Anti-TdT (E ) IVD

Transcription

1 3/ IVD Anti-TdT (E ) Monoclonal mouse anti-human reagent for identification of cells expressing this enzymatic marker Form Catalog No. FITC PE APC 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 1. INTENDED USE Anti-terminal-deoxynucleotidyl transferase (TdT) is intended for in vitro diagnostic use in the identification of cells expressing this enzymatic marker, 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 for data acquisition and analysis. Refer to your instrument user s guide for instructions. Applications Expression of TdT in the characterization of hematologic neoplasia COMPOSITION Anti-TdT, clone E , is generated from the fusion of FO mouse myeloma cells with spleen cells from BALB/c mice immunized with purified TdT enzyme. Anti-TdT is composed of mouse IgG 1 heavy chains and kappa light chains. Each of the following reagents is supplied in phosphate-buffered saline (PBS) formulated as described in Table 1. Table 1 Bottling concentrations Form Amount provided Conc a (µg/ml) FITC PE APC a. Conc = concentration 12.5 µg in 1.0 ml of PBS with gelatin and 0.1% sodium azide 6 µg in 1.0 ml of PBS with gelatin and 0.1% sodium azide 12.5 µg in 0.5 ml of PBS with gelatin and 0.1% sodium azide bdbiosciences.com ClinicalApplications@bd.com 1

2 Antibody purity is as follows. FITC: 5% free fluorophore at bottling, as measured by sizeexclusion chromatography (SEC) PE, APC: 20% free fluorophore at bottling, as measured by 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 BD FACS lysing solution (10X) (Catalog No ). For dilution instructions and warnings, refer to the instructions for use (IFU). BD FACS permeabilizing solution 2 (Catalog No or ) Centrifuge Bovine serum albumin (BSA) * Falcon is a registered trademark of Corning Incorporated. BD CellWASH (Catalog No ) or a wash buffer of PBS with 0.1% sodium azide. BD FACS brand flow cytometer. Refer to the appropriate instrument user s guide for information. 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. 10,11 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. 10 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 1. Lyse 100 µl of whole blood with 1.0 ml of BD FACS lysing solution for 10 minutes. 2. Centrifuge and remove the supernatant. 2

3 3. Add 0.5 ml of BD FACS permeabilizing solution 2 and wait for 10 minutes. 4. Wash with 0.5% BSA in 1X PBS and 0.1% sodium azide. 5. Add the appropriate volume of Anti-TdT conjugated monoclonal antibody. 6. Mix thoroughly and incubate for 30 minutes in the dark at room temperature (20 C 25 C). 7. Wash with 1X PBS with 0.1% sodium azide, add 0.5 ml of 1% paraformaldehyde, mix thoroughly, and analyze. Store at 2 C 8 C until analyzed. We recommend analyzing within 24 hours of staining. 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. 14 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 a mixture of REH cells and leucocytes from 50 ml of whole blood and gated on lymphocytes (including REH cells). Laser excitation is at 488 nm and 635 nm. Figure 1Representative data analyzed with a BD FACS brand flow cytometer using BD FACS permeabilizing solution 2 FITC APC Internal Quality Control Cytometer setup depends on the cytometer you are using in your experiment. See the appropriate cytometer IFU for the correct setup method. 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-TdT recognizes a 60-kilodalton (kda) polymerase, a nuclear enzyme that catalyzes the template-independent addition of nucleotides to single-stranded DNA primers. 1-6,16 It has been reported that TdT is involved in the regulation or translocation or both of DNA and gene rearrangement during normal T- and B-cell development. 6,17-19 TdT is present in nuclei of immature T and B lymphocytes found in normal thymus and bone marrow. 20 TdT-positive cells represent 2% to 7% of bone marrow cells in neonates and children up to 5 years of age and 1% to 2% of normal adult bone marrow cells. 21,22 All PE 3

4 malignant lymphoblasts of precursor B and T cells of acute lymphoblastic lymphoma/leukemia (ALL) and cases of chronic myelogenous leukemia (CML) in lymphoid blast crisis express TdT. 7-9,23-28 Mature lymphocytes from normal peripheral blood or peripheral lymphoid tissue usually do not contain the enzyme. Sensitivity Sensitivity is defined as resolution of the TdT + population from the TdT population. Sensitivity was measured by evaluating a range of antibody concentrations. Each concentration of the reagent was tested on peripheral blood and REH cells. The separation of TdT + from TdT was determined for each sample and averaged within each concentration. The bottled antibody concentration for each reagent provided optimum sensitivity in resolving the TdT + cells from the negative. See Table 1. Reproducibility Several studies have shown the expression of TdT in essentially all cases of B- and T-cell lymphoblastic leukemia/ lymphoma. 7-9,26-28 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 REH cells 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 PE APC 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. 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. 11 4

5 Since 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 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. REFERENCES 1. Alt FW, Oltz EM, Young F, Gorman J, Taccioli G, Chen J. VDJ recombination. Immunol Today. 1992;13: Alt FW, Baltimore D. Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions. Proc Natl Acad Sci USA. 1982;79: Baltimore D. Is terminal deoxynucleotidyl transferase a somatic mutagen in lymphocytes? Nature. 1974;248: Desiderio SV, Yancopoulos GD, Paskind M, et al. Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells. Nature. 1984;311:

6 5. Landau NR, Schatz DG, Rosa M, Baltimore D. Increased frequency of N-region insertion in a murine pre B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector. Mol Cell Biol. 1987;7: Bonati A, Zanelli P, Ferrari S, et al. T-cell receptor beta-chain gene rearrangement and expression during human thymic ontogenesis. Blood. 1992;79: McCaffrey R, Smoler DF, Baltimore D. Terminal deoxynucleotidyl transferase in a case of childhood acute lymphoblastic leukemia. Proc Natl Acad Sci U S A. 1973;70: Murphy S, Jaffe ES. Terminal transferase activity and lymphoblastic neoplasms. N Engl J Med. 1984;311: Weiss LM, Bindl JM, Picozzi VJ, Link MP, Warnke RA. Lymphoblastic lymphoma: an immunophenotype study of 26 cases with comparison to T cell acute lymphoblastic leukemia. Blood. 1986;67: 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: Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline Second Edition. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI document H42-A Fuller SA, Phillips A, Coleman MS. Affinity purification and refined structural characterization of terminal deoxynucleotidyl transferase. Biochem J. 1985;231: Komori T, Okada A, Stewart V, Alt FW. Lack of N regions in antigen receptor variable regions of TdTdeficient lymphocytes. Science. 1993;261: Waldmann TA. The arrangement of immunoglobulin and T cell receptor genes in human lymphoproliferative disorders. Adv Immunol. 1987;40: Clevers H, Alarcón B, Wileman T, Terhorst C. The T cell receptor/cd3 complex: a dynamic protein ensemble. Ann Rev Immunol. 1988;6: Gore SD, Kastan MB, Civin CI. Normal human bone marrow precursors that express terminal deoxynucleotidyl transferase include T-cell precursors and possible lymphoid stem cells. Blood. 1991;77: Bearman RM, Winberg CD, Maslow WC, et al. Terminal deoxynucleotidyl transferase activity in neoplastic and nonneoplastic hematopoietic cells. Am J Clin Pathol. 1981;75: Muehleck SD, McKenna RW, Gale PF, Brunning RD. Terminal deoxynucleotidyl transferase (TdT)- positive cells in bone marrow in the absence of hematologic malignancy. Am J Clin Pathol. 1983;79: Horvatinovich JM, Sparks SD, Borowitz MJ. Detection of terminal deoxynucleotidyl transferase by flow cytometry: a three color method. Cytometry. 1994;18: Paietta E, Meenan B, Heavey C, Thomas D. Detection of terminal transferase in acute myeloid leukemia by flow cytometry. Cytometry. 1994;16: Roma AO, Kutok JL, Shaheen G, Dorfman DM. A novel, rapid, multiparametric approach for flow cytometric analysis of intranuclear terminal deoxynucleotidyl transferase. Am J Clin Pathol. 1999;112: Bollum FJ. Terminal deoxynucleotidyl transferase as a hematopoietic cell marker. Blood. 1979;54: McCaffrey R, Harrison TA, Parkman R, Baltimore D. Terminal deoxynucleotidyl transferase activity in human leukemic cells and in normal human thymocytes. N Engl J Med. 1975;292:

7 28. Kung PC, Long JC, McCaffrey RP, Ratliff RL, Harrison TA, Baltimore D. Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma. Am J Med. 1978;64: