Anti-Lambda ( )

Transcription

1 Anti-Lambda ( ) Monoclonal mouse anti-human reagent for identification of cells expressing lambda light chains 10/ IVD Form Catalog No. FITC PE Not for sale in the US 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-Lambda is intended for in vitro diagnostic use in the identification of cells expressing lambda light chains, 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 FACSDiva, BD CellQuest Pro, or BD CellQuest software) for data acquisition and analysis. See your instrument user s guide for instructions. Applications Expression of lambda light chains in the characterization of hematologic neoplasia 1 2. COMPOSITION Anti-Lambda, clone , * is derived from hybridization of mouse P3-X63- Ag8.653 myeloma cells with cells from BALB/cJ mice immunized with human IgA 1 -λ myeloma protein. Anti-Lambda is composed of mouse IgG 1 heavy chains and lambda light chains. The FITC reagent is supplied in phosphate-buffered saline (PBS) containing gelatin and 0.1% sodium azide. The PE reagent is supplied in buffer containing bovine serum albumin (BSA) and 0.1% sodium azide. Concentrations are listed in Table 1. Table 1 Bottling concentrations Form Amount provided Conc a (µg/ml) FITC 12.5 µg in 1.0 ml of PBS 12.5 PE 12.5 µg in 1.0 ml of buffer 12.5 bdbiosciences.com ClinicalApplications@bd.com * This clone has not been submitted to any previous Workshop on Human Leucocyte Differentiation Antigens. 1

2 a. Conc = concentration Antibody purity is as follows. FITC: 5% free fluorophore at bottling, as measured by size-exclusion chromatography (SEC) PE: 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 Centrifuge BD FACS lysing solution (10X) (Catalog No ) For dilution instructions and warnings, see the instructions for use (IFU). 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 See 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. 2,3 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. 2 WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 4,5 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. Falcon is a registered trademark of Corning Incorporated. 2

3 6. PROCEDURE 1. To avoid serum interference when using this reagent, prewash the whole blood 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. 2. Pellet cells by centrifugation and resuspend in 1X PBS with 0.1% sodium azide to the original volume. 3. Add the appropriate volume of Anti-Lambda fluorochromeconjugated monoclonal antibody to 100 µl of cell suspension in a 12 x 75-mm tube. See the vial label for volume. 4. Vortex gently and incubate 15 to 30 minutes in the dark at room temperature (20 C 25 C). 5. Add 2 ml of 1X BD FACS lysing solution. 6. Vortex gently and incubate for 10 minutes in the dark at room temperature. 7. Centrifuge at 300g for 5 minutes. Remove the supernatant. 8. Add 2 to 3 ml of BD CellWASH solution (or wash buffer) and centrifuge at 200g for 5 minutes. Remove the supernatant. 9. 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. 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 to perform appropriate analysis. Flow Cytometry Vortex the cells thoroughly at low speed to reduce aggregation before running them on the flow cytometer. 6 Acquire and analyze list-mode data using appropriate software. Before acquiring samples, adjust the threshold to minimize debris and ensure that populations of interest are included. Figure 1 displays representative data from pre-washed normal peripheral blood and gated on CD20 + B cells. Laser excitation is at 488 nm. 3

4 Figure 1 Representative data analyzed using a BD FACS brand flow cytometer CD20 PerCP-Cy5.5 CD20 PerCP-Cy5.5 Anti-Lambda FITC Anti-Lambda PE Internal Quality Control To set photomultiplier tube (PMT) voltages, fluorescence compensation, and to check instrument sensitivity before acquisition, use one of the following combinations: For BD FACSCanto and BD FACSCanto II cytometer setup, use BD FACS 7-color setup beads and BD FACSCanto clinical software. See the IFU and instrument user s guide. For BD FACSCalibur cytometer setup, use BD Calibrite beads and BD FACSComp software. See the IFU and instrument 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 The Anti-Lambda antibody is specific for lambda light chains of human immunoglobulins. 8 Immunoglobulins bearing lambda light chains are present on approximately 40% of normal B lymphocytes and on Igλ + leukemic cells In serum, Anti-Lambda reacts with immunoglobulins bearing lambda light chains as well as free lambda light chains. Sensitivity Sensitivity is defined as resolution of the Anti-Lambda + population from the Anti-Lambda population. Sensitivity was measured by evaluating a range of antibody concentrations. Each concentration of reagent was tested on lymphocytes. The separation of Anti- Lambda + from Anti-Lambda was determined for each sample and averaged within each concentration. The bottled antibody concentration for each reagent provided optimum sensitivity in resolving the Anti-Lambda + cells from the negative. See Table 1. Reproducibility Estimates of assay reproducibility were determined at BD Biosciences using peripheral whole blood samples stained in duplicate with the reagent and acquired on a BD FACSCalibur flow cytometer. 4

5 The components of reproducibility include instrument-to-instrument, operator-to-operator, run-to-run, day-today, and lot-to-lot (inter-assay precision). The average signal to noise (S/N) ratio, standard deviation (SD), and coefficients of variation (CVs), excluding the day-today component, are provided in Table 2. Table 2 Inter-assay precision of the assay without day-to-day component (S/N ratio) Average df a S/N ratio SD %CV PE a. df = degrees of freedom When the day-to-day component was included, the reproducibility of the reagent was 86.1 for the averages with an SD of 33.9 and a %CV of 39.4 due to the degradation of the target molecule in whole blood. However, when the reproducibility of the assay for the percent positive (% positive) was determined, the inter-assay precision was acceptable (Table 3). Table 3 Inter-assay precision of the assay with day-to-day component (% positive) Average % df a positive SD %CV PE a. df = degrees of freedom Repeatability Estimates of assay repeatability were determined at BD Biosciences using peripheral whole blood samples stained in duplicate with the reagent and acquired on a BD FACSCalibur flow cytometer. The components of repeatability include tube-to-tube (intra-assay precision). The average S/N ratio, SD, and CVs are provided in Table 4. Table 4 Intra-assay precision of the assay Average df a S/N Ratio SD %CV PE a. df = degree of freedom 8. 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. 3 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. 5

6 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) 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. 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. Fukushima PI, Nguyen PKT, O'Grady P, Stetler- Stevenson M. Flow cytometric analysis of Kappa and Lambda light chain expression in evaluation of specimens for B-cell neoplasia. Cytometry. 1996;26: 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; M29-A3. 5. 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; H42-A2. 8. Kubagawa H, Gathings WE, Levitt D, Kearney JF, Cooper MD. Immunoglobulin isotype expression of normal pre-b cells as determined by immunofluorescence. J Clin Immunol. 1982;2:

7 9. Ault KA. Flow cytometric evaluation of normal and neoplastic B cells. In: Rose NR, Friedman H, Fahey JL, eds. Manual of Clinical Laboratory Immunology. Washington, DC: American Society for Microbiology; 1986: 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: Têtu B, Manning JT Jr, Ordóñez NG. Comparison of monoclonal and polyclonal antibodies directed against immunoglobulin light and heavy chains in non-hodgkin's lymphoma. Am J Clin Pathol. 1986;85: Harris NL, Data RE. The distribution of neoplastic and normal B-lymphoid cells in nodular lymphomas: use of an immunoperoxidase technique on frozen sections. Hum Pathol. 1982;13: Meis JM, Osborne BM, Butler JJ. A comparative marker study of large cell lymphoma, Hodgkin's disease, and true histiocytic lymphoma in paraffinembedded tissue. Am J Clin Pathol. 1986;86: Picker LJ, Weiss LM, Medeiros LJ, Wood GS, Warnke RA. Immunophenotypic criteria for the diagnosis of non-hodgkin's lymphoma. Am J Pathol. 1987;128: Foon KA, Todd RF III. Immunologic classification of leukemia and lymphoma. Blood. 1986;68: Tubbs RR, Sheibani K, Weiss RA, Sebek BA, Deodhar SD. Tissue immunomicroscopic evaluation of monoclonality of B-cell lymphomas: comparison with cell suspension studies. Am J Clin Pathol. 1981;76: Smith BR, Weinberg DS, Robert NJ, et al. Circulating monoclonal B lymphocytes in non- Hodgkin's lymphoma. N Engl J Med. 1984;311: