BD Oncomark CD15/CD117

Transcription

1 2/ IVD BD Oncomark CD15/CD117 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 CD15 FITC/CD117 PE is intended for in vitro flow cytometric immunophenotyping. CD15/CD117 assays are used in the diagnosis of hematologic disorders COMPOSITION CD15, clone MMA, is derived from the hybridization of mouse P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with the U-937 histiocytic cell line. CD117, clone 104D2, is derived from fusion of Sp2/0 myeloma cells with splenocytes from BALB/c mice immunized with the megakaryoctic cell line MOLM-1. 8,9 CD15 is composed of mouse IgM heavy chains and kappa light chains. CD117 is composed of mouse IgG 1 heavy chains and kappa light chains. This reagent is supplied as a combination of CD15 FITC and CD117 PE, in 1 ml of phosphate-buffered saline (PBS) containing gelatin and 0.1% sodium azide. Antibody purity is as follows. FITC, PE: 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. 1

2 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 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 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 CD15 FITC/CD117 PE can be used for immunophenotyping by flow cytometry with peripheral blood and bone marrow aspirates collected in * Falcon is a registered trademark of Corning Incorporated. BD Vacutainer EDTA tubes. Each type of specimen can have different storage conditions and limitations that should be considered prior to collection and analysis. 10,11 WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 12,13 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. 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. 10 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 CD15/ CD117 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. 2

3 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 quadrants Dot plots displaying region R1 and 6. Determine antigen expression based on the sample negative population. 7. PERFORMANCE CHARACTERISTICS Specificity CD15 recognizes the human myelomonocytic antigen lacto-nfucopentose III. 15 CD117 binds to the receptor for stem cell factor (SCF-R) It selectively recognizes NIH-3T3 cells transfected with human c-kit, the gene that codes for SCF- R. Antigen Distribution The CD15 antigen, present on greater than 95% of mature peripheral blood eosinophils and neutrophils, is present at low density on circulating monocytes. 23 The CD117 antigen (SCF-R) is expressed primarily on hematopoietic progenitor cells in the bone marrow. The receptor is highly expressed at similar levels on primitive, erythroid, and granulomonocytic CD34 + cell subsets, whereas B-lymphoid committed progenitor cells (CD34 + CD19 + ) express low levels of SCF-R. 21 Among CD34 bone marrow cells, only a small number of cells (mostly erythroid) express the receptor. 21 3

4 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. 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. Schwartz S, Heinecke A, Zimmermann M, et al. Expression of the C-kit receptor (CD117) is a feature of almost all subtypes of de novo acute myeloblastic leukemia (AML), including cytogenetically good-risk AML, and lacks prognostic significance. Leuk Lymphoma. 1999;34: Cascavilla N, Musto P, D'Arena G, et al. CD117 (c-kit) is a restricted antigen of acute myeloid leukemia and characterizes early differentiative levels of M5 FAB subtype. Haematologica. 1998;83: Macedo A, Orfao A, Gonzalez M, et al. Immunological detection of blast cell subpopulations in acute myeloblastic leukemia at diagnosis: implications for minimal residual disease studies. Leukemia. 1995;9: Wells SJ, Bray RA, Stempora LL, Farhi DC. CD117/ CD34 expression in leukemic blasts. Am J Clin Pathol. 1996;106:

5 5. Terstappen LWMM, Safford M, Könemann S, et al. Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis. Leukemia. 1992;6: Cohen PL, Hoyer JD, Kurtin PJ, Dewald GW, Hanson CA. Acute myeloid leukemia with minimal differentiation. A multiple parameter study [see comments]. Am J Clin Pathol. 1998;109: Sun T, Sangaline R, Ryder J, et al. Gating strategy for immunophenotyping of leukemia and lymphoma. Am J Clin Pathol. 1997;108: Matsuo Y, Adachi T, Tsubota T, Imanishi J, Minowada J. Establishment and characterization of a novel megakaryoblastoid cell line, MOLM-1, from a patient with chronic myelogenous leukemia. Human Cell. 1991;4: Ashman LK, Cambareri A, Nguyen L, Bühring H-J. CD117 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: 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-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: Skubitz K, Balke J, Ball E, et al. Report on the CD15 cluster workshop. In: Knapp W, Dörken B, Gilks W, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT. Functional isolation and characterization of human hematopoietic stem cells. Science. 1995;267: Gunji Y, Nakamura M, Osawa H, et al. Human primitive hematopoietic progenitor cells are more enriched in KIT low cells than in KIT high cells. Blood. 1993;82: Broudy VC, Lin N, Zsebo KM, et al. Isolation and characterization of a monoclonal antibody that recognizes the human c-kit receptor. Blood. 1992;79: Briddell RA, Broudy VC, Bruno E, Brandt JE, Srou EF, Hoffman R. Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a monoclonal antibody to the c-kit receptor. Blood. 1992;79: Simmons P, Aylett G, Niutta S, To L, Juttner C, Ashman L. c-kit is expressed by primitive human hematopoietic cells that give rise to colony-forming cells in stroma-dependent or cytokine-supplemented culture. Exp Hematol. 1994;22: Olweus J, Terstappen LWMM, Thompson PA, Lund-Johansen F. Expression and function of receptors for stem cell factor and erythropoietin during lineage commitment of human hematopoietic progenitor cells. Blood. 1996;88: Yarden Y, Kuang W-J, Yang-Feng T, et al. Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 1987;6: Hanjan SN, Kearney JF, Cooper MD. A monoclonal antibody (MMA) that identifies a differentiation antigen on human myelomonocytic cells. Clin Immunol Immunopathol. 1982;23: