BD Oncomark CD45/GlyA/CD41a
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1 2/ IVD BD Oncomark CD45/GlyA/CD41a 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 CD45 FITC/Anti glycophorin A PE/CD41a PerCP- Cy 5.5 * is intended for in vitro flow cytometric immunophenotyping of the development in the erythroid, leucocytic, and megakaryocytic cell lineages in peripheral blood and bone marrow. Megakaryocytes co-express the plateletspecific glycoprotein gpiib/iiia (CD41a) and CD45 throughout their differentiation. 1 In contrast, glycophorin A expression is present during erythroid cell development, with a gradual decrease in CD45 intensity during erythroid maturation. Using this approach, erythroid and lymphoid cells can be clearly distinguished based upon their antigenic expression. 2 CD45/Anti glycophorin A/CD41a assays are used in the diagnosis of hematologic disorders. 1,2 2. COMPOSITION CD45, clone 2D1, is derived from the hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood mononuclear cells (PBMCs). Anti glycophorin A is clone GAR- 2(HIR-2). * 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
2 CD41a, clone HIP8, is derived from the hybridization of mouse P3X63 myeloma cells with spleen cells from BALB/c mice immunized with purified platelet membrane glycoproteins. CD45 and CD41a are each composed of mouse IgG 1 heavy chains and kappa light chains. Anti glycophorin A is composed of mouse IgG 2b heavy chains and kappa light chains. This reagent is supplied as a combination of CD45 FITC, Anti glycophorin A PE, and CD41a PerCP-Cy5.5 in 1 ml of phosphate-buffered 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 Falcon is a registered trademark of Corning Incorporated. Micropipettor with tips Vortex mixer 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 CD45 FITC/Anti glycophorin A PE/CD41a 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. 3,4 WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 5,6 and dispose of with proper precautions in 2
3 accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, 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. 3 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. CAUTION The Anti glycophorin A antibody concentration is appropriate for staining samples containing mature red blood cells. If these cells are removed from the sample before staining, the remaining nucleated, immature red cells will stain too brightly. If this occurs, it is necessary to reoptimize the reagent concentration or instrument settings. 1. Add 20 µl of BD Oncomark CD45/ Anti glycophorin A/CD41a 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). 3
4 Figure 1 quadrants Dot plots displaying region R1 and 6. Determine antigen expression based on the sample negative population. 7. PERFORMANCE CHARACTERISTICS Specificity CD45 (Anti HLe-1) recognizes human leucocyte antigens, with molecular weights from 180 to 220 kilodaltons (kda), that are members of the T200 family. 8 Anti glycophorin A reacts with a major sialoglycoprotein present on the human erythrocyte membrane. 9 Glycophorin A is a transmembrane dimeric complex of 31 kda with its caboxyterminal ends extending into the cytoplasm of the red cell. 10 CD41a recognizes the calcium-dependent gpiib/iiia complex, 140 kda. 11 Antigen Distribution The CD45 antigen is present on all human leucocytes, including lymphocytes, monocytes, granulocytes, eosinophils, and basophils in peripheral blood and has a role in signal transduction, modifying signals from other surface molecules. 8 The CD45 antibody has been reported to react weakly with mature circulating erythrocytes and platelets. 8,12 Glycophorin A is expressed on human red blood cells, normoblasts, and erythroid precursor cells. 13 It is also found on erythroid leukemias and some CD34 + M7 megakaryoblastic leukemias Mature non-nucleated red blood cells are characteristically glycophorin A positive but CD45 negative. 17 Glycophorin A was reported to serve as a receptor for several infectious disease agents The CD41a antigen (gpiib/iiia complex) is found on platelets and platelet precursors. It acts as a receptor for fibrinogen, von Willebrand factor (vwf), fibronectin, and vitronectin, and it mediates platelet adhesion and aggregation 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. 4
5 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. TROUBLESHOOTING Problem Possible Cause Solution Poor resolution between debris and lymphocytes Cell interaction with other cells and platelets Rough handling of cell preparation Inappropriate instrument settings Prepare and stain another sample. Check cell viability; centrifuge cells at lower speed. Follow proper instrument setup procedures; optimize instrument settings as required. Problem Possible Cause Solution Staining dim or fading REFERENCES 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 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. Qiao X, Loudovaris M, Unverzagt K, et al. Immunocytochemistry and flow cytometry evaluation of human megakaryocytes in fresh samples and cultures of CD34+ cells. Cytometry. 1996;23: Loken MR, Shah VO, Dattilio KL, Civin CI. Flow cytometric analysis of human bone marrow. I. Normal erythroid development. Blood. 1987;69: 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; Approved Guideline Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI document M29-A3. 6. 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:
6 7. 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: Schwinzer R. Cluster report: CD45/CD45R. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: Gahmberg CG, Jokinen M, Andersson LC. Expression of the major sialoglycoprotein (glycophorin) on erythroid cells in human bone marrow. Blood. 1978;52: Wise GE, Oakford LX, Dzandu JK. Ultrastructure of a transmembrane glycoprotein, glycophorin A. Tissue & Cell. 1988;20: von dem Borne AEGKr, Modderman PW, Admiraal LG, Nieuwenhuis HK. Platelet antibodies, the overall results. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: Jackson A. Basic phenotyping of lymphocytes: selection and testing of reagents and interpretation of data. Clin Immunol Newslett. 1990;10: Rogers CE, Bradley MS, Palsson BO, Koller MR. Flow cytometric analysis of human bone marrow perfusion cultures: erythroid development and relationship with burst-forming units-erythroid. Exp Hematol. 1996;24: Bain B, Catovsky D. Current concerns in haematology 2: classification of acute leukaemia. In: J Clin Pathol. 1990;43: Helleberg C, Knudsen H, Hansen PB, et al. CD34 + megakaryoblastic leukaemic cells are CD38, but CD61 + and glycophorin A + : improved criteria for diagnosis of AML-M7? Leukemia. 1997;11: Bain BJ. Immunological markers in acute myeloid leukaemia. In: Leukaemia Diagnosis: A Guide to the FAB Classification. London: Gower Medical Publishing; 1990: de Vries E, De Bruin-Versteeg S, Comans- Bitter WM, et al. Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes. Arch Dis Child Fetal Neonatal Ed. 1999;80: Jokinen M, Ehnholm C, Väisänen-Rhen V, et al. Identification of the major human sialoglycoprotein from red cells, glycophorin A M, as the receptor for Escherichia coli IH and characterization of the receptor site. Eur J Biochem. 1985;147: Templeton TJ, Keister DB, Muratova O, Procter JL, Kaslow DC. Adherence of erythrocytes during exflagellation of plasmodium falciparum microgametes is dependent on erythrocyte surface sialic acid and glycophorins. J Exp Med. 1998;187: Wybenga LE, Epand RF, Nir S, et al. Glycophorin as a receptor for sendai virus. Biochem. 1996;35: de Haas M, von dem Borne A. CD41/CD61 Workshop Panel report. In: Kishimoto T, Kikutani H, von dem Borne AEGK, et al, eds. Leucocyte Typing VI: White Cell Differentiation Antigens. New York, NY: Garland Publishing, Inc.; 1997: Feng R, Shimazaki C, Inaba T, et al. CD34 + /CD41a + cells best predict platelet recovery after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant. 1998;21: Jennings LK, Ashmun RA, Wang WC, Dockter ME. Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry. Blood. 1986;68:
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