Anti-Kappa (TB 28-2) IVD

Transcription

1 10/ IVD Anti-Kappa (TB 28-2) Monoclonal mouse anti-human reagent for identification of cells expressing kappa light chains Form Catalog No. FITC PE APC 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-Kappa is intended for in vitro diagnostic use in the identification of cells expressing kappa 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 CellQuest, BD CellQuest Pro, or BD FACSDiva software) for data acquisition and analysis. See your instrument user s guide for instructions. Applications Expression of kappa light chains in the characterization of hematologic neoplasia 1 2. COMPOSITION Anti-Kappa, clone TB 28-2, * is derived from hybridization of mouse P3-X63- Ag8.653 myeloma cells with cells from CB6 (BC57b x BALB/c) mice immunized with human IgG-κ myeloma protein. Anti-Kappa is composed of mouse IgG 1 heavy chains and kappa light chains. 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 25 µg in 1.0 ml PBS 25 PE 12.5 µg in 1.0 ml PBS 12.5 APC 25 µg in 0.5 ml PBS 50 a. Conc = concentration bdbiosciences.com ClinicalApplications@bd.com * This clone has not been submitted to any previous Workshop on Human Leucocyte Differentiation Antigens. 1

2 Antibody purity is as follows. FITC: 5% free fluorophore at bottling, as measured by size-exclusion 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, see 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. 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 these reagents, 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-Kappa fluorochrome-conjugated 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 for 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 lymphocytes. Laser excitation is at 488 nm and 635 nm. 3

4 Figure 1 Representative data analyzed using a BD FACS brand flow cytometer and gated on CD19 + CD19 PerCP-Cy5.5-A CD19 PerCP-Cy5.5-A CD19 PerCP-Cy5.5-A Anti-Kappa FITC-A Anti-Kappa PE-A Anti-Kappa APC-A 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-Kappa antibody is specific for kappa light chains of human immunoglobulins. Immunoglobulins bearing kappa light chains are present on approximately 60% of normal B lymphocytes and on Igκ + leukemic cells In serum, Anti-Kappa reacts with immunoglobulins bearing kappa light chains as well as free kappa light chains. Sensitivity Sensitivity is defined as resolution of the Anti-Kappa + population from the Anti-Kappa population. Sensitivity was measured by evaluating a range of antibody concentrations. Each concentration of reagent was tested on lymphocytes. The separation of Anti-Kappa + from Anti-Kappa was determined for each sample and averaged within each concentration. The bottled antibody concentration for each reagent provided optimum sensitivity in resolving 4

5 the Anti-Kappa + cells from the negative. See Table 1. Reproducibility The Anti-Kappa clone, TB 28-2, has been used in studies to determine that the normal level of the Kappa-positive population within the B-cell subset is ~60%. 1,18 This clone produces consistent results within normal populations across sites and has been used to demonstrate clonality in multiple studies. 1,14,18-22 Furthermore, Kappa/Lambda ratios produced using the TB 28-2 clone are consistent with historic Kappa/Lambda ratios Consistent results across multiple studies are indications of reproducibility of the clone. 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 FACSCanto II flow cytometer. 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 coefficient of variation (CV), excluding the day-to-day 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 FITC When the day-to-day component was included, the reproducibility of the reagent was 20.6 for the averages with an SD of 7.3 and a %CV of 35.5 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 day-to-day 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 FITC 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 FACSCanto II flow cytometer. The components of repeatability include tube-to-tube (intra-assay precision). The average S/N ratio, SD, and CV are provided in Table 4. Table 4 Intra-assay precision of the assay without day-to-day component (S/N ratio) Average df a S/N Ratio SD %CV FITC a. df = degrees of freedom a. df = degrees of freedom 5

6 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 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 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. 6

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