BD Simultest IMK Plus

BD Simultest IMK Plus Catalog No. 349217 IVD BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. 2015 BD 02/2015 23-3178-04 Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA 95131 USA Benex Limited Pottery Road, Dun Laoghaire, Co. Dublin, Ireland Tel +353.1.202.5222 Fax +353.1.202.5388 BD Biosciences European Customer Support Tel +32.2.400.98.95 Fax +32.2.401.70.94 help.biosciences@europe.bd.com Becton Dickinson Pty Ltd, 4 Research Park Drive, Macquarie University Research Park, North Ryde NSW 2113, Australia Becton Dickinson Limited, 8 Pacific Rise, Mt. Wellington, Auckland, New Zealand bdbiosciences.com ClinicalApplications@bd.com

CONTENTS 1. INTENDED USE... 5 2. SUMMARY AND EXPLANATION... 5 Clinical Applications... 6 3. PRINCIPLES OF THE PROCEDURE... 8 4. REAGENTS... 11 Reagents Provided, Sufficient for 50 Tests... 11 Precautions... 17 5. INSTRUMENT... 18 6. SPECIMEN AND COLLECTION PREPARATION... 19 Interfering Conditions... 20 7. PROCEDURE... 20 Reagents Provided... 20 Reagents and Materials Required But Not Provided... 20 Staining and Fixing the Cells... 21 Flow Cytometry... 23 Quality Control... 27 8. RESULTS... 29 Calculation of Corrected Counts... 29 Calculation of Absolute Counts... 33 Three-Part Differential... 34 9. LIMITATIONS... 35 iii

10. EXPECTED VALUES... 38 Leucocyte Subsets... 38 Absolute Counts... 41 11. PERFORMANCE CHARACTERISTICS... 43 Within-Sample Reproducibility... 43 Between-Instrument Reproducibility... 44 Between-Laboratory Reproducibility... 44 BD Simultest IMK Plus vs Comparative Methods... 44 Stability of Stained Cell Preparations... 46 Cross-Reactivity... 47 Linearity-Recovery... 48 12. TROUBLESHOOTING... 49 REFERENCES... 52 WARRANTY... 58 iv

1. INTENDED USE The BD Simultest IMK Plus reagent kit is a two-color direct immunofluorescence method for enumerating percentages of the following mature (nonblast) human lymphocyte subsets in erythrocytelysed whole blood (LWB): T (CD3 + ) and activated T (CD3 + HLA-DR + ) lymphocytes, B (CD19 + ) lymphocytes, helper/inducer (CD4 + ) and suppressor/cytotoxic (CD8 + ) lymphocytes, and natural killer (NK) (CD16 + or CD56 + or both) lymphocytes. The helper/suppressor ratio (CD4 + /CD8 + ) can also be determined. 2. SUMMARY AND EXPLANATION Human lymphocytes can be divided into three major populations based on their biologic function and cell-surface antigen expression that correlates with function: T lymphocytes, B lymphocytes, and NK lymphocytes. T lymphocytes participate in antigen-specific cellmediated immunity and regulate the secretion of immunoglobulin by B lymphocytes. T lymphocytes can also be classified based on their functional properties as helper/inducer, suppressor/cytotoxic, or activated T lymphocytes. 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 T lymphocytes by rosetting them with sheep red blood cells (SRBCs) 1 and identifying B lymphocytes using SRBCs coated with fluorochromelabeled polyclonal antibodies to surface immunoglobulin. 1 Cytolytic lymphocyte assays included the hemolytic plaque assay 2 and a cytotoxicity assay to detect the release of radioactive chromium. 3 Antibody reagents have been used to distinguish cell-surface antigens by fluorescence microscopy, electron microscopy, and radioactive label assay. 4 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 5

disposal considerations. Flow cytometry using indirect immunofluorescence, while providing assay sensitivity, 5 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. 6 8 The LWB method used with this kit does not require density-gradient separation and therefore allows for shorter sample preparation time and less handling of whole blood 6 (see the BD Monoclonal Antibodies Source Book, Section 2.2). The development of monoclonal antibody 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 simultaneous quantification of two or more leucocyte subpopulations. Immune monitoring is facilitated by flow cytometers, such as the BD FACScan, which accurately characterize cells by means of fourparameter analysis. The BD FACScan, when used with BD Simultest IMK Plus software, characterizes up to 50,000 cells in a single sample by simultaneous analysis of forward scatter (FSC), side scatter (SSC), and multicolor fluorescence. Clinical Applications* Total T- and B-lymphocyte percentages are used to characterize some forms of immunodeficiency diseases 9,10 and some types of autoimmune disease. 11,12 * Not all studies cited in this section employed BD reagents. 6

Activated T lymphocytes (HLA-DR + T lymphocytes) can be elevated in states of immune activation, 13 which can be caused by infection, 14 or impending rejection in the case of transplant monitoring. 15 The actual cause of immune activation must be verified by additional clinical and laboratory tests. Helper/inducer lymphocytes are a subset of T (CD3 + ) lymphocytes, which also express the CD4 antigen. Suppressor/cytotoxic lymphocytes express the CD8 antigen and are principally a subset of T (CD3 + ) lymphocytes, although a subset of NK lymphocytes is also CD8 +. 16 Determining the percentages of CD4 + and CD8 + lymphocytes 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 is depressed and the relative percentage of the CD8 + subset is elevated in many patients with congenital or acquired immunodeficiencies 9 such as severe combined immunodeficiency (SCID) 9 and acquired immune deficiency syndrome (AIDS). 17 The percentage of suppressor/cytotoxic cells can be outside the normal reference range in some autoimmune diseases 11 and in certain immune reactions such as acute graft-versus-host disease (GVHD) 18 and transplant rejection. 15 The relative percentage of the CD8 + lymphocyte population can often be decreased in active systemic lupus erythematosus (SLE) but can also be increased in SLE patients undergoing steroid therapy. 1 The CD4 + /CD8 + (helper/suppressor) lymphocyte ratio, quantified as the ratio of CD4 FITC-positive lymphocytes to CD8 PE-positive lymphocytes, has been used to evaluate the immune status of patients with, or suspected of developing, autoimmune disorders 11,12 or immune deficiencies. 1,17 In many cases, the relative percentages of helper lymphocytes decline and suppressor lymphocytes increase in immune deficiency states. These states can also be marked by T-cell lymphopenia. 10,19 In addition, the ratio has been used to monitor bone 7

marrow transplant patients for onset of acute GVHD. 18 While a useful indicator, use of the CD4 + /CD8 + (helper/suppressor) lymphocyte ratio has specific limitations discussed in items Section 9, Limitations, of this instructions for use (IFU). NK lymphocytes, identified as being CD3-negative and CD16- or CD56-positive or both, 20 have been shown to mediate cytotoxicity against certain tumors and virus-infected target cells. NK-mediated cytotoxicity does not require class I or class II major histocompatibility complex (MHC) molecules to be present on the target cell. 21 NKlymphocyte activity is depressed in AIDS patients and, in some instances, in AIDS-related complex and lymphadenopathy syndrome. 22 3. PRINCIPLES OF THE PROCEDURE The kit contains six pairs of BD Simultest murine monoclonal antibody reagents conjugated with FITC and PE that include BD Leucogate (CD45/CD14) (Reagent A) for establishing a lymphocyte acquisition gate and BD Simultest Control IgG 1 FITC/IgG 2a PE (Reagent B) for setting fluorescence markers around the negative population and detecting nonantigen-specific antibody binding. The kit also contains BD FACS lysing solution (Reagent G) for preparing cells by the LWB method. We recommend the BD FACScan for flow cytometry and BD Simultest IMK Plus software for evaluating the data. A fresh peripheral blood sample is collected by venipuncture and stained within 6 hours with each of the six antibody reagents from the BD Simultest IMK Plus kit. When the 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 then treated with BD FACS lysing solution to lyse erythrocytes, and washed prior to flow cytometric analysis. 8

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 excited at 488 nm by an argon-ion laser. The FITC-stained lymphocytes emit yellow-green light (emission maximum approximately 515 nm) while the PE-stained lymphocytes emit redorange light (emission maximum approximately 580 nm). The cells also interact with the laser beam by scattering the light. The forward-scattered light provides a measure that correlates well with cell size, while the side-scattered light is an indicator of cellular granularity. The BD FACScan flow cytometer used with BD Simultest IMK Plus software counts a sufficient number of cells to ensure that a minimum of 2,000 lymphocytes are included in the acquisition gate. The data files should be saved in list mode and given logical names to aid in retrieval for subsequent analysis by BD Simultest IMK Plus software. BD Simultest IMK Plus software uses BD Leucogate, Reagent A, to establish a lymphocyte acquisition gate that includes greater than or equal to 98% of the normal mature (nonblast) lymphocytes in the sample. However, if the gate contains greater than or equal to 3% monocytes, the software automatically reduces or tightens the lightscatter 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 BD Leucogate lymphocyte acquisition gate on the basis of SSC, FSC, and fluorescence properties. Refer to the BD Simultest IMK Plus User s Guide for details on how the software sets the gates and automatically 9

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 therefore require testing by other methods. If the software is unable to set a gate, a message will appear on the printout. See the BD Simultest IMK Plus 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 present, particularly that caused by Fc receptors. When greater than 5% of the 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 the laboratory printout for the Control Tube B. When the negative Control tube is being processed by the software, the operator should check for error messages that would indicate nonantigen-specific antibody binding. The negative Control is a mixture of conjugated monoclonal antibodies with the same fluorochromes (FITC and PE) as the test reagents. The Control antibodies are specific to 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 acquisition gate set with BD Leucogate (Tube A) and the fluorescence markers determined using the Control (Tube B) are used to analyze the subsequent tubes (C through F). When the Quadrant Correction software option has been selected from the Main Menu of BD Simultest IMK Plus software, the lymphocyte subpopulations in Tubes C through F are enumerated and then expressed as percentages of lymphocytes in the acquisition gate. BD Simultest IMK Plus software provides a report quantifying these 10

immunologically significant lymphocyte subsets in LWB as percentages of total circulating (nonblast) human lymphocytes and reports a CD4 + / CD8 + lymphocyte ratio. If this software option is not selected, results will be expressed as percentages of the total gated events. For quality control purposes, a three-part differential for percentages of monocytes, granulocytes, and lymphocytes is determined automatically by the software and is printed only for comparison with results from a standard laboratory differential white count. 23 To obtain the three-part differential, the software uses an algorithm to identify 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 and granulocytes can also be distinguished on the basis of their SSC signal. Lymphocytes exhibit a low SSC signal and granulocytes a high SSC signal. NOTE The differential count provided by BD Simultest IMK Plus 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 nor entered into BD Simultest IMK Plus software to obtain absolute counts. 4. REAGENTS Reagents Provided, Sufficient for 50 Tests All monoclonal antibody reagents contain murine immunoglobulins conjugated to either FITC or PE in 1.0 ml of buffered saline with gelatin and 0.1% sodium azide. The monoclonal antibodies are derived from fusion of mouse myeloma cells with spleen cells or lymph node cells of BALB/c mice. The fluorescein-to-protein ratio (F:P) for BD monoclonal antibody reagents is within the range of 2 to 10. The F:P ratio for each reagent has been optimized for its intended use. 11

Reagent A, BD Leucogate (CD45/CD14), 1.0 ml BD Leucogate is used to define and evaluate the light-scatter gate that distinguishes lymphocytes from granulocytes, monocytes, unlysed or nucleated red blood cells (RBCs), and debris. This reagent contains FITC-labeled CD45, clone 2D1, 24 26 for identification of leucocytes, and PE-labeled CD14, clone MφP9, 27 29 for identification of monocytes. The CD45 30 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 composed of IgG 1 heavy chains and kappa light chains. The CD45 antigen is present on all human leucocytes and has a role in signal transduction, modifying signals from other surface molecules. 30 The molecular weight of the antigen recognized by this antibody is 170 to 220 kilodaltons (kda). 30 The CD14 27 antibody was derived from 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 antibody is composed of mouse IgG 2b heavy chains and kappa light chains. The molecular weight of the CD14 antigen recognized by this antibody is 53 kda. 31 The antigen is a myeloid differentiation antigen 31 and is present on 75% to 90% of human monocytes. 32 The CD14 antibody reacts weakly with granulocytes. 33 Reagent B, Control, 1.0 ml The isotype control reagent is used to set the FL1 and FL2 quadrant markers around the unstained (negative) lymphocyte population to establish a boundary between negative and positive cell populations and estimates nonantigen-specific antibody binding, in particular that caused by Fc receptors. It contains FITC-labeled IgG 1, clone X40, and PE-labeled IgG 2a, clone X39, murine monoclonal antibodies that react 12

specifically with keyhole limpet hemocyanin (KLH), an antigen not present on human leucocytes. Reagent C, CD3/CD19, 1.0 ml CD3/CD19 is used to enumerate T and B lymphocytes. It contains FITC-labeled CD3, clone SK7, 34 37 for the identification of T lymphocytes, and PE-labeled CD19, clone 4G7, 38 for the identification of B lymphocytes. The CD3 antibody 36 is derived from hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human thymocytes. This antibody is composed of mouse IgG 1 heavy chains and kappa light chains. CD3 reacts with the epsilon chain of the CD3 antigen/t-cell antigen receptor (TCR) complex. This complex is composed of at least four proteins that range in molecular weight from 20 to 30 kda. 39 The antigen recognized by CD3 antibodies is non covalently associated with either α/β or γ/δ TCR (70 to 90 kda). 40 The CD19 antibody 38 is derived from hybridization of mouse P3-X63-Ag8.653 myeloma cells with spleen cells of BALB/c mice immunized with human chronic lymphocytic leukemia (CLL) cells. The antibody is composed of IgG 1 heavy chains and kappa light chains. The CD19 antigen is present on human B lymphocytes at all stages of maturation but is lost on plasma cells. The antigen can be involved in activation and proliferation of B lymphocytes. 41 The molecular weight of the antigen recognized by this antibody is 90 kda. 41 Reagent D, CD4/CD8, 1.0 ml CD4/CD8 is used to simultaneously characterize helper/inducer and suppressor/cytotoxic lymphocytes. It contains FITC-labeled CD4, clone SK3, 42,43 for the identification of helper/inducer lymphocytes, and PElabeled CD8, clone SK1, 42,43 for the identification of suppressor/ cytotoxic lymphocytes. 13

The CD4 antibody 42 is derived from hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood T lymphocytes. It is composed of mouse IgG 1 heavy 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). 44,45 The antigen has a molecular weight of 59 kda. The cytoplasmic portion of the antigen is associated with the protein tyrosine kinase p56 lck. The CD4 antigen can regulate the function of the CD3 antigen/ TCR complex. 46 CD4 reacts with monocytes/macrophages and helper/ inducer T lymphocytes. 47 The CD8 antibody 42 is derived from hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood T lymphocytes. It is composed of mouse IgG 1 heavy chains and kappa light chains. The CD8 antigen is present on the human suppressor/cytotoxic T-lymphocyte subset 35,43 as well as on a subset of NK lymphocytes. 16 The CD8 antigenic determinant interacts with class I MHC molecules, resulting in increased adhesion between the CD8 + T lymphocytes and the target cells. 48-50 Binding of the CD8 antigen to class I MHC enhances the activation of resting T lymphocytes. 48 51 The CD8 antigen is expressed as a disulfide-linked bimolecular complex with a 32-kDa α subunit. 52,53 The CD8 antigen is coupled to p56 lck. The CD8:p56 lck complex can play a role in T- lymphocyte activation through mediation of the interactions between the CD8 antigen and the CD3 antigen/tcr complex. 50,51 Reagent E, CD3/Anti HLA-DR, 1.0 ml CD3/Anti HLA-DR is used to enumerate T lymphocytes, DR + non- T lymphocytes (primarily B lymphocytes), and activated T lymphocytes. It contains FITC-labeled CD3, clone SK7, 34 37 for the identification of T lymphocytes, and PE-labeled Anti HLA-DR, clone L243, 54 for the identification of DR + non-t lymphocytes and activated T lymphocytes. 14

The Anti HLA-DR antibody is derived from hybridization of mouse NS1/1-AG4 myeloma cells with spleen cells from BALB/c mice immunized with the human lymphoblastoid B-cell line RPMI 8866. It is composed of mouse IgG 2a heavy chains and kappa light chains. The HLA-DR antigen (human leucocyte antigen, D-related) is a human class II MHC molecule. The antigen is a transmembrane glycoprotein composed of α- and β subunits that have molecular weights of 36 and 27 kda, respectively. 54 55 The antibody reacts with a nonpolymorphic HLA-DR epitope. 54 56 The antigen is expressed on B lymphocytes, monocytes, macrophages, activated T lymphocytes, activated NK lymphocytes, and human progenitor cells. 14,19,57 60 Reagent F, CD3/CD16+CD56, 1.0 ml CD3/CD16+CD56 is used to identify T and NK lymphocytes. It contains FITC-labeled CD3, clone SK7, 34 37 to identify T lymphocytes. It also contains PE-labeled CD16, clone B73.1, 61 64 and PE-labeled CD56, clone MY31, 63,65 to identify NK-lymphocyte populations as well as T-lymphocyte subsets. The CD16 antibody 64 was derived from hybridization of mouse P3X-63-Ag8.653 myeloma cells with spleen cells of BALB/c mice immunized with NK lymphocytes. The antibody is composed of IgG 1 heavy chains and kappa light chains. 61 63 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. 20 The CD56 antibody 65 was derived from hybridization of mouse Sp2/0 myeloma cells with cells of (B6xBALB/c)F 1 mice immunized with the KG1a cell line. The antibody is composed of IgG 1 heavy chains and kappa light chains. The molecular weight of the glycosylated antigen recognized by this antibody ranges from 175 to 220 kda 20,66 (or 137 kda when deglycosylated 67 ) and is present on NK lymphocytes. The CD56 antigen is involved in neuronal homotypic cell adhesion and cell differentiation during embryogenesis. 65 15

Reagent G, 10X BD FACS lysing solution, 60 ml Reagent G contains 10X buffered BD FACS 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 shown on the label. For use, dilute 1:10 with room temperature (20 C 25 C) reagent-grade water. Store in a glass container at room temperature. The prepared solution is stable for 1 month at room temperature. Concentration values are listed in the following table: Reagent Component Concentration (µg/ml) A: BD Leucogate CD45 FITC 25 CD14 PE 12.5 B: Control IgG 1 FITC 25 IgG 2a PE 12.5 C: CD3/CD19 CD3 FITC 25 CD19 PE 6 D: CD4/CD8 CD4 FITC 1.5 CD8 PE 25 E: CD3/HLA-DR CD3 FITC 25 Anti-HLA-DR PE 3.1 F: CD3/CD16+CD56 CD3 FITC 25 CD16 PE 12.5 CD56 PE 25 16

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 date. The antibody reagents should not be frozen or exposed to direct light during storage or during incubation with cells. 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 the reagents, such as precipitation or discoloration, indicates instability or deterioration. In such cases, the reagents 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, 10% formaldehyde, CAS number 50-00-0, and 3.51% methanol, CAS number 67-56-1. Danger 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. 17

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. WARNING All biological specimens and materials coming into contact with them are considered biohazards. Handle as if capable of transmitting infection 68,69 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. INSTRUMENT The BD Simultest IMK Plus kit 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. 18

The following instrument system is recommended: BD FACScan flow cytometer system equipped for three-color fluorescence detection and two-parameter light-scatter detection. For detailed information on use, refer to the BD FACScan User s Guide. BD CONSORT 30 or CONSORT 32 computer system and peripherals (included in the BD FACScan system). For detailed information on use, refer to the BD CONSORT 30 Software User s Guide or the BD CONSORT 32 System User s Guide. BD Simultest IMK Plus software. For detailed information on use, refer to the BD Simultest IMK Plus User s Guide. BD Calibrite beads (Catalog No. 349502). These beads can be used for setting the photomultiplier tube (PMT) voltages, setting the fluorescence compensation, and checking instrument sensitivity on the BD FACScan flow cytometer. For detailed information on use, refer to the BD Calibrite Beads IFU. BD Autocomp software. For detailed information on use, refer to the BD Autocomp Software User s Guide. All performance characteristics were obtained using the above instrument system. Other systems can have different characteristics. 6. SPECIMEN AND COLLECTION PREPARATION Collect blood aseptically by venipuncture 5,70 into a sterile EDTA (lavender top) BD Vacutainer blood collection tube. A minimum of 1 ml of whole blood is required for this procedure. Blood should be stained within 6 hours of drawing 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 count (WBC) and a differential white count should be obtained from the same sample of whole blood before staining. An 19

acceptable WBC concentration range is from 3.5 x 10 3 to 9.8 x 10 3 WBC/µL. Samples with counts greater than 9.8 x 10 3 WBC/µL must be diluted with 1X phosphate-buffered saline (PBS) containing 0.1% sodium azide (see Section 9, Limitations). For samples with counts less than 3.5 x 10 3 WBC/µL, more blood might be needed and a separation procedure can be required to concentrate the cells. Interfering Conditions Previously fixed and stored cells 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 abnormal (blast) cells or unlysed or nucleated RBCs can interfere with test results. Hemolyzed samples or samples with less than 1 ml of whole blood in the collection tube should be rejected. CAUTION Use standard precautions when obtaining, handling, and disposing of all human blood samples and potentially carcinogenic reagents. 7. PROCEDURE Reagents Provided See Reagents Provided, Sufficient for 50 Tests, and Precautions in Section 4, Reagents. Reagents and Materials Required But Not Provided BD Vacutainer EDTA blood collection tubes or equivalent. Falcon * disposable 12 x 75-mm polystyrene test tubes (or equivalent. Vortex mixer. * Falcon is a registered trademark of Corning Incorporated. 20

Low-speed centrifuge (200g) with swinging bucket rotor and 12 x 75-mm tube carriers. Vacuum aspirator with trap. Micropipettor with tips. BD CellWASH (Catalog No. 349524) or a wash buffer of PBS with 0.1% sodium azide. BD CellFIX (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 1 week. BD FACSFlow sheath fluid (Catalog No. 342003). CAUTION Use only BD FACSFlow sheath fluid diluent to dilute BD Calibrite beads. Reagent-grade (both distilled and deionized) water. Staining and Fixing the Cells Whole blood samples are stained with Reagents A through F. Diluted Reagent G, 1X BD FACS lysing solution, is then used to lyse RBCs following staining. If the WBC count is sufficient, cell separation is not required prior to staining. See Section 6, Specimen and Collection 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 A through 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, 20 µl of Reagent E into tube E, and 20 µl of Reagent F into tube F. 3. For each patient sample, use a fresh micropipettor tip and carefully add 100 µl of the correct concentration of well-mixed, 21

anticoagulated whole blood patient sample into the bottom of each of the six labeled tubes. The required WBC concentration is 3.5 x 10 3 to 9.8 x 10 3 WBC/µL. Vortex thoroughly at low speed for 3 seconds and incubate for 15 to 30 minutes at room temperature (20 C 25 C). NOTE Protect 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 might not be stained with the reagent. 4. Dilute 10X BD FACS lysing solution to 1X following the instructions for Reagent G under Reagents Provided, Sufficient for 50 Tests, in Section 4, Reagents. Add 2 ml of room temperature (20 C 25 C) 1X BD FACS lysing solution to each tube. Immediately vortex thoroughly at low speed for 3 seconds and incubate for 10 to 12 minutes at room temperature (20 C 25 C) in the dark. Do not exceed 12 minutes. NOTE Avoid prolonged exposure of the cells to lytic reagents, which can cause white cell destruction. See Section 9, Limitations. 5. Immediately after incubation, centrifuge tubes at 300g for 5 minutes at room temperature (20 C 25 C). 6. Aspirate the supernatant, leaving approximately 50 µl of residual fluid in each tube to avoid disturbing the pellet. 7. Vortex thoroughly at low speed to resuspend the cell pellet in the residual fluid and then add 2 ml of BD CellWASH solution or PBS with 0.1% sodium azide to each tube. Vortex thoroughly at low speed for 3 seconds. Centrifuge at 200g for 5 minutes at room temperature (20 C 25 C). 8. Aspirate the supernatant, leaving approximately 50 µl of residual fluid in the tube to avoid disturbing the pellet. 22

9. Vortex thoroughly at low speed to resuspend the cell pellet in the residual fluid and then add 0.5 ml of BD CellFIX solution or 1% paraformaldehyde to each tube. Vortex thoroughly at low speed for 3 seconds. Make sure that the cells are well mixed with the fixing solution. 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) before putting them through the flow cytometer to help reduce aggregation. Flow Cytometry Follow the BD instructions for two-color flow cytometric analysis and refer to Section 5, Instrument. The following general approach is recommended. The BD FACScan flow cytometer is first prepared for sample analysis using BD Calibrite beads and BD Autocomp software. The stained sample tubes are then run on the flow cytometer and analyzed with the BD Simultest IMK Plus software. Refer to the BD FACScan User s Guide, the BD Calibrite Beads IFU, the BD Autocomp Software User s Guide, and the BD Simultest IMK Plus User s Guide for detailed instructions for use. We recommend that patient data be stored to allow subsequent analysis of data files. Set up and adjust compensation of the BD FACScan flow cytometer. Using BD Calibrite beads and BD Autocomp software, set photomultiplier tube (PMT) voltages, adjust fluorescence compensation, and check detector sensitivity. Refer to the BD Calibrite Beads IFU, the BD Autocomp Software User s Guide, and the BD FACScan User s Guide for details. Run all sample tubes through the flow cytometer and acquire data in list mode files using BD Simultest IMK Plus software. Refer to the BD Simultest IMK Plus User s Guide for detailed instructions for use of the software. The software automatically collects a sufficient 23

number of events to obtain a minimum of 2,000 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 (Q1) (low yellow-green/high red-orange), Q2 (high yellow-green/high red-orange, or dual-fluorescence), Q3 (low yellow-green/low red-orange), and Q4 (high yellow-green/low redorange) (see Figure 1). Figure 1 The fluorescence display quadrants (indicated as Q1 Q4) and the corresponding colors. Q1 Low yellowgreen/high red-orange Q2 High yellow-green/high red-orange (dual fluorescence) Q3 Low yellowgreen/low red-orange Q4 High yellow-green/low red-orange The BD Leucogate tube A is used to gate on lymphocytes. The BD FACScan with BD Simultest IMK Plus software automatically sets a lymphocyte acquisition gate to eliminate most debris, monocytes, and granulocytes (see Figure 2 and Figure 3). Refer to the BD Simultest IMK Plus 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. Errors in sample gating can be caused by the sample preparation or instrument setup. The Control tube B is used to set fluorescence intensity markers. BD Simultest IMK Plus software automatically uses the lymphocyte 24

acquisition gate set with BD Leucogate and then establishes FL1 and FL2 markers. These markers define the boundaries between positively stained and unstained events 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. Figure 2 BD FACScan LWB sample from a hematologically normal male patient stained with BD Simultest IMK Plus reagents. BD Leucogate was used to reduce debris, monocytes, and granulocytes in the gate shown under tube A. Dot plot displays of FL1 (x-axis) versus FL2 (y-axis) are shown for tubes B through F. Tube A Tube B Tube C CD8 SSC IgG 2a CD19 FSC IgG 1 CD3 Tube D Tube E Tube F Anti HLA- DR CD16+CD56 CD4 CD3 After the markers are set, if more than 5% of the total counts for tube B remain in Q1 (low yellow-green/high red-orange), Q2 (high yellowgreen/high red-orange), and Q4 (high yellow-green/low red-orange), nonantigen-specific antibody binding is suspected. The software notifies the operator with a message too much nonspecific staining. 25 CD3

If this error message appears, a new sample should be obtained from the original aliquot of anticoagulated whole blood and the entire staining procedure should be repeated. Figure 3 Mononuclear cell populations in Q1 through Q4 for tubes C through F. The populations represent the primary reagent reactivity. (For a complete list of reactivity, see the reagent descriptions under Reagents Provided, Sufficient for 50 Tests, in Section 4, Reagents.) Tube C (CD3/CD19) Tube D(CD4/CD8) Q1 CD3 CD19 + B lymphocytes Q2 Nonantigen-specific antibody binding a Q1 CD4 CD8 + suppressor/ cytotoxic and NK lymphocytes Q2 CD4 + CD8 + lymphocytes Q3 Unstained lymphocytes; contaminating monocytes, granulocytes, and debris Q4 CD3 + CD19 T lymphocytes Q3 Unstained lymphocytes; granulocytes and debris Q4 CD4 + CD8 helper/inducer lymphocytes; monocytes a. See Section 9, Limitations. Tube E (CD3/Anti HLA-DR) Tube F (CD3/CD16+CD56) Q1 CD3 - Anti HLA-DR + B lymphocytes, activated NK lymphocytes; monocytes/ macrophages Q3 Unstained lymphocytes; contaminating monocytes, granulocytes, and debris Q2 CD3 + Anti HLA-DR + activated T lymphocytes Q4 CD3 + Anti HLA-DR T lymphocytes Q1 CD3 CD16 + CD56 + NK lymphocytes Q3 Unstained lymphocytes; contaminating monocytes, granulocytes, and debris Q2 CD3 + CD16 + CD56 + T lymphocyte subset Q4 CD3 + CD16 CD56 T lymphocytes 26

Data for tubes C, D, E, and F is acquired and analyzed by BD Simultest IMK Plus software using the gates and markers established with tubes A and B. Analyze these results using the criteria provided under Quality Control and according to the instructions in Section 8, Results. Quality Control For optimal results, we recommend using BD Calibrite beads and BD Autocomp software for setting the PMT voltages, setting the fluorescence compensation, and checking instrument sensitivity prior to use of BD Simultest IMK Plus reagents on the BD FACScan flow cytometer. Refer to Appendix A in the BD Simultest IMK Plus User s Guide for information on optimizing the flow cytometer prior to analyzing patient samples. The negative Control provided in the BD Simultest IMK Plus reagent kit is run with each patient sample to set FL1 and FL2 markers between negatively and positively stained lymphocyte clusters and to detect the presence of nonantigen-specific antibody binding 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 quadrant 3 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, Section 12, 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 nonantigen-specific antibody binding. 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 sample are shown in Figure 2. 27

If there is poor separation between negative and positive clusters as seen on visual inspection of the dot plots or contour displays, nonantigen-specific antibody binding can be inferred and the run should be rejected. Nonantigen-specific antibody binding might be seen because of poor condition of the cells. Consult 12.Troubleshooting, Section 12, if nonantigen-specific antibody binding is observed. BD Simultest IMK Plus software will automatically inspect the data and alert the operator with a number of possible error messages. Refer to Appendix D of the BD Simultest IMK Plus User s Guide for a list of possible messages. The software uses the following criteria for inspection of the dot plots obtained for each sample to evaluate the quality of the data obtained. 1. The operator should reject any results if any one 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 20%); or excessive monocyte (greater than 4%) or granulocyte (greater than 4%) contamination of the lymphocyte gate. 2. If there is no obvious reason for the normal control to fail, a sample from another normal control should be restained and rerun 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 Reagent G, BD FACS lysing solution. Too much debris can also occur when assaying blood samples from patients with certain hematologic disorders where red cells are difficult to lyse, for example, myelofibrosis, spherocytosis. Nucleated erythrocytes will be counted as debris and, if debris exceeds 20%, the software will flag 28

the sample as too many nonlymphs in gate and the sample results should be rejected. 4. The operator should check that the BD Simultest IMK Plus lymphocyte differential count is within ±10% of the independent differential white count. If the BD Simultest IMK Plus count is outside the range, a discrepancy exists and the run should be questioned. Either the BD Simultest IMK Plus count is in error or the independent laboratory differential white count is incorrect. See Section 9, Limitations. 5. The operator should check that there is agreement in T-lymphocyte percentages between tubes C, E, and F, which contain CD3 +. The run should be rejected if the total T-lymphocyte values in any two of the three tubes differ by more than 8%. 6. Helper/inducer and suppressor/cytotoxic lymphocytes will not add to total T lymphocytes because the CD8 antigen is also expressed on NK lymphocytes. Internal assay consistency can be indicated when the sum of the percentages of T, B, and NK lymphocytes totals 100% ±5% when the Quadrant Correction option of BD Simultest IMK Plus software is selected from the Main Menu. Refer to the BD Simultest IMK Plus User s Guide for instructions on selecting this option. See also Troubleshooting, Section 12, at the end of this IFU. 8. RESULTS Calculation of Corrected Counts When the Quadrant Correction software option is selected, the BD FACScan system with BD Simultest IMK Plus software automatically calculates each reported lymphocyte subset as a percentage of total lymphocytes in the lymphocyte acquisition gate. The software first subtracts nonlymphocytes from quadrant 3 (Q3) and 29

then reports the results as percentages of lymphocytes in the lymphocyte acquisition gate set using the BD Leucogate tube (A). When all quality control criteria listed under Quality Control in Section 7, Procedure, have been met, the Quadrant Correction software option provides appropriate estimations of the true subset values. However, if the quality control criteria are not met (for example, because of excessive nonlymphocyte contamination of the gate), results can be suspect. In this case, the data should be analyzed manually to determine the effect of cross-reacting nonlymphocytes on results. If the Quadrant Correction option is turned off, results will be reported as a percentage of the total gated events. See the BD Simultest IMK Plus User s Guide. The principle of the computation follows (Q values refer to counts in quadrants): Equation 1 computes the percentage of lymphocyte events within the BD Leucogate lymphocyte acquisition gate. This value is given as %L in tube A in Table 1 and reflects the purity of lymphocytes within the gate drawn using the BD Leucogate tube (A). 30

Table 1 Summary of the representative data from Figure 2 and identification of quadrants used to compute subsets Tube %L * %M * %G * %D * % of Total Gated Lymphs A (BD Leucogate) 95 1 3 1 99 Tube Cell Type Quadrant (s) % of Lymphocytes( Corrected) C (CD3/CD19) Total T lymphocytes Q4 82 Total B Lymphocytes Q1 9 D (CD4/CD8) Helper/Inducer lymphocytes; monocytes Q2, Q4 50 E (CD3/Anti HLA-DR) F (CD3/ CD16+CD56) Suppressor/cytotoxic and NK lymphocytes Helper/ Suppressor Ratio Q1, Q2 41 1.2 Total T lymphocytes Q4 81 Activated T Q2 18 lymphocytes Total T lymphocytes Q4 81 NK lymphocytes Q1 7 * %L, %M, %G, and %D are lymphocytes, monocytes, granulocytes, and debris in the gate defined by BD Leucogate expressed as percentages of all the events in that gate. These values reflect the quality and purity of the gate. Percent of total gated lymphocytes are the lymphocytes in the gate defined by BD Leucogate expressed as a percentage of all lymphocytes in the ungated sample. This value measures the proportion of all lymphocytes included in the gate. Helper/suppressor ratio = %CD4 + lymphocytes/%cd8 + lymphocytes 31

N = total number of events in the lymphocyte gate defined by BD Leucogate = L + M + G + D where: L = number of lymphocytes in the gate M = number of monocytes in the gate G = number of granulocytes in the gate D = number of debris events in the gate. Equation 1. Percent gated lymphocytes (%L) = L/N x 100 Equation 2a. Percent gated, corrected lymphocytes in Q1, Q2, or Q4 = --------------------------------------------------------------------------------------------- Number of gated events in Q1, Q2, or Q4 100 N (%L/100) In Equation 2a, percentages of positive cells in each of the three positive quadrants (Q1, Q2, and Q4) are expressed as corrected percentages of lymphocytes, assuming all the non-lymphocytes in the BD Leucogate-defined gate are unstained cells. To calculate the correction factor in these quadrants, the software uses Equation 2a for Q1, Q2, and Q4. NOTE The Quadrant Correction option does not correct for nonlymphocyte events that can occur in Q1, Q2, and Q4. Therefore, CD4-positive monocyte events are not corrected in Q4 by the software. On the Laboratory Report sheet, values for unstained cells (Q3) are reported under Corr %L as percent lymphocytes corrected in Q3. Q3 represents unstained cells and, presumably, all the nonlymphocyte events in the gate as well as unstained lymphocytes. Therefore, in addition to converting the results from the percentage of total events to the percentage of lymphocytes in the gate, Equation 2b also subtracts the gated nonlymphocyte events from the Q3-gated events. 32

Equation 2b. Percent gated, corrected, unstained lymphocytes in Q3 (Corr %L) = (Number gated events in Q3 Number gated nonlymphocytes) 100 --------------------------------------------------------------------------------------------------------------------------------------------------------------------- N x (%L/100) where: Number of gated nonlymphocytes = number of gated debris events + number of gated granulocytes + number of gated monocytes. See Table 1, tube A. Calculation of Absolute Counts An absolute cell count can be computed if a WBC count and the lymphocyte percentage from a differential white count are obtained using standard laboratory procedures. The BD FACScan with BD Simultest IMK Plus software automatically calculates an absolute count for each BD Simultest IMK Plus parameter 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 BD Simultest IMK Plus 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 a laboratory differential white cell count obtained for the same sample. Do not use the BD Simultest IMK Plus software three-part differential values. 3. The absolute counts for each subset are then computed as follows: Absolute count of CD4 + cells/µl = %CD4 + ---------------------- %L --------- (WBCs/µL) 100 100 33

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 the WBC count, (b) percent lymphocytes, and (c) the normal reference range and precision for percent lymphocytes positive for the specific marker. Three-Part Differential For lysed whole blood, it is possible to determine monocytes, lymphocytes, and granulocytes as a percentage of leucocytes using the BD Leucogate tube A (see Table 2). The BD FACScan with BD Simultest IMK Plus software automatically calculates a three-part differential (Table 2). Refer to the BD Simultest IMK Plus User s Guide for representative data printouts. Table 2. Three-part differential data from Figure 2 Three-Part Differential a % Leucocytes Lymphocytes 25 Monocytes 5 Granulocytes 70 a. 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. NOTE The differential count provided by BD Simultest IMK Plus 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 nor entered into BD Simultest IMK Plus software to obtain absolute counts. 34

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) because 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 staining. Whole blood samples with counts of less than 3.5 x 10 3 WBC/µL and greater than 9.8 x 10 3 WBC/µL will require special handling to obtain correct results. Samples with counts of greater than 9.8x10 3 WBC/µL will need to be diluted with PBS containing 0.1% sodium azide. Samples with counts of less than 3.5 x 10 3 WBC/µL can require more blood and a separation procedure to concentrate the cells. Confounding variables such as medications that affect properties of blood cells can yield inaccurate results. For example, poor resolution between positive and negative cells has been observed with transplant patients receiving immunosuppressive drugs. If there is poor separation between negative and positive clusters, the run should be rejected (see Troubleshooting, Section 12, at the end of this IFU). If the difference in the total T-cell percentage between any two of the three tubes containing CD3 is greater than 8%, the run should be rejected. Laboratories must establish their own normal reference ranges for each of the BD Simultest IMK Plus parameters, which can be affected by sex, 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 count from 35