Anti TCR-α/β (WT31) IVD

Transcription

1 11/ IVD Anti TCR-α/β (WT31) Monoclonal mouse anti-human reagent for identification of cells expressing TCR-α/β antigen Form Catalog No. FITC 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 Anti TCR-α/β is intended for in vitro diagnostic use in the identification of cells expressing TCR-α/β antigen, using a BD FACS brand flow cytometer. The flow cytometer must be equipped to detect light scatter and the appropriate fluorescence, and be equipped with appropriate analysis software (such as BD CellQuest or BD LYSYS II software) for data acquisition and analysis. Refer to your instrument user s guide for instructions. Applications Expression of TCR-α/β antigen in the characterization of hematologic neoplasia 1,2 2. COMPOSITION Anti TCR-α/β, clone WT31, is derived from hybridization of mouse Sp2/0-Ag14 myeloma cells with spleen cells from BALB/c mice immunized with human thymocytes. 3 Anti TCR-α/β is composed of mouse IgG 1 heavy chains and kappa light chains. 3 Each reagent is supplied in phosphatebuffered saline (PBS) containing gelatin and 0.1% sodium azide. Concentrations are listed in Table 1. Table 1 Bottling concentrations Form Amount provided Conc a (µg/ml) FITC 100 µg in 2.0 ml of PBS 50 a. Conc = concentration Antibody purity is as follows. FITC: 5% free fluorophore at bottling, as measured by size-exclusion chromatography (SEC) 1

2 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 CD3 PE (Catalog No ) 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. BD FACS brand flow cytometer. Refer to the appropriate instrument user s guide for information. * Falcon is a registered trademark of Corning Incorporated. 5. SPECIMEN(S) Reagents can be used for immunophenotyping by flow cytometry with a variety of specimen types, including peripheral blood, bone marrow aspirates or biopsies, and other body fluids or tissues. Each type of specimen can have different storage conditions and limitations that should be considered prior to collection and analysis. 4,5 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. Viability of samples should be assessed and a cut-off value established. A cut-off value of at least 80% viable cells has been suggested. 4 WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 6,7 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 1. In a 12 x 75-mm tube, add 20 µl of reagent to 10 6 peripheral blood mononuclear cells (PBMCs) in 50 µl of medium containing 0.1% sodium azide. 2. Mix thoroughly and incubate for 15 minutes in the dark at 2 C 8 C. 3. Add 20 µl of CD3 PE. 4. Mix thoroughly and incubate again for 15 minutes at 2 C 8 C. 5. Wash with BD CellWASH solution (or wash buffer). 2

3 6. Add 0.5 ml of BD CellFIX solution (or 1% paraformaldehyde solution) and mix thoroughly. Store at 2 C 8 C until analyzed. We recommend analyzing within 24 hours of staining. CAUTION Anti TCR-α/β is partially inhibited by the binding of antibodies against the CD3 antigen. 8 Anti TCR-α/β FITC and Anti-CD3, clone SK7, PE at the supplied concentrations have been tested and are recommended for two-color immunofluorescence studies. Problems can result from the use of other antibodies directed against the CD3 antigen when used in conjunction with Anti TCR-α/β. The reciprocal combination, Anti-CD3 FITC and Anti TCR-α/β Biotin with a second-step reagent, is likewise not recommended due to steric hindrance considerations. Since the TCR-1 antigen is labile in tissue section preparations, the performance of Anti TCR-α/β in immunohistologic applications is suboptimal. Analytical Results Abnormal numbers of cells expressing this antigen or aberrant expression levels of the antigen can be expected in some disease states. It is important to understand the normal expression pattern for this antigen and its relationship to expression of other relevant antigens in order to perform appropriate analysis. Flow Cytometry Vortex the cells thoroughly at low speed to reduce aggregation before running them on the flow cytometer. 9 Acquire and analyze list-mode data using appropriate software. Before acquiring samples, adjust the threshold to minimize debris and ensure populations of interest are included. Figure 1 displays representative data performed on normal blood and gated on lymphocytes. Laser excitation is at 488 nm. Figure 1 Representative data analyzed with a BD FACS brand flow cytometer Number of Cells Mouse IgG1 FITC 69% Anti-TCR-α/β FITC Internal Quality Control We recommend using BD Calibrite beads and BD FACSComp software to set photomultiplier tube (PMT) voltages, fluorescence compensation, and to check instrument sensitivity prior to use. Refer to the BD Calibrite Beads instructions for use (IFU) and the BD FACSComp Software User s Guide. We recommend running 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 PERFORMANCE CHARACTERISTICS Specificity Anti TCR-α/β recognizes a conformational epitope formed by the T-cell receptor (TCR) for antigen and the CD3 epsilon chain. 8,11 The α/β TCR is a disulfide-linked 80-kilodalton (kda) heterodimer consisting of a 44-kDa α chain and a 37-kDa β chain. 8,12 3

4 WT31 (Anti TCR-α/β) identifies approximately 97% of normal peripheral blood T lymphocytes that express the CD3 antigen. 13 The TCR-α/β antigen is also expressed on 50% to 70% of thymocytes. 13 A small subset of normal peripheral blood T lymphocytes (1% to 9%) and thymocytes (less than 2%) lacks reactivity or reacts weakly with Anti TCR-α/β. 3,13,14 This CD3 + TCR-α/β subset predominantly expresses the γ/δ chains of the TCR complex. 3,13,14 NOTE The weak reactivity described is caused by WT31 recognizing a conformational epitope that exists when γ or δ subunits are associated with the CD3ε chain. 11 Figure 2 (plots A and B) show the effects of the absence or presence of CD3 on WT31 and TCR-γ/δ staining patterns. In cases where a CD3ε transfectant (or other system) is employed, the WT31 antibody might fail to detect the presence of α/β subunits. Anti TCR-α/β is mitogenic for resting peripheral blood T lymphocytes from responding individuals Figure 2 Two-parameter analysis of lysed whole blood stained with WT31 (Anti TCR-α/β-1) FITC and TCR-γ/δ PE TCR-γ/δ PE Plot A WT31 FITC TCR-γ/δ PE Plot B WT31 FITC Sensitivity Sensitivity is defined as resolution of the TCR-α/β + population from the TCR-α/β population. Sensitivity was measured by evaluating a range of antibody concentrations. Each concentration of reagent was tested on PBMCs. The separation of TCR-α/β + from TCR-α/β was determined for each sample and averaged within each concentration. The bottled antibody concentration for each reagent provided optimum sensitivity in resolving the TCR-α/β + cells from the negative. See Table 1. Repeatability To determine the repeatability of staining with each reagent, samples were stained with multiple lots of reagents. The different samples used in the evaluation provided an average mean fluorescence intensity (MFI) value as shown in Table 2. For each sample, two different lots of reagents generated a pair of results. Individual standard deviations (SDs) were determined from the paired results for each sample. Individual SDs were combined to derive a pooled SD for each reagent that provides an estimate of within-sample repeatability. Table 2 Repeatability of MFI of Anti TCR-α/β + lymphocytes across different lots and across multiple donors (N) N a a. N = number of samples b. CV = coefficient of variation Average MFI Pooled SD Pooled %CV b FITC LIMITATIONS Conjugates with brighter fluorochromes (PE, APC) will give greater separation than those with other dyes (FITC, PerCP). When populations overlap, calculation of the percentage of cells positive for the marker can be affected by the choice of fluorochrome. 4

5 Use of 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. Single reagents can provide only limited information in the analysis of leukemias and lymphomas. Using combinations of reagents can provide more information than using the reagents individually. Multicolor analysis using relevant combinations of reagents is highly recommended. 5 As reagents can be used in different combinations, laboratories need to become familiar with the properties of each antibody in conjunction with other markers in normal and abnormal samples. Reagent performance data was collected typically with EDTA-treated blood. Reagent performance can be affected by the use of other anticoagulants. 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 Improper medium preparation (sodium azide omitted) 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. Use sodium azide in staining medium and washing steps. Resuspend fresh sample at a higher concentration; repeat staining and analysis. Troubleshoot instrument. 1. Spits H, Paliard X, Vandekerckhove Y, van Vlasselaer P, de Vries JE. Functional and phenotypic differences between CD4+ and CD4 T cell receptorgamma delta clones from peripheral blood. J Immunol. 1991;147: Knowles DM. Immunophenotypic markers useful in the diagnosis and classification of hematopoietic neoplasms. In: Knowles DM, Thompson DD, eds. Neoplastic Hematopathology. 2nd ed. Philadelphia: Lippincott Williams & Wilkins; 2001: Weiss A, Newton M, Crommie D. Expression of T3 in association with a molecule distinct from the T cell antigen receptor heterodimer. Proc Natl Acad Sci USA. 1986;83:

6 4. 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. 7. 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: Spits H, Borst J, Tax W, Capel P, Terhorst C, de Vries J. Characteristics of a monoclonal antibody (WT-31) that recognizes a common epitope on the human T cell receptor for antigen. J Immunol. 1985;135: 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: Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline Second Edition. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI document H42-A Salmerón A, Sanchez-Madrid F, Ursa M, Fresno M, Alarcón B. A conformational eptitope expressed upon association of CD3ε with either CD3-δ or CD3-γ is the main target for recognition by Anti-CD3 monoclonal antibodies. J Immunol. 1991;147: Oettgen H, Kappler J, Tax W, Terhorst C. Characterization of the two heavy chains of the T3 complex on the surface of human T lymphocytes. J Biol Chem. 1984;259: Lanier LL, Weiss A. Presence of Ti (WT31) negative T lymphocytes in peripheral blood and thymus. Nature. 1986;324: Brenner MB, McClean J, Dialynas DP, et al. Identification of a putative second T cell receptor. Nature. 1986;322: Gupta S, Shimizu M, Ohira K, Vayuvegula B. T-cell activation via the T-cell receptor: a comparison between WT31 (defining alpha/beta TCR)-induced and anti-cd3-induced activation of human T lymphocytes. Cell Immunol. 1991;132: Tax W, Leeuwenberg H, Willems H, Capel P, Koene R. Monoclonal antibodies reactive with OKT3 antigen or OKT8 antigen. In: Bernard A, Boumsell L, Dausset J, Milstein C, Schlossman S, eds. Leucocyte Typing. New York, NY: Springer- Verlag; 1984: Tax WJ, Willems HW, Reekers PP, Capel PJ, Koene RA. Polymorphism in mitogenic effect of IgG 1 monoclonal antibodies against T3 antigen on human T cells. Nature. 1983;304: