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iii CONTENTS1. INTENDED USE............................................................................52. SUMMARY AND EXPLANATION................................................5Clinical Applications....................................................................73. PRINCIPLES OF THE PROCEDURE................................................84. REAGENTS.................................................................................11Reagents Provided, Sufficient for 50 Tests..................................11Precautions.................................................................................175. INSTRUMENT.............................................................................186. SPECIMEN COLLECTION AND PREPARATION..........................19Interfering Conditions................................................................207. PROCEDURE..............................................................................20Reagents Provided......................................................................20Reagents and Materials Required But Not Provided...................20Staining and Fixing the Cells......................................................21Flow Cytometry..........................................................................23Quality Control..........................................................................288. RESULTS.....................................................................................30Percent Lymphocyte Conversion.................................................30Three-Part Differential................................................................33Absolute Counts.........................................................................349. LIMITATIONS.............................................................................35 iv 10. EXPECTED VALUES...................................................................39Leucocyte Subsets.......................................................................39Absolute Counts.........................................................................4111. PERFORMANCE CHARACTERISTICS..........................................43Within-Sample Reproducibility...................................................43Between-Instrument Reproducibility...........................................44Between-Laboratory Reproducibility..........................................46Simultest IMK-Lymphocyte vs Comparative Method.................46Stability of Stained Cell Preparations..........................................47Cross-Reactivity.........................................................................48Linearity-Recovery.....................................................................4812. TROUBLESHOOTING.................................................................49WARRANTY.....................................................................................52REFERENCES....................................................................................52 5 1.INTENDED USEBDSimultest™ IMK-Lymphocyte is a two-color direct immunofluorescence reagent kit for enumerating percentages of the following mature human leucocyte subsets in erythrocyte-lysed whole blood (LWB): T(CD3) lymphocytes, B(CD19) lymphocytes, helper/inducer T(CD3T(CD3) lymphocytes, and natural killer (NK) (CD3and/or CD56) lymphocytes. The helper/suppressor T-lymphocyte ratio (CD3/ CD32.SUMMARY AND EXPLANATIONHuman lymphocytes can be divided into three major populations based on their biologic function and cell-surface antigen expression: Tlymphocytes, Blymphocytes, and NK lymphocytes. Tlymphocytes participate in antigen-specific cell-mediated immunity and regulate the secretion of immunoglobulin by Blymphocytes. Tlymphocytes can also be classified based on their functional properties as helper/inducer and suppressor/cytotoxic. NK lymphocytes mediate cytotoxicity against certain tumors and virus-infected target cells, without requiring the presence of class I or II major histocompatibility complex (MHC) molecules on the target.Historical methods for assaying lymphocyte subsets were problematic because they were technique-dependent, time-consuming, or not highly cell lineage–specific. Classic methods included identifying Tlymphocytes by rosetting them with sheep red blood cells (SRBCs)2-4and identifying Blymphocytes with fluorochrome-labeled polyclonal 5-7Antibody reagents have been used to distinguish cell-surface antigens by fluorescence microscopy, electron microscopy, and radioactive label assay. Microscopy methods are not highly sensitive because of the limited number of cells that can be analyzed, while methods employing the use of radioactive isotopes require additional safety and waste 6 disposal considerations. Cytolytic lymphocyte assays included the hemolytic plaque assay and a cytotoxicity assay to detect the release The development of monoclonal antibodies to human leucocyte antigens provided reagents of single-defined specificity, a major improvemFlow cytometry improved the sensitivity of antigen detection and the reproducibility of leucocyte subset identification compared to microscopy. Flow cytometry using indirect immunofluorescence, while providing assay sensitivity, requires an additional step to add the fluorochrome-labeled secondary reagent.Lymphocytes from human whole blood traditionally were prepared for flow cytometric analysis using density-gradient separation methods. Studies have shown that these methods were time-consuming, involved multiple blood-handling steps, and could result in the loss of lymphocyte subsets.12,14-16 The LWB method used with BDSimultest IMK-Lymphocyte does not require density-gradient separation and therefore allows for shorter sample preparation time and less handling of whole blood (refer to the BD Monoclonal Antibodies Source Section 2.2). Studies have also shown that the LWB method is less likely to show loss of lymphocyte subsets and can help improve assay reproducibility when compared to earlier methods.12,15,16The development of monoclonal antibodies and flow cytometry technology has made possible new approaches to leucocyte subset identification and immune monitoring. Fluorochrome-labeled monoclonal antibodies and multicolor flow cytometry permit o or more leucocyte subpopulations. Immune monitoring is facilitated by flow cytometers, such as the BDFACScan™ and BDFACStrak™, which characterize cells by means of four-parameter analysis. The BDFACScan and BDFACStrak, when used with BDSimultest™ IMK-Lymphocyte software, characterize up to 50,000 cells in a single sample by simultaneous analysis of forward scatter (FSC), side scatter (SSC), and multicolor fluorescence. 7 Clinical Applications*Total T- and B-lymphocyte percentages can be used to characterize some forms of immunodeficiency17-19 and autoimmune diseases.Determining percentages of CD4and CD8lymphocytes can be useful in monitoring the immune status of patients with immune deficiency diseases, autoimmune diseases, or immune reactions. The relative percentage of the CD4 subset has been reported to be depressed and the relative percentage of the CD8 subset to be elevated in many patients with congenital or acquired immune deficiencies such as severe combined immunodeficiency (SCID)deficiency syndrome (AIDS).oxic T lymphocytes can lie outside the normal reference range in some autoimmune diseasescertain immune reactions such as acute graft-versus-host disease (GVHD) The relative percentage of the lymphocyte population can often be decreased in active systemic lupus erythematosus (SLE) but can also be increased in SLE patients undergoing steroid therapy.Helper/inducer Tlymphocytes are a subset of T (CD3) lymphocytes that also express the CD4 antigen. Suppressor/cytotoxic Tlymphocytes express the CD8 antigen and are principally a subset of T(CD3lymphocytes, although a subset of NK lymphocytes is also CD8 lymphocyte ratio can be quantified as the ratio of CD4 FITC–positive lymphocytes to CD8 PE–positive lymphocytes. The most common method that has been used to determine the CD4/CD8 ratio has been a single two-color reagent containing CD4 and CD8 antibodies. The CD4ratio has been used to evaluate the immune status of patients with autoimmune disorders or those suspected of developing immune disorders21,22 *Not all the studies in this section employed BD reagents. 8 immunodeficiencies.4,22 In addition, the ratio has been used to monitor bone marrow transplant patients for onset of acute GVHD.The conventional helper/suppressor ratio (CD4/CD8) does not distinguish between T- and NK-lymphocyte CD8 antigen expression, or between helper/inducer Tlymphocytes and monocytes that express the CD4 antigen in low copy numbers.The reagent combinations provided in the kit allow the CD3 helper/inducer Tlymphocytes and CD3 suppressor/cytotoxic Tlymphocytes to be identified and enumerated separately from contaminating CD3 monocytes and CD3 NK lymphocytes.Instead of using a single reagent containing CD4 and CD8, the BDSimultest IMK-Lymphocyte kit uses two monoclonal antibody reagents (CD3/CD4 and CD3/CD8) to determine the true helper/suppressor T-lymphocyte ratio (CD3/CD3). The true helper/suppressor T-lymphocyte ratio does not include CD4-positive monocytes and CD8-positive NK lymphocytes.NK lymphocytes, identified as being CD3have been shown to mediate cytotoxicity against certain tumors and virus-infected target cells.3.PRINCIPLES OF THE PROCEDUREWhen monoclonal antibody reagents are added to human whole blood, the fluorochrome-labeled antibodies bind specifically to antigens on the surface of leucocytes. The stained samples are treated with BDFACS™ lysing solution (reagent G) to lyse erythrocytes. Samples are then washed and fixed prior to flow cytometric analysis.An aliquot of the stained patient sample is introduced into the flow cytometer and passed in a narrow stream through the path of a laser beam. The stained cells fluoresce when excited by the laser beam and the emitted light is collected and processed by the flow cytometer. The use of two fluorochromes permits simultaneous two-color analysis because each fluorochrome emits light at a different wavelength when 9 excited at 488nm by an argon-ion laser. The FITC-stained lymphocytes emit yellow-green light (emission maximum approximately 515nm) while the PEorange light (emission maximum approximately 580nm).The cells also interact with the laser beam by scattering the light. The FSC light provides a measure that correlates well with cell size, while the SSC light is an indicator of cellular granularity. The BDFACScan and BDFACStrak flow cytometers used with BDSimultest IMK-Lymphocyte software count a sufficient number of cells to ensure that a minimum of 2,000 lymphocytes are included in the analysis gate. The data files should be saved in list mode and given logical names to aid in retrieval for subsequent analysis by the BDSimultest IMK-Lymphocyte software.The BDSimultest IMK-Lymphocyte software uses the BDLeucogate™ sample, Reagent A, to automaticallygate that includes greater than or equal to 98% of the normal mature lymphocytes in the sample. However, if the gate contains greater than or equal to 3% monocytes, the sotightens the light-scatter gate to collect greater than or equal to 95% of the lymphocytes contained in the sample.The software identifies and calculates the percentages of contaminating monocytes, granulocytes, and debris that are included within the BDLeucogate lymphocyte analysis gate on the basis of FSC, SSC, and fluorescence properties. Refer to the BDSimultest IMK-Lymphocyte Software User’s Guide for details on how the software sets the gate and automatically adjusts subset percentages by using the percent purity of the gated lymphocyte population.The presence of blast cells can interfere with the gating procedure and result in a processing failure. Samples containing blast cells can set a gate, a message will appear on the printout. Refer to the 10 BDSimultest IMK-Lymphocyte Software User’s Guide for a complete listing of software error messages.The software uses the negative control, Reagent B, to set fluorescence-1 (FL1) and fluorescence-2 (FL2) markers around the negative lymphocyte population and to assess the amount of nonantigen-specific antibody binding (nonspecific staining) present, particularly that caused by Fc receptors. When greater than 5% of the negative control events are above the FL1 or FL2 negative control markers, an error message of “too much nonspecific staining” will appear on the computer display screen and on the laboratory printout for the negative control tube. When the negative control tube is being processed by the software, the operator should check the screen for error messages that would indicate nonspecific staining.The negative control is a mixture of conjugated monoclonal antibodies with the same fluorochromes (FITC and PE) as the test reagents. The negative control antibodies are specific for antigens not present on human leucocytes. The Control should be used to stain a separate aliquot of each patient sample.For each patient sample, the lymphocyte gate set with BDLeucogate and the fluorescence markers determined using the Control are used to analyze the remaining tubes C through F. When the Percent Lymphocyte Conversion option has been selected from the Main Menu of the BDSimultest IMK-Lymphocyte software, the results are es in the analysis gate. If this software option is not selected, results will be expressed as percentages of the total gated events. BDSimultest IMK-Lymphocyte software provides a report quantifying immunologically significant lymphocyte rculating human lymphocytes and the helper/suppressor T-lymphocyte ratio (CD3CD8For the purposes of quality control, a three-part differential with percentages of monocytes, granulocytes, and lymphocytes is automatically determined by the software and printed only for 11 comparison with results from a laboratory differential. To obtain the three-part differential, the software uses an algorithm to distinguish the three cell populations based on FSC, SSC, and fluorescence. Monocytes are CD14-positive and have an SSC signal intermediate between that of lymphocytes and granulocytes. Lymphocytes exhibit a low and granulocytes a high SSC signal.NOTEThe three-part differential provided by BDSimultest IMK-Lymphocyte software should be used only for comparison with an independent differential white cell count for quality control purposes and should not be used in place of an independent laboratory differential white cell count in patient charts or entered into BDSimultest IMK-Lymphocyte software to obtain absolute counts.4.REAGENTSReagents Provided, Sufficient for 50 TestsReagents A through F contain two murine monoclonal antibodies, one conjugated with FITC, and one conjugated with PE. Each antibody reagent, sufficient for 50tests, is provided in 1mL of buffered saline with gelatin and 0.1% sodium azide. The monoclonal antibodies are produced by cell lines derived from the fusion of mouse myeloma cells with spleen cells of BALB/c mice. The fluorescein-to-protein ratio (F:P) for BD IgG monoclonal antibody reagents is 2 to 5. The F:P ratio for each reagent has been optimized for its intended use.Reagent A, BDLeucogate (CD45/CD14), 1 mLBDLeucogate is used to define and evaluate the light-scatter gate that anulocytes, monocytes, debris, and unlysed or nucleated red blood cells. This reagent contains FITC-labeled CD45, clone 2D1,28-30 for identification of leucocytes, and PE-labeled CD14, clone M31-33 for identification of monocytes. 12 The CD45 antibody was derived from hybridization of mouse NS-1 myeloma cells with spleen cells of BALB/c mice immunized with human peripheral blood mononuclear cells (PBMCs). The antibody is heavy chains and kappa light chains. The antigen is present on all human leucocytes, including lymphocytes, monocytes, granulocytes, eosinophils, and basophils in peripheral blood and has a role in signal transduction, modifying signals from other surface molecules. The CD45 antibody has been reported to react weakly with mature circulating erythrocytes and platelets.The CD45 antibody recognizes human leucocyte antigens, 180 to 220 kilodaltons (kDa), that are members of the T200 family.The CD14 antibody was derived from the hybridization of mouse Sp2/0 myeloma cells with spleen cells of BALB/c mice immunized with peripheral blood monocytes from a rheumatoid arthritis patient. The heavy chains and kappa light chains. The CD14 antibody recognizes a human monocyte antigen of 35 The CD14 antigen is present on the majority of normal peripheral blood monocytes.36 The CD14 antibody reacts weakly with granulocytes.Reagent B, Control, 1mLThe Control reagent is used to set FL1 and FL2 quadrant markers around the reference unstained (negative) lymphocyte population and to estimate the amount of nonantigen specific antibody binding (nonspecific staining), present, in particular that caused by Fc receptors. It contains FITC-labeled IgG, clone X40, and PE-labeled , clone X39. 13 Both X40 and X39 react specifically with keyhole limpet hemocyanin (KLH), an antigen not expressed on human cells or human cell lines. Clones X40 and X39 are both derived from hybridization of mouse Sp2/0-Ag14 myeloma cells with spleen cells from BALB/c mice immunized with KLH. Clone X40 is composed of mouse IgG heavy chains and kappa light chains. Clone X39 is composed of mouse IgGCD3/CD19 is used to identify T and B lymphocytes. It contains FITC-labeled CD3, clone SK7,38-41 for identification of Tlymphocytes, and PE-labeled CD19, clone 4G7, for identification of B lymphocytes.The CD3 antibody was derived from the hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human thymocytes. The antibody is composed of mouse IgG heavy chains and kappa light chains. The CD3 antibody reacts with the epsilon chain of the CD3/T-cell antigen receptor (TCR) complex.This complex is composed of at molecular weight from 20 to 30 kDa.The antigen recognized by CD3 antibodies is noncovalently associated with either or TCR The CD19 antibody was derived from hybridization of mouse P3-X63-Ag8.653 myeloma cells with spleen cells of BALB/c mice immunized with human chronic lymphocytic leukemia cells. The heavy chains and kappa light chains. The antigen recognized by CD19 antibodies is present on human B lymphocytes at all stages of maturation, but is lost on plasma cells. The CD19 antigen can be involved in activation and proliferation of B lymphocytes. The molecular weight of the antigen recognized by this antibody is 90 kDa. 14 Reagent D, CD3/CD4, 1 mLCD3/CD4 is used to identify helper/inducer Tlymphocytes. This reagent combination permits CD3 monocytes in quadrant1 (see Figure1 on page24 for quadrant definition) to be separated from the helper/inducer T lymphocytes in quadrant 2 (See Figure2 on page25). It contains FITC-labeled CD3, clone SK7, for the identification of Tlymphocytes, and PE-labeled CD4, clone SK3,for the identification of helper/inducer T lymphocytes.The CD4 antibody was derived from the hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood Tlymphocytes. It is composed of mouse IgGheavy chains and kappa light chains. The CD4 antibody recognizes the CD4 antigen, which interacts with class II MHC molecules and is the primary receptor for the human immunodeficiency virus (HIV).The cytoplasmic portion of the antigen is associated with the protein lck. The CD4 antigen can regulate the function of the CD3 antigen/TCR complex. The CD4 antibody reacts with monocytes/macrophages, which have a CD4 antigen density lower than that on helper/inducer Tlymphocytes.Reagent E, CD3/CD8, 1 mLCD3/CD8 is used to identify suppressor/cytotoxic Tlymphocytes. This reagent combination permits CD3 NK lymphocytes in quadrant 1 to be separated from the CD3suppressor/cytotoxic Tlymphocytes in quadrant 2 (see Figure2 on page25). It contains FITC-labeled CD3, clone SK7, for identifying Tlymphocytes, and PE-labeled CD8, clone SK1, for identifying suppressor/cytotoxic Tlymphocytes.myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood T lymphocytes. It is composed of mouse IgGheavy chains and kappa light chains. The CD8 antigen is present on the 15 human suppressor/cytotoxic T-lymphocyte subset39,47 as well as on a subset of NK lymphocytes. The CD8 antigenic determinant interacts with class I MHC molecules, resulting in increased adhesion between the CD8 T lymphocytes and the target cells.52-54 Binding of the CD8 antigen to class I MHC molecules enhances the activation of resting Tlymphocytes.52-55 The CD8 antigen is expressed as a disulfide-linked bimolecular complex with a 32-kDa subunit.56,57domain of the subunit of the CD8 antigen is associated with the protein tyrosine kinase p56lck54,55Reagent F, CD3/CD16+CD56, 1 mLThe CD3/CD16+CD56 reagent is used to identify T and NK lymphocytes. It contains PE-labeled CD16, clone B73.1,58-61, and PE-labeled CD56, clone MY31,, to ensure identification of the NK lymphocyte population. Since some Tlymphocytes also express the CD16 and/or CD56 antigens, the reagent also contains FITC-labeled CD3 to identify T lymphocytes and the T-lymphocyte subset that expresses the CD16 and/or CD56 antigens.The CD16 antibody was derived from the hybridization of mouse P3-X63-Ag8.653 myeloma cells with spleen cells of BALB/c mice immunized with NK lymphocytes. The antibody is composed of mouse heavy chains and kappa light chains.58-60 The antigen recognized by CD16 antibodies is a 50 to 65-kDa protein that is the IgG Fc III receptor present on NK lymphocytes and neutrophils.The CD56 antibody was derived from the hybridization of mouse Sp2/0 myeloma cells with spleen cells of (B6xBALB/c)Fwith the KG-1a cell line. The anti. The CD56 antigen is present on NK lymphocytes. The antibody recognizes the 140-kDa isoform of the neural cell adhesion molecule (NCAM).61,63 The 140-kDa core protein is extensively glycosylated to give a mature antigen.62,64 CD56 antigen is present on essentially all resting and activated CD16 16 lymphocytes. CD56 antigen density increases when NK lymphocytes are activated.CD56 Tlymphocytes compose a unique subset of cytotoxic Tlymphocytes thatcytotoxicity. The CD56 antigen is involved in neuronal homotypic cell adhesion and cell differentiation during embryogenesis.Reagent G, 10X BDFACS Lysing Solution, 60 mLReagent G contains 10X buffered BDFACS lysing solution, with less than 50% diethylene glycol and less than 15% formaldehyde. When stored at 2°C–25°C, the 10X concentrate is stable until the expiration date on the label. For use, dilute 1:10 with room temperature (20°C–25°C) reagent-grade water. The prepared solution is stable for 1month when stored in a glass container at room temperature.ReagentComponentConcentration (µg/mL)ACD45 25CD14 12.5BIgG12.512.5CCD325CD193.1DCD325CD41.5ECD325CD86.25FCD325CD166.25CD5612.5 17 PrecautionsFor In Vitro Diagnostic Use.When stored at 2°C–8°C, antibody reagents are stable until the expiration date shown on the label. Do not use after the expiration The antibody reagents should not be frozen or exposed to direct light during storage or during incubation with cells. Keep the reagent vial dry.Incubation or centrifugation times or temperatures other than those specified can be a source of error.For optimal results, stain blood samples within 6 hours of venipuncture.Alteration in the appearance of any of the reagents, such as precipitation or discoloration, indicates instability or deterioration. In such cases, the reagent should not be used.The antibody reagents contain sodium azide as a preservative; however, care should be taken to avoid microbial contamination, which can cause erroneous results.Reagent G contains 30.0% diethylene glycol, CAS number 111-46-6, 9.99% formaldehyde, CAS number 50-00-0, and 3.51% methanol, H311 Toxic in contact with skin. H331 Toxic if inhaled. H341 Suspected of causing genetic defects. H350 May cause cancer. Route of exposure: Inhalative.H371-H335 May cause damage to organs. May cause respiratory irritation.H373 May cause damage to the kidneys through prolonged or repeated exposure. Route of exposure: Oral. 18 WARNINGcontact with them are considered biohazards. Handle as if capable of transmitting infection67,68 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.5.INSTRUMENTBDSimultest IMK-Lymphocyte is designed for use on a BD flow cytometer equipped with appropriate computer hardware, software, and gating electronics. The flow cytometer must be equipped to detect two-color fluorescence, FSC, and SSC.All performance characteristics were obtained using the BDFACScan and verified on the BDFACStrak. Other systems can have different characteristics and should be verified by the user.The following instrument system is recommended:BDFACScan or BDFACStrak flow cytometer system equipped for two-color fluorescence detection and two-parameter light-scatter H318 Causes serious eye damage.H302 Harmful if swallowed.H315 Causes skin irritation.H317 May cause an allergic skin reaction.Wear protective clothing / eye protection. Wear protective gloves.Avoid breathing mist/vapours/spray. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing. IF SWALLOWED: Immediately call a doctor. 19 detection. For detailed information on use, refer to the BDFACScan User’s GuideBDFACStrak User’s GuideBDCONSORT™ 30 or CONSORT 32 computer system and peripherals (included in the BDFACScan system) or CONSORT 31 computer system and peripherals (included in the BDFACStrak system). For detailed information on use, refer to the CONSORT 30 Software User’s Guide, the CONSORT 32 System User’s , or the CONSORT31 System User’s GuideBDSimultest IMK-Lymphocyte software. For detailed information BDSimultestIMK-Lymphocyte User’s GuideBDCalibrite™ beads (Catalog No. 349502). These beads are used for setting the photomultiplier tube (PMT) voltages, setting the fluorescence compensation, checking instrument sensitivity, and monitoring the performance of the flow cytometer. For detailed information on use, refer to the BDCalibrite Beads instructions for use (IFU).BDFACSComp™ software. For detato the BDFACSComp Software User’s Guide.6.SPECIMEN COLLECTION AND PREPARATIONCollect blood aseptically by venipuncture into a sterile EDTA (lavender top) BDVacutainer® blood collection tube. A minimum of 1 mL of whole blood is required for this procedure. Blood should be stained within 6 hours of venipuncture for optimal results. Anticoagulated blood can be stored at room temperature (20°C–25°C) for up to 6 hours until ready for staining. Blood samples refrigerated prior to staining can give aberrant results.A white blood cell (WBC) count and a differential white count should be obtained from the same sample of whole blood before staining. An acceptable WBC concentration range is from 3.5 x 10to 9.4 x 10WBC/µL. Samples with counts greater than 9.4 x 10WBC/µL might need to be diluted with 1X phosphate buffered saline (PBS) containing 20 0.1% sodium azide, or BDCellWASH™ solution; check with your local BDBiosciences representative for availability. For samples with counts less than 3.5x10WBC/µL, more blood might be needed and a separation procedure can be required to concentrate the cells.Interfering ConditionsPreviously fixed and stored patient specimens should not be used. Whole blood samples refrigerated prior to staining can give aberrant results. For optimal results, blood samples should be stained within 6 hours of venipuncture. Samples obtained from patients taking immunosuppressive drugs can yield poor resolution. The presence of blast cells or unlysed or nucleated red blood cells (RBCs) can interfere with test results. Hemolyzed samples should be rejected. Follow the collection tube manufacturer’s guidelines for the minimum volume of blood to be collected.CAUTION obtaining, handling, and disposing of all human blood samples and potentially carcinogenic reagents.7.PROCEDUREReagents ProvidedSee Reagents Provided and Precautions in Section 4, Reagents.Reagents and Materials Required But Not ProvidedBDVacutainer EDTA blood collection tubes or equivalent.Falcon®* disposable 12 x 75-mm polystyrene test tubes or equivalent.Vortex mixer.Low-speed centrifuge (minimum speed 200) with swinging bucket rotor and 12 x 75-mm tube carriers.Vacuum aspirator with trap. *Falcon is a registered trademark of Corning Incorporated. 21 Micropipettor with tips.BDCellWASH (Catalog No. 349524) or a wash buffer of PBS with 0.1% sodium azide.BDCellFIX™ (Catalog No. 340181) or 1% paraformaldehyde solution in PBS with 0.1% sodium azide. Store at 2°C–8°C in amber glass for up to 1week.BDFACSFlow™ sheath fluid (Catalog No. 342003), or equivalent. CAUTIONUse only BDFACSFlow sheath fluid diluent to dilute BDCalibrite beads.Reagent-grade (both distilled and deionized) water.Staining and Fixing the CellsWhole blood samples are first stained with Reagents A through F. Diluted Reagent G is used to lyse RBCs following staining. See Interfering Conditions under Section 6, Specimen Collection and Preparation, and Section 9, Limitations. Use care to protect the tubes from direct light. Perform the procedure at room temperature (20°C–25°C) using room temperature reagents. Refer to Precautions in Section 4, Reagents.1.For each patient sample, label six 12 x 75-mm tubes with A, B, C, D, E, and F. Also label each tube with the sample identification number.2.Place 20 µL of Reagent A into tube A, 20 µL of Reagent B into tube B, 20 µL of Reagent C into tube C, 20 µL of Reagent D into tube D, 20µL of Reagent E into tube E, and 20 µL of Reagent F into tube F.3.For each sample tube, use a fresh micropipettor tip and carefully add 100µL of well-mixed, anticoagulated whole blood into the bottom of each of the labeled tubes. The recommended WBC concentration is 3.5x 10to 9.4 x 10WBC/µL. Vortex 22 thoroughly at low speed for 3seconds and incubate for 15 to 30 minutes at room temperature (20°C–25°C). NOTEProtect samples from direct light during this incubation procedure and use care to prevent blood from running down the side of the tube. If whole blood remains on the side of the tube, it will not be stained with the reagent.4.Dilute 10X lysing solution to 1X following the instructions in Section 4, Reagent G. Add 2mL of room temperature (20°C–25°C) 1X lysing solution to each tube. Immediately vortex thoroughly at low speed for 3seconds and incubate for 10 to 12 0°C–25°C) in the dark. Do not exceed 12minutes.NOTEAvoid prolonged exposure of the cells to lytic reagents, which can cause white cell destruction. See Section 9, Limitations on page35.5.Immediately after incubation, centrifuge tubes at 300 for 5 minutes at room temperature (20°C–25°C).6.Aspirate the supernatant, leaving approximately 50µL of residual fluid in the tube to avoid disturbing the pellet.7.Vortex thoroughly at low speed to resuspend the cell pellet in the residual fluid and then add 2mL of BDCellWASH solution or PBS with 0.1% sodium azide to each tube. Vortex thoroughly at low speed for 3 seconds. Centrifuge at 200temperature (20°C–25°C).8.Aspirate the supernatant, leaving approximately 50µL of residual fluid in the tube to avoid disturbing the pellet.9.Vortex thoroughly at low speed to resuspend the cell pellet in the residual fluid and then add 0.5mL of BDCellFIX solution or 1% paraformaldehyde to each tube. Vortex thoroughly at low speed 23 for 3 seconds. Make sure that the cells are well mixed with the 10.The cells are now ready to be analyzed on the flow cytometer. Cap or cover the prepared tubes and store at 2°C–8°C in the dark until flow cytometric analysis. Analyze the fixed cells within 24 hours after staining. Vortex the cells thoroughly (at low speed) to reduce aggregation before running them through the flow cytometer.13 Flow CytometryFollow the BD instructions for two-color flow cytometric analysis and see Section 5, Instrument. The following general approach is recommended. The BDFACScan or BDFACStrak flow cytometer is first prepared for sample analysis using BDCalibrite beads and BDFACSComp software. The stained samples are then run on the flow cytometer and analyzed with BDSimultest IMK-Lymphocyte software. Refer to the BDFACScan User’s Guide BDFACStrak User’s Guidethe BDCalibrite Beads IFU, the BDFACSComp Software User’s Guideand the BDSimultestIMK-Lymphocyte Software User’s Guidedetailed instructions on use. We recommend that patient data be stored in list mode to allow subsequent analysis of data files.Set up and adjust compensation of the BDFACScan or BDFACStrak flow cytometer. Using BDCalibrite beads and BDFACSComp software, set PMT voltages, adjust fluorescence compensation, and check detector sensitivity. For details, refer to the BDCalibrite Beads IFU, the BDFACSCompSoftware User’s Guideand either the BDFACScanUser’s Guide or the BDFACStrak User’s GuideRun all samples through the flow cytometer and acquire data in list mode files using BDSimultest IMK-Lymphocyte software. Refer to the BDSimultest IMK-Lymphocyte Software User’s Guide for detailed instructions for use of the software. The software automatically collects a sufficient number of events to obtain a minimum of 2,000 24 lymphocytes within the lymphocyte gate. The software counts the number of events in each quadrant and then computes a percentage of positive lymphocyte events for quadrant 1 (low yellow-green/high red-orange), quadrant 2 (high yellow-green/high red-orange, or dual-fluorescence), quadrant 3 (low yellow-green/low red-orange), and quadrant 4 (high yellow-green/low red-orange) (see Figure1).Figure1The fluorescence-display quadrants (indicated as Q1 through Q4) and the corresponding colors Q1Q2Q3Q4 Low yellow-green/high red-orange High yellow-green/high red-orange (dual fluorescence) Low yellow-green/low red-orange High yellow-green/low red-orange 25 The BDLeucogate tube (A) is used to gate on lymphocytes. The BDFACScan or BDFACStrak with BDSimultest IMK-Lymphocyte software automatically sets a lymphocmost debris, monocytes, and granulocytes (see Figure2). Refer to the BDSimultestIMK-Lymphocyte Software User’s Guide for information on how the gate is set. If there is inadequate separation between populations, the sample will be flagged to alert the operator and a message will appear. Poor sample preparation or incorrect instrument setup can contribute to errors in sample gating.Figure2BDFACScan LWB sample, from a hematologically normal patient, stained with BDSimultest IMK-Lymphocyte reagents. BDLeucogate was used to reduce debris, monocytes, and granulocytes in the gate shown under tube A. Dot plot displays of FL1 (x-axis) vs FL2 (y-axis) are shown for tube B through F. 26 Tube E Tube BIgGIgGTube CCD19Tube DCD4 Tube FCD16+CD56Tube ASSCFSC CD3 27 The Control tube (B) is used to set fluorescence intensity markers and gives an indication of nonspecific staining. BDSimultest IMK-Lymphocyte software automatically uses the lymphocyte analysis gate set with BDLeucogate and then establishes FL1 and FL2 marker placement. These markers define the boundaries between negative ents in the lymphocyte gate. Fluorescence markers should be set around the negative population that appears as the cluster of events that are low in both yellow-green and red-orange fluorescence.Data for tubes C, D, E, and F is acquired and analyzed by BDSimultest IMK-Lymphocyte software using the gate and markers established with tubes A and B. The lymphocyte populations present in each quadrant for each tube are shown in Table1. See the BDSimultest IMK-Lymphocyte Software User’s Guide for detailed instructions on use of the software. Analyze these results using the criteria provided under Quality Control and according to the instructions in Section 8, Table1.Mononuclear cell populations separated by quadrant for tubes C through FTubeQuadrantQ1Q2Q3Q4C:CD3/CD19CD3CD19BlymphocytesCD3CD19Nonspecific stainingCD3lymphocytes, monocytes, granulocytes, and debrisCD3CD19TlymphocytesD: CD3/CD4CD3CD4monocytesCD3helper/inducer TlymphocytesCD3lymphocytes, granulocytes, and debrisCD3TlymphocytesE: CD3/CD8CD3CD8NK lymphocyte subsetCD3suppressor/ cytotoxic TlymphocytesCD3lymphocytes, monocytes, granulocytes, and debrisCD3T lymphocytes 28 Quality ControlFor optimal results, we recommend using BDCalibrite beads and BDAutocomp™ software for setting the PMT voltages, setting the fluorescence compensation, and checking instrument sensitivity prior to use of BDSimultest IMK-Lymphocyte on the BDFACScan or BDFACStrak flow cytometer.We recommend that a control sample from a normal adult subject be run daily to optimize instrument settings and as a quality control check of the system. Correct results for a hematologically normal patient are illustrated in Figure2.Negative controls are run with each patient sample to set FL1 and FL2 markers between negative and positively stained lymphocyte clusters and to detect the presence of nonspecific staining that would indicate erroneous patient results. Examine the computer screen display and the Laboratory Report for Control tube B. If more than 5% of the events occur outside of Q3 for the Control tube, a message of “too much nonspecific staining” will be reported for tube B. In this case, the results for tubes C through F should be considered suspect. See Troubleshooting at the end of this IFU. Visual inspection of the dot plots obtained for tubes C through F is necessary to ensure that fluorescence markers are correctly set and that there is minimal F: CD3/CD16+ /CD16 and/or CD56NKlymphocytes, granulocytesCD3and/or CD56T-lymphocyte subsetCD3CD16CD56lymphocytes, monocytes, and debrisCD3 and CD56Tlymphocytesa.See section 9, Limitations.Table1.Mononuclear cell populations separated by quadrant for tubes C through FTubeQuadrantQ1Q2Q3Q4 29 Visually inspect the dot plot for Control tube B. If the negative cluster is diffuse and smeared over the FL2 intensity range, the marker might not be set correctly and results can be suspect.If poor separation between positive and negative clusters is seen on visual inspection of the dot plots for the normal control, especially for CD3 and CD4, nonspecific staining or incomplete staining can be inferred and the run should be rejected. Nonspecific staining might be seen because of poor condition of the cells. Consult Section 12, Troubleshooting, if nonspecific observed.BDSimultest IMK-Lymphocchecks to see that there is agreement in T-lymphocyte percentages between tubes containing CD3 (tubes C, D, E, and F). The run will be rejected if the total T-lymphocyte value in any two tubes differs by Internal assay consistency can be indicated when the total percentages of T, B, and NK lymphocytes total 100% ±5% when using the Percent Lymphocyte Conversion option with BDSimultest IMK-Lymphocyte software. Refer to the BDSimultest IMK-Lymphocyte User's Guideinstructions when selecting this option.BDSimultest IMK-Lymphocyte software will automatically inspect the data and alert the operator with a number of possible error messages. Refer to the BDSimultest IMK-Lymphocyte Software User’s Guide a list of possible messages. Use the following criteria for inspection of the dot plots obtained for each sample to evaluate the quality of the 1.The operator should reject the results if one or more of the following error messages is received for the normal control: no separation between cellular populations; too few lymphocytes (less than 500); excessive RBC or nucleated RBC contamination and debris (greater than 10%); or excessive monocyte (greater than 3%) or granulocyte (greater than 6%) contamination of the lymphocyte gate. 30 2.If there is no obvious reason for the normal control to fail, a sample from another normal control should be stained and run, and the entire staining procedure repeated on all subsequent samples.3.Samples with nucleated RBCs can contain too much debris because of incomplete lysis of nucleated erythrocytes with BDFACS lysing solution (reagent G). Too much debris can also occur when assaying blood samples from patients with certain hematologic disorders for which red cells are difficult to lyse, as in myelofibrosis and spherocytosis. Nucleated erythrocytes will be counted as debris and, if debris exceeds 10%, the software will flag the sample as “too many nonlymphs in gate” and the sample results should be rejected.4.The tube-to-tube difference in gated events must be less than or equal to 500 events for all tubes in the panel except the BDLeucogate tube (A). (However, if the Control tube is problematic, then the panel is suspect.) See also Troubleshooting at the end of this IFU.8.RESULTSPercent Lymphocyte ConversionWhen the Percent Lymphocyte Conversion software option is selected, BDSimultest IMK-Lymphocyte software automatically calculates each reported lymphocyte subset as a percentage of lymphocytes in the lymphocyte analysis gate based on the lymphocyte percentage determined with the BDLeucogate tube A. The software also subtracts nonlymphocytes from Q3. Refer to the BD Source Book, Section 8.3.When all quality control criteria listed in Section 7, Procedure, have been met, the Percent Lymphocprovides appropriate estimations of the true subset values. However, if the quality control criteria are not met (for example, excessive 31 nonlymphocyte contamination of the gathis case, the data should be analyzed manually to determine the effect of cross-reacting nonlymphocytes on results. If the Percent Lymphocyte Conversion option is turned off, results will be reported as a percentage of the total gated events. (Refer to the BDSimultest IMK-Lymphocyte Software User’s Guide or the BD Monoclonal AntibodiesSection 8.3).The principle of the computation follows (Q values refer to counts in quadrants):N = total number of events in the lymphocyte gate defined by BDLeucogate = L + M + G + D where:L = number of lymphocytes in the gateM = number of monocytes in the gateG = number of granulocytes in the gateD = number of debris events in the gateEquation 1. Percent gated lymphocytes (%L) = L/N x 100Equation 1 computes the percentage of lymphocyte events within the BDLeucogate lymphocyte analysis gate. This value is given as “%L” in tube A in Table2 and reflects the purity of lymphocytes within the gate drawn using the BDLeucogate tube (A).Equation 2a. Percent gated converted lymphocytes (Conv%L) in Q1, The software uses Equation 2a to calculate the converted percentage of lymphocytes for Q1, Q2, and Q4. Itare unstained and appear in Q3. Number of gated events in Q1, Q2, or Q4N x (%L/100)--------------------------------------------------------------------------------------------------- 32 NOTEThe Percent Lymphocyte Conversion option does not correct for nonlymphocyte events that can occur in Q1, Q2, and Q4. Therefore, CD16 granulocyte events are not corrected in Q1 by the software.Equation 2b. Percent gated converted lymphocytes (Conv%L) in Q3 =where:Number of gated nonlymphocytes = number of gated debris events + number of gated granulocytes + number of gated monocytes. See Table2, tube A.The software uses Equation 2b to calculate the converted percentage of lymphocytes for Q3. Since it assumes that all nonlymphocytes are in this quadrant, it subtracts them from the number of gated events in Q3 and expresses the remainder as a percentage of the lymphocytes in the gate.Table2 summarizes the data in Figure2.Table2.Summary of the representative data from Figure 2 and identification of quadrants used to compute subsetsTube%La.%L, M, G, and D are lymphocytes, monocytes, granulocytes, and debris in the lymphocyte gate defined by BDLeucogate, expressed as percentages of gated events. These values reflect the quality and purity of the gate. Data was collected on a BDFACScan flow cytometer.% of Total Gated Lymphsb.% of total gated lymphocytes are the lymphocytes in the gate defined by BDLeucogate, expressed as a percentage of all lymphocytes in the ungated sample. This value measures the proportion of all lymphocytes included in the gate.A9513199 (Number of gated events in Q3 - Number of gated nonlymphs)N x (L/100)-----------------------------------------------------------------------------------------------------------------------------------------------------100 33 Table3 gives representative data for the three-part differential obtained on a healthy adult donor using the BDFACScan with the BDSimultest IMK-Lymphocyte software.Three-Part DifferentialFor lysed whole blood, it is possible to estimate monocytes, lymphocytes, and granulocytes as a BDLeucogate tube (A) (see Table2). BDSimultest IMK-Lymphocyte software automatically calculates a three-part differential (Table3). Refer to the BDSimultestIMK-Lymphocyte Software User’s Guide for TubeCell TypeQuadrant(s)% of Lymphocytesa.See Section 8, Results, for an explanation of Percent Lymphocyte Conversion.Ratiob.Helper/suppressor T-lymphocyte ratio = (CD3CD4)/(CD3CD8CD3/CD19Total T lymphocytesQ474Total B lymphocytesQ17CD3/CD4Total T lymphocytesQ2 and Q475Helper/inducer TlymphocytesQ252CD3/CD8Total T lymphocytesQ2 and Q472Suppressor/cytotoxic TlymphocytesQ224CD3/CD4;CD3/CD8Helper/inducer; suppressor/cytotoxic TlymphocytesQ2—2.2§CD3/CD16 + CD56Total T lymphocytesQ2 and Q474NK lymphocytesQ116 34 NOTEThe differential provided by BDSimultest IMK-Lymphocyte software should be used only for comparison with an independent differential white cell count for quality control purposes and should not be used in place of an independent laboratory differential white cell count in patient charts or entered into BDSimultest IMK-Lymphocyte software to obtain absolute counts.Absolute CountsAn absolute cell count can be computed if a WBC count and the lymphocyte percentage from an independent differential white cell count are obtained using standard laboratory procedures. BDSimultest IMK-Lymphocyte software automatically calculates an absolute count for each subset if the operator enters a WBC count and the lymphocyte percentage from a differential white cell count for each patient sample of whole blood. Refer to the BDSimultest IMK-Lymphocyte Software User’s Guide. The principle of the computation follows:1.Enter the WBC count in WBC/µL obtained on the same blood sample.2.Enter the percentage of lymphocytes from an independent laboratory differential white cell count obtained for the same Table3.Representative data for the three part differential obtained on a healthy adult donor using the BDFACScan with BDSimultest IMK-LymphocytesoftwareThree-Part Differentiala.The three-part differential is obtained by expressing lymphocytes, monocytes, and granulocytes in the entire ungated sample as a percentage of the sum of lymphocytes, monocytes, and granulocytes in the entire sample.% LeucocytesLymphocytes21Monocytes5Granulocytes74 35 sample. Do not use the BDSimultest IMK-Lymphocyte three-part differential values.3.The absolute counts for each subset are then computed as in the Absolute count of CD3 cells/µL =If the data is presented in absolute counts, the value and precision of the absolute count reference range will be a function of (a) the laboratory normal reference range and precision for WBC count, (b) percent lymphocytes, and (c) the normal reference range and precision for percent lymphocytes positive for the specific marker.9.LIMITATIONSUse freshly drawn blood and stain within 6 hours of venipuncture. Prior to staining, store blood at room temperature (20°C–25°C). Cells that have been refrigerated before staining can give aberrant results. Previously fixed cells are not recommended for use.Stained and fixed cells should be assayed within 24 hours of Whole blood samples with counts less than 3.5 x 10WBC/µL and greater than 9.4 x 10WBC/µL can require special handling to obtain correct results. Samples with counts greater than 9.4x10WBC/µL might need to be diluted with PBS containing 0.1% sodium azide, or BDCellWASH. Samples with counts less than 3.5x10WBC/µL can require more blood and a separation procedure to concentrate the cells.Confounding variables such as mediof blood cells can yield inaccurate results. For example, poor resolution between positive and negative cells has been observed %CD3events--------------------------------------------------100--------(WBC/ 36 with transplant patients receiving immunosuppressive drugs. If there is poor separation between negative and positive clusters, the run should be rejected (see Troubleshooting, at the end of this IFU).If the difference in the total T-lymphocyte percentage for any tube differs from the average total T-lymphocyte percentage by greater than 8%, the software will print a notice on the Physician Report. In this case, the run should be rejected.Laboratories must establish their own normal reference ranges for each of the BDSimultest IMK-Lymphocyte parameters that can be affected by sex of patient, age of patient, and preparative technique. Race of patient can also have an effect, although sufficient data is not available to establish this. Age, sex, clinical status, and race of subjects should be known when a reference range is determined.If the results are to be expressed in absolute counts, an independent differential white cell count and a WBC count must also be run on the same sample of blood. Do not use the differential from the BDSimultest IMK-Lymphocyte report to compute absolute counts. The differential provided by BDSimultest IMK-Lymphocyte software is printed only for comparison with results from an independent laboratory differential white cell count for quality control purposes and should not be used in place of an independent laboratory differential white cell count in patient charts or entered into BDSimultest IMK-Lymphocyte software to obtain absolute counts.Variation in either automatic or manual lymphocyte analysis gate settings will change the relative amounts of subsets assayed. BDSimultest IMK-Lymphocyte software uses the BDLeucogate tube (A) to include at least 95% of the total lymphocytes in the inspection of the gate setting for validation. 37 Abnormal states of health are not always represented by abnormal percentages of lymphocyte subsets. That is, an individual who might be in an abnormal state of health can exhibit the same T-, B-, or NK-lymphocyte percentages asfrom BDSimultest IMK-Lymphocyte must be used in conjunction with other information available from the clinical evaluation and additional independent diagnostic procedures.A normal subject has been reported to have no reaction with However, this lack of reactivity has not been observed in The information obtained from the use of this reagent must be combined with other information and interpreted by a medically qualified diagnostician to establish presence or absence of specific disease states.The BDSimultest IMK-Lymphocyte Percent Lymphocyte Conversion software option assumes that all nonlymphocytes are unstained and appear in quadrant 3. Therefore CD16granulocytes that appear in quadrant 1 will be included as CD16lymphocytes. The sample is flagged if the granulocyte contamination is greater than 6%. Potentially interfering granulocytes should be minimized by eliminating as many as possible from the lymphocyte gate using their light-scatter properties. Refer to the BDSimultestIMK-Lymphocyte Software User’s Guide and the BD Monoclonal Antibodies Source BookSection 8.3, for more information on Percent Lymphocyte Samples with nucleated RBCs can show incomplete lysis because BDFACS lysing solution (reagent G) might not lyse nucleated erythrocytes. This can also occur when assaying blood samples from patients with certain hematologic disorders in which red cells are difficult to lyse, as in myelofibrosis and spherocytosis. Nucleated erythrocytes will be counted as debris and, if debris is greater than 10%, the software w 38 nonlymphs in gate.” We recommend that such sample results be rejected. When the Percent Lymphocyte Conversion software option has been selected, nonlymphocytes such as nucleated or unlysed red cells identified using BDLeucogate are assumed to be unstained and are subtracted BDSimultest IMK-Lymphocyte software checks for nonspecific staining in Control tube B only. The marker settings from tube B should be verified using the negative population from tube C. If the markers appear to be set inappropriately, reset them using the negative population from tube C. The operator should examine the printout for any evidence of nonspecific antibody staining in indicated when there is poor separation between positive and negative clusters on the fluorescence dot plot for tubes C through F. The presence of such nonspecific staining can reflect a change in the reagent or an error in the preparation of the samples. Such samples should be restained.BDSimultest IMK-Lymphocyte is not intended for screening samples for the presence of leukemic cells or for use in phenotyping samples from leukemia patients. The presence of blast cells might not allow BDLeucogate to set an adequate lymphocyte analysis gate. The software will flag the sample and results will not be Samples containing leukemic cells with volume and scatter characteristics similar to mature cells will be counted as such. Examples of these are chronic lymphocytic leukemia (CLL), FAB-L1 type acute lymphoblastic leukemia (ALL), and adult T-cell leukemia/lymphoma (ATL).For whole blood samples, exposure of cells to BDFACS lysing solution (reagent G) beyond 12 minutes can cause white cell destruction due to osmotic shock when the wash solution is added (see item 4 under Staining and Fixing the Cells in Section7, 39 The ability of the flow cytometer to select lymphocytes and to eliminate platelets, red cells, debris, granulocytes, and monocytes from the lymphocyte gate depends on the existence of a clear separation between these formed elements and lymphocytes on a display of FSC vs SSC. For some patients, this separation is not clear and lymphocyte gating will be less effective.Performance characteristics have been determined with a BDFACScan using BDSimultest IMK-Lymphocyte software, and verified on the BDFACStrak. Performance characteristics have not been established with any other instrument.Absolute count values of lymphocyte subsets might not be comparable across laboratories due to variations in methods for determining white blood cell counts and/or white cell differential values.Note that some BDSimultest IMK-Lymphocyte antibodies react with other nonlymphocyte-formed elements in blood (See Cross-Reactivity under Performance Characteristics, Section11).Note that normal reference ranges established by BD for the (CD3CD4)/(CD3) lymphocyte ratio extend below a ratio of 1.0 (Table4 and Table5).10.EXPECTED VALUESLeucocyte SubsetsData for four different quadrants is collected and displayed for each tube on the BDFACScan and BDFACStrak display (Figure2).The cell populations present in each quadrant for each tube are shown in Table1 and in the BDSimultestIMK-Lymphocyte Software User’s . The clinically significant results are found in the Physician BD has investigated the normal reference ranges for BDSimultest IMK-Lymphocyte parameters in 160 normal male and female subjects using the BDFACScan flow cytometer at three sites (two US clinical 40 sites and BD in San Jose, California). The expected normal reference ranges for LWB are shown in Table4. Ranges are presented as percentages of lymphocytes in the analysis gate using the BDLeucogate tube and the Percent Lymphocyte Conversion option in BDSimultest IMK-Lymphocyte software. The ranges obtained were tested for differences by clinical site, by sex of subject, and by age of subject. For each cell population, nonparametric comparisons were made between values obtained at each study site, between values obtained for males and females, and between values obtained for subjects ages 18 to 70. When the comparison indicated a significant difference, the data from the groups was not pooled, and separate reference ranges are given. The normal reference range for percentages of positive cells is calculated from a Table4.Representative reference ranges for BDSimultest IMK-Lymphocyte parameters in hematologically normal adults as percentages of total gated lymphocytes (converted) (data pooled from three clinical sites)a.This data was collected on a BDFACScan flow cytometer using BDSimultest IMK-Lymphocyte reagents and software. Software employed was BDSimultest IMK-Lymphocyte software, one of the BDSimulset™ family of software.ParameterSexAgenMean (%)95% b.The 95% range is obtained by fitting an appropriate distribution to the data and then estimating the central 95% area of the fitted distribution.Total T lymphocytesBoth18–701607259–85Total B lymphocytesBoth18–70160136.4–23Helper/inducer TlymphocytesMale18–70844329–57Female18–70754631–61Suppressor/cytotoxic TlymphocytesBoth18–701592511–38Helper/suppressor T-lymphocyte Both18–701591.90.9–3.6NK lymphocytesBoth18–70160145.6–31 41 fitted distribution.73 The ability to pool reference ranges for the BDSimultest IMK-Lymphocyte parameters (Table4) is an indication of between-laboratory reproducibility.There are small but significant differences between the sexes for helper/inducer Tlymphocytes. More than one upper and lower limit for helper/inducer T-lymphocyte ranges are presented (Table4 and Table5) because of sex differences that have been observed.Adult reference ranges should not be used with pediatric blood samples.Race can be a variable in the establishment of normal reference ranges, although insufficient data was collected by BD to determine NOTEExpected normal values can vary depending upon age, sex, or race of patient. Because of thesnce range for each parameter.Absolute CountsThe BDFACScan/CONSORT 30 or 32 system and BDFACStrak/CONSORT31 system with BDSimultest IMK-Lymphocyte software automatically calculate the absolute count if a WBC count and percentage of lymphocytes or an absolute lymphocyte count are obtained and entered into the software.Table5.Representative reference ranges (absolute counts from one clinical site) for BDSimultest IMK-Lymphocyte parameters in hematologically normal adults (ages 18SexnMean95% Range Both635.663.50–9.40Lymphocyte percentageBoth6229.819.0–43.0Total T lymphocytesBoth621.200.72–2.33 42 NOTEDo not use the BDSimultest IMK-Lymphocyte three-part differential values for absolute count determinations. The differential provided by BDSimultest IMK-Lymphocyte software should be used only for comparison with an independent differential white cell count d not be used in place of an independent laboratory differential white cell count in patient charts or entered into BDSimultest IMK-Lymphocyte software to obtain absolute counts.Absolute counts will exhibit significant interlaboratory variation depending upon the procedure employed for obtaining the WBC count and differential white count.Representative absolute counts are shown in Table5. The table is generated from data acquired at one US clinical site. BDSimultest IMK-Lymphocyte parameters are expressed as a percentage of lymphocytes in the lymphocyte analysis gate. Refer to the BD Total B lymphocytesBoth620.230.10–0.43Helper/inducer TlymphocytesMale420.670.43–1.01Female200.900.50–1.76Suppressor/cytotoxic TlymphocytesBoth610.440.17–1.05Helper/suppressor T-lymphocyte ratioBoth621.890.87–3.40NK lymphocytesBoth620.290.09–0.43a.This data was collected on a BDFACScan flow cytometer using BDSimultest IMK-Lymphocyte reagents and software. Software employed was BDSimultest IMK-Lymphocyte software, one of the BDSimulset family of software. Absolute counts are expressed as 10b.The 95% range is estimated directly from the data using the 2.5 and 97.5 percentiles.c.Hematology data were obtained from a Sysmex K1000 hematology analyzer (Sysmex Corporation of Japan)Table5.Representative reference ranges (absolute counts from one clinical site) for BDSimultest IMK-Lymphocyte parameters in hematologically normal adults (ages 18SexnMean95% Range 43 11.PERFORMANCE CHARACTERISTICSPerformance of the BDSimultest IMK-Lymphocyte reagent kit was established by testing at two US clinical sites and at BD in San Jose, Within-Sample ReproducibilityBlood samples from each of six normal and four abnormal subjects were obtained, aliquoted (two times for both normals and abnormals), stained with the BDSimultest IMK-Lymphocyte reagents, lysed, washed, and fixed within 6hours of sample collection. Flow cytometric analysis was performed within 24 hours on a BDFACScan flow cytometer in the same laboratory. Table6 and Table7 show the average within-sample reproducibility obtained for both normal and abnormal subjects.Table6.Within-sample reproducibility for BDSimultest IMK-Lymphocyte parameters (6 normal subjects) as percentages of gated lymphocytes (converted)a.This data was collected on a BDFACScan flow cytometer using BDSimultest IMK-Lymphocyte reagents and software. Software employed was BDSimultest IMK-Lymphocyte software, one of the BDSimulset family of software.ParameterMean as % LymphocytesSD as an Estimate of Within-Stain ReproducibilitySD as an Estimate of Between-Stain ReproducibilitydfTotal T lymphocytes72.70.845120.85815.6Total B lymphocytes13.50.816120.81618.0Helper/inducer Tlymphocytes45.81.080121.48610.4Suppressor/cytotoxic Tlymphocytes27.20.817121.06111.1Helper/suppressor T-lymphocyte ratio1.720.063120.07115.0NK lymphocytes13.31.384121.38417.8 44 Between-Instrument ReproducibilityUsing a protocol similar to that above, samples of whole blood from five normal subjects were divided inb.Mean is the pooled mean, Y = the mean of the individual means. Means are expressed as percentages of lymphocytes (except for helper/suppressor ratio).c.SD = standard deviation, the pooled standard deviation.d.df (normal) = degrees of freedom, the number of observations (4) minus the number of means (2), multiplied by the number of subjects (6) = 12.e.df = degrees of freedom using Satterthwaite’s method.Table7.Within-sample reproducibility for BDSimultest IMK-Lymphocyte parameters (4abnormal subjects) as percentages of gated lymphocytes (converted)a.This data was collected on a BDFACScan flow cytometer using BDSimultest IMK-Lymphocyte reagents and software. Software employed was BDSimultest IMK-Lymphocyte software, one of the BDSimulset family of software.ParameterMeanas % Lymphocytesb.Mean is the pooled mean, Y = the mean of the individual means. Means are expressed as percentages of lymphocytes (except for helper/suppressor ratio).SD as an Estimate of Within-Stain Reproducibilityc.SD = standard deviation, the pooled standard deviation.d.df (abnormal) = degrees of freedom, the number of observations (4) minus the number of means (2), multiplied by the number of subjects (4) = 8.SD as an Estimate of Between-Stain Reproducibilitydfe.df = degrees of freedom using Satterthwaite’s method.Total T lymphocytes82.90.76680.9278.1Total B lymphocytes8.810.55980.6628.4Helper/inducer Tlymphocytes31.61.06181.1189.9Suppressor/cytotoxic Tlymphocytes51.41.36981.5618.8Helper/suppressor T-lymphocyte ratio0.690.03180.03111.8NK lymphocytes8.000.70780.70711.5 45 BDSimultest IMK-Lymphocyte reagents, lysed, washed, and fixed. Samples were each run twice on two different BDFACScan flow cytometers. Results are shown in Table8.Table8.Between-instrument reproducibility for BDSimultest IMK-Lymphocyte parameters* (5 normal subjects and 2 instruments) as percentages of gated lymphocytes a.This data was collected on a BDFACScan flow cytometer using BDSimultest IMK-Lymphocyte reagents and software. Software employed was BDSimultest IMK-Lymphocyte software, one of the BDSimulset family of software.IMK-Lymphocyte ParameterInstrumentMean (%)b.Mean is the pooled mean, Y = the mean of the individual means. Means are expressed as percentages of lymphocytes (except for helper/suppressor ratio).Total T lymphocytes168.20.771.13268.70.711.04Total B lymphocytes117.20.804.66217.80.884.97Helper/inducer Tlymphocytes138.61.042.68238.10.882.32Suppressor/cytotoxic Tlymphocytes128.91.043.58227.61.003.62Helper/suppressor T-lymphocyte ratio11.510.063.7421.540.074.47NK lymphocytes112.90.463.58213.40.413.06 46 Between-Laboratory ReproducibilityBetween-laboratory reproducibility is indicated by the ability to pool the normal reference ranges for BDSimultest IMK-Lymphocyte parameters as shown in Table4.Simultest IMK-Lymphocyte vs Comparative MethodAliquots from the same blood samples from both the normal and abnormal subjects used to determine normal reference ranges were analyzed with the BDSimultest IMK-Lymphocyte reagents (using BDSimultest IMK-Lymphocyte software) and the BDSimultest IMK Plus reagents (using BDSimultest™ IMK Plus software.)Samples were stained, lysed, washed, and fixed and flow cytometric analysis performed using the BDFACScan. Results were compared using linear regression. A summary of the results is presented in Table9.c.SD = standard deviation, the pooled standard deviation.SD = = variance of the ith sample for 1k = number of samples.n = number of observations in a sample.d.CV = average coefficient of variation (= SD/Y x 100).Table9.BDSimultest IMK-Lymphocyte vs comparative method (BDSimultest IMK-Lymphocyte reagent panel and BDSimultest IMK Plus reagent panel)ParameterSlopeInterceptrnIMK PlusIMK-LymphocyteTotal T lymphocytes0.943.660.968550–9747–96Total B lymphocytes0.980.620.97851–251–29 +++---------------------------------------------------------------------------------------------------------------- 47 Stability of Stained Cell PreparationsCell preparations from six normal and four abnormal subjects were acquired and analyzed on the BDFACScan. The preparations were stored at 2°C–8°C in the dark for 24 hours and flow cytometric analysis repeated. Results demonstrate small but statistically significant differences for helper/inducer T-lymphocyte values and for helper/suppressor T-lymphocyte ratio values between day 0 and day 1. All other differences were not significant. We recommend analyzing samples within 24 hours of staining.Helper/inducer Tlymphocytes0.95–0.270.96848–688–65Suppressor/cytotoxic Tlymphocytes1.01–6.030.938419–7412–67Helper/suppressor T-lymphocyte ratio1.25–0.080.90840.17–3.00.21–3.17NK lymphocytes0.960.370.96852–321–33a.This data was collected on a BDFACScan flow cytometer using BDSimultest IMK-Lymphocyte reagents and software. Software employed was BDSimultest IMK-Lymphocyte software, one of the BDSimulset family of software.b.The two methods differ in that the BDSimultest IMK Plus kit uses CD4/CD8 to identify helper/inducer and suppressor/cytotoxic T lymphocytes, whereas the BDSimultest IMK-Lymphocyte uses CD3/CD4 and CD3/CD8 to distinguish true helper/inducer and true suppressor/cytotoxic T-lymphocyte subsets.c.Includes monocytes.d.Includes NK lymphocytes.e.Includes monocytes and NK lymphocytes.Table9.BDSimultest IMK-Lymphocyte vs comparative method (BDSimultest IMK-Lymphocyte reagent panel and BDSimultest IMK Plus reagent panel)ParameterSlopeInterceptrnIMK PlusIMK-Lymphocyte 48 Cross-ReactivityThe specificity of these monoclonal antibodies has been established by blind testing at a number of laboratories by the International Leucocyte Workshop Group.29,31,38,42,61,62The CD45 antibody has been reported to react weakly with mature circulating erythrocytes and platelets. The CD14 antibody reacts with granulocytes, as well as monocytes/macrophages. The CD4 antibody reacts with monocytes as well as helper/inducer Tlymphocytes.The CD8 antigen is present on a subset of NK lymphocytes. The CD16 antibody reacts weakly with neutrophils.Potentially interfering granulocytes should be eliminated from the lymphocyte gate using their light-scatter properties.Although the Control reagent (Reagent B) is designed to detect nonspecific staining, nonspecific staining due to poor condition of cells be inferred if no clear separation between negative and positive cells is seen on the fluorescence plot or if more than 5% of events for the Control tube appear outside Q3. Consult the Troubleshooting section at the end of this IFU if nonspecific staining is seen.Linearity-RecoveryAll lots of BDSimultest IMK-Lymphocyte reagents are tested on cell suspensions to ensure optimal performance with 100 µL of whole blood. For this study, the normal range was 3.5 x 10WBC/µL. Results are expected to be linear from 3.5 x 10to 9.4 x 10WBC/µL. 49 12.TROUBLESHOOTINGProblemPossible CausesInsufficient separation between negative and positive populations.1) Decreased fluorescence due to incorrect PMT voltage or amplifier gain.2) Insufficient antibody reagent. No separation found between debris and lymphocyte clusters on light-scatter dot plot.1) Presence of excess platelets or nucleated red blood cells or unlysed red blood cells.2) FSC amplifier gain improperly set.3) Sample not stained within 6 hours of collection.4) FSC threshold set too high or too low.Suboptimal antibody-staining performance.1) Sample not stained within 6 hours of collection.2) Analysis gate selected by software included too many nonlymphocyte events.3) Fluorescence markers set incorrectly.Solution1) Run BDAutocomp software with BDCalibrite beads. Must meet specifications for intensity in fluorescence channels.2) Check pipet calibration, reagent volume added, as well as volume and concentration of cells added. Check reagent storage conditions.1) Restain a fresh sample. Wash cells at lower speed, for example, 100 for 15 minutes at room temperature (202) Adjust FSC amplifier gain such that the left edge of the lymphocyte cluster begins at approximately channel 50 (256-channel scale). This is especially important when going from setup/compensation with BDAutocomp software to whole blood determinations. Refer to the appropriate user’s guide or instrument manual.3) Collect a fresh sample to stain.4) Adjust FSC threshold so that there are approximately 10 channels of debris (256-channel scale). 50 1) Collect a fresh sample to stain.2) Reprocess data using CONSORT 30, BDLysys™ II, or BDSimulset software and set a manual gate. NOTE: A tight lymphocyte gate might exclude large lymphocytes, for example, NK lymphocytes, lymphoblasts. If possible, reacquire samples after optimization of scatter, adjust SSC PMT, FSC amplifier gain, and FSC threshold to separate lymphocyte clusters from debris. Refer to the appropriate software user’s guide.3) Reprocess data using CONSORT 30, BDLysysII, or BDSimulset software and set manual fluorescence markers.ProblemPossible CausesSoftware unable to set an acceptable lymphocyte gate.1) Sample is lymphopenic.2) FSC threshold set too high or too low.3) FSC and/or SSC amplifier gain improperly set.4) Sample not stained within 6 hours of collection.5) Software marker setting not appropriate.Unstained cells in the stained sample move over marker set with Control tube (B) into Q4 or Q1.1) Nonspecific staining due to sample containing dead or damaged cells.2) Nonspecific staining due to Fc receptors.Staining is dim or inconsistent.1) Cell concentration too high at staining step.2) Insufficient reagent.3) Cells not analyzed within 24 hours of staining.4) Improper medium preparation (sodium azide omitted).5) Buffer or fixative at improper pH.6) Possible contamination of PBS, or fixative.Solution 51 1) Concentrate cells using a density-gradient method or reacquire a larger number of events using CONSORT 30 or BDLysysII.2) Adjust FSC threshold so that there are approximately 10 channels of debris (256-channel scale).3) Adjust FSC amplifier gain such that the left edge of the lymphocyte cluster begins at approximately channel 50 (256-channel scale). Adjust SSC amplifier gain in order to optimize cell populations. This is especially important when going from setup/compensation with BDAutocomp software to whole blood determinations. Refer to the appropriate user’s guide.4) Collect a fresh sample to stain.5) Reacquire data using CONSORT 30 or BDLysysII.1) Collect a fresh sample to stain.2) Check for excessive monocyte, granulocyte, or debris contamination in the lymphocyte analysis gate.1) Check and adjust cell concentration; repeat staining with fresh sample.2) Repeat staining with proper amount of reagent.3) Repeat staining with fresh sample; analyze promptly.4) Use sodium azide in staining medium and washing steps.5) Prepare freshly diluted PBS with reagent-grade water and fresh fixative with PBS with sodium azide, or prepare fresh BDCellWASH. Adjust to the proper pH. Filter through a 0.2-µm filter.6) See 5 above.ProblemPossible CausesFew or no cells.1) Cell concentration too low.2) Centrifuge speed too low.3) Flow cytometer malfunctioning. 52 WARRANTYUnless otherwise indicated in any applicable BD general conditions of sale for non-US customers, the following warranty applies to the purchase of these products.PRODUCTSSOLDHEREUNDERWARRANTEDONLYCONFORMANDCONTENTSSTATEDTHEPRODUCTLABELINGTHEDELIVERYTHECUSTOMERDISCLAIMSOTHERWARRANTIESEXPRESSEDIMPLIEDINCLUDINGWARRANTIESMERCHANTABILITYANDFORANYPARTICULARPURPOSEANDNONINFRINGEMENTSOLELIABILITYREPLACEMENTPRODUCTSREFUNDPURCHASEPRICENOTPROPERTYDAMAGEINCIDENTALCONSEQUENTIALDAMAGESINCLUDINGPERSONALINJURYECONOMICLOSSCAUSEDPRODUCTREFERENCES1Fitzgerald-Bocarlsly P, Herberman R, Hercend T, et al. A definition of natural killer cells. In: Ades E, Lopez C, eds. Natural Killer Cells and Host Defense. Basel: Karger; 1989:1.2Brain P, Gordon J, Willets W. Rosette formation by peripheral lymphocytes. Clin Exp Immunol. 1970;6:681-688.Increased autofluorescence.1) Possible contamination of PBS, or fixative. 2) Poor sample preparation.Solution1) Resuspend fresh sample at a higher concentration. Repeat staining and analysis.2) Check centrifuge calibration. Repeat prep using correct centrifuge speed.3) Troubleshoot instrument.1) Prepare freshly diluted PBS with reagent-grade water and fresh fixative; filter PBS and fixative through a 0.2-µm filter.2) Collect a fresh sample and stain.ProblemPossible Causes 53 3Jondal M, Holm G, Wigzell H. Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J Exp Med. 1972;136:207-215.4Wolde-Mariam W, Peter J. Recent diagnostic advances in cellular immunology. Diagnost Med. 1984;7:25-32.5Froland S, Natvig J, Berdal P. Surface-bound immunoglobulin as a marker of Blymphocytes in man. Nature. 1971;234:251-252.6Bellanti J, Peters S. Diagnostic applications of immunology. 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J Clin Lab Anal1990;4:193-195.73Reichert T, DeBruyère M, Deneys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopathol. 1991;60:190-208.74Prince H, Hirji K, Waldbeser L, Plaeger-Marshall S, Kleinman S, Lanier L. Influence of racial background on the distribution of T cell subsets and Leu-11-positive lymphocytes in healthy blood donors. Diag Immunol. 1985;3:33-37. 02/201523-2568-06 BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2015 BD Becton, Dickinson and CompanyBD Biosciences2350 Qume DriveSan Jose, CA 95131 USABenex LimitedPottery Road, Dun Laoghaire,Co. Dublin, IrelandTel +353.1.202.5222Fax +353.1.202.5388BD BiosciencesEuropean Customer SupportTel +32.2.400.98.95Fax +32.2.401.70.94help.biosciences@europe.bd.comBecton Dickinson Pty Ltd,4 Research Park Drive,Macquarie University Research Park,North Ryde NSW 2113, AustraliaBecton Dickinson Limited,8 Pacific Rise, Mt. Wellington,Auckland, New Zealandbdbiosciences.comClinicalApplications@bd.com BDSimultest™ IMK-LymphocyteCatalog No. 340182