 40% of intestinal lamina propria T lymphocytes, and <3% of resting peripheral blood lymphocytes. 17 The CD22 antigen is expressed in the cytoplasm of all B lymphocytes and is only present on the cell surface of mature B lymphocytes. 18 In contrast with the CD10, CD19, and CD20 antigens, the CD22 antigen is still present on lymphoplasmacytoid cells but is diminished on the fully matured plasma cell. 19 The CD22 antigen is expressed in most B-cell leukemias, including hairy-cell leukemia, 7 and nearly all (97%) B-cell lymphomas, 20 but not in T-cell leukemias or T-cell lymphomas. 21 Antigen density is markedly increased in hairy-cell leukemia. 7,15 The CD20 antigen is expressed on B lymphocytes synchronous with the expression of surface IgM. 16,22 The antigen is present on both resting and activated B lymphocytes but is lost prior to differentiation into plasma cells. 16 The CD20 antigen is found in both mantlezone and germinal center areas of secondary follicles of lymphoid tissue and can be expressed on follicular dendritic cells (FDCs) in germinal centers. 16 Lowlevel expression of the CD20 antigen has been detected on a subpopulation of T 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, software, and quality control procedures used by your laboratory. 4

5 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. 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 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. Problem Possible Cause Solution Few or no cells Cell concentration too low REFERENCES Cytometer malfunctioning Resuspend fresh sample at a higher concentration; repeat staining and analysis. Troubleshoot instrument. 1. Hoffkes HG, Schmidtke G, Uppenkamp M, Schmucker U. Multiparametric immunophenotyping of B cells in peripheral blood of healthy adults by flow cytometry. Clin Diagn Lab Immunol. 1996;3: Weinberg DS, Pinkus GS, Ault KA. Cytofluorometric detection of B cell clonal excess: a new approach to the diagnosis of B cell lymphoma. Blood. 1984;63: Braylan RC, Benson NA, Iturraspe J. Analysis of lymphomas by flow cytometry: current and emerging strategies. Ann NY Acad Sci. 1993;677: Letwin BW, Wallace PK, Muirhead KA, Hensler GL, Kashatus WH, Horan PK. An improved clonal excess assay using flow cytometry and B-cell gating. Blood. 1990;75: Cepek K, Wong D, Brenner M, Springer T. CD103 (α E ) cluster report. In: Schlossman S, Boumsell L, Gilks W, et al, eds. Leucocyte Typing V: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1995;2: Kruschwitz M, Fritzsche G, Schwarting R, et al. Ber- ACT8: new monoclonal antibody to the mucosa lymphocyte antigen. J Clin Pathol. 1991;44: Schwarting R, Stein H, Wang CY. The monoclonal antibodies as-hcl 1 (aleu-14) and as-hcl 3 (aleu-m5) allow the diagnosis of hairy cell leukemia. Blood. 1985;65: 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:

6 10. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI document M29-A 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: 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: Micklem KJ, Dong Y, Willis A, et al. HML-1 antigen on mucosa-associated T cells, activated cells, and hairy leukemic cells is a new integrin containing the beta 7 subunit. Am J Pathol. 1991;139: Parker CM, Cepek KL, Russell GJ, et al. A family of β 7 integrins on human mucosal lymphocytes. Proc Natl Acad Sci USA. 1992;89: Nadler LM. B Cell/Leukemia Panel Workshop: summary and comments. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human B Lymphocytes. New York, NY: Springer-Verlag; 1986;2: Dörken B, Möller P, Pezzutto A, Schwartz-Albiez R, Moldenhauer G. B-cell antigens: CD20. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. 1 ed. New York, NY: Oxford University Press; 1989: Tanaka T, Goda K, Takeuchi E, Miyasaka M. CD103 Workshop Panel report. In: Kishimoto T, Kikutani H, von dem Borne AEG, et al, eds. Leucocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing, Inc.; 1997: Loken MR, Shah V, Hollander Z, Civin C. Flow cytometric analysis of normal B lymphoid development. Pathol Immunopathol Res. 1988;7: Terstappen LWMM, Johnsen S, Segers-Nolten IMJ, Loken MR. Identification and characterization of plasma cells in normal human bone marrow by high resolution flow cytometry. Blood. 1990;76: Borowitz M, Bonsvaros A, Brynes R, et al. Monoclonal antibody phenotyping of B cell non- Hodgkins lymphoma: The Southeastern Cancer Study Group experience. Am J Pathol. 1985;121: Mason DY, Stein H, Gerdes J, et al. Values of monoclonal anti-cd22 (p135) antibodies for the detection of normal and neoplastic B lymphoid cells. Blood. 1987;69: Loken MR, Shah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development. Blood. 1987;70: Hultin LE, Hausner MA, Hultin PM, Giorgi JV. CD20 (Pan-B cell) antigen is expressed at a low level on a subpopulation of human T lymphocytes. Cytometry. 1993;14: