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[email protected]

IVD

BD Simultest™ IMK Plus

Catalog No. 349217

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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

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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

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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 erythrocyte-lysed 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 cell-mediated 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 fluorochrome-labeled polyclonal antibodies to surface immunoglobulin.1 Cytolytic lymphocyte assays included the hemolytic plaque assay2 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

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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 blood6 (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 four-parameter 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 diseases9,10 and some types of autoimmune disease.11,12

* Not all studies cited in this section employed BD reagents.

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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 immunodeficiencies9 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 diseases11 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 disorders11,12 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

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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 NK-lymphocyte 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 IgG1 FITC/IgG2a 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.

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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 red-orange 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 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 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

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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

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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.

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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 CD4530 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 IgG1 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 CD1427 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 IgG2b 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 antigen31 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 IgG1, clone X40, and PE-labeled IgG2a, clone X39, murine monoclonal antibodies that react

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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 antibody36 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 IgG1 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 antibody38 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 IgG1 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 PE-labeled CD8, clone SK1,42,43 for the identification of suppressor/cytotoxic lymphocytes.

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The CD4 antibody42 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 IgG1 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 p56lck. 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 antibody42 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 IgG1 heavy chains and kappa light chains. The CD8 antigen is present on the human suppressor/cytotoxic T-lymphocyte subset35,43 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 p56lck. The CD8:p56lck 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.

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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 IgG2a 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 antibody64 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 IgG1 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 antibody65 was derived from hybridization of mouse Sp2/0 myeloma cells with cells of (B6xBALB/c)F1 mice immunized with the KG1a cell line. The antibody is composed of IgG1 heavy chains and kappa light chains. The molecular weight of the glycosylated antigen recognized by this antibody ranges from 175 to 220 kDa20,66 (or 137 kDa when deglycosylated67) and is present on NK lymphocytes. The CD56 antigen is involved in neuronal homotypic cell adhesion and cell differentiation during embryogenesis.65

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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 IgG1 FITC 25

IgG2a 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

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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.

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WARNING All biological specimens and materials coming into contact with them are considered biohazards. Handle as if capable of transmitting infection68,69 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.

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.

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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 venipuncture5,70 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

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acceptable WBC concentration range is from 3.5 x 103 to 9.8 x 103 WBC/µL. Samples with counts greater than 9.8 x 103 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 103 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.

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• 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,

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anticoagulated whole blood patient sample into the bottom of each of the six labeled tubes. The required WBC concentration is 3.5 x 103 to 9.8 x 103 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.

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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

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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 red-orange) (see Figure 1).

Figure 1 The fluorescence display quadrants (indicated as Q1–Q4) and the corresponding colors.

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

Q1Low yellow-green/highred-orange

Q2High yellow-green/highred-orange(dual fluorescence)

Q3Low yellow-green/low red-orange

Q4High yellow-green/low red-orange

25

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.

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 yellow-green/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.”

C D 3 C D 3

C D 3

C D 4

CD

16+

CD

56

Anti–

HLA

-DR

CD

8S

SC

FSC Ig G 1

IgG

2a

CD

19

Tube A Tube B Tube C

Tube D Tube E Tube F

26

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.)

Q1CD4–CD8+ suppressor/ cytotoxic and NK lymphocytes

Q2CD4+CD8+ lymphocytes

Q3Unstained lymphocytes; granulocytes and debris

Q4CD4+CD8– helper/inducer lymphocytes; monocytes

Q1CD3–CD19+ B lymphocytes

Q2Nonantigen-specific antibody bindinga

a. See Section 9, Limitations.

Q3 Unstained lymphocytes; contaminating monocytes, granulocytes, and debris

Q4CD3+CD19– T lymphocytes

Q1 CD3-Anti–HLA-DR+ B lymphocytes, activated NK lymphocytes; monocytes/macrophages

Q2CD3+Anti–HLA-DR+ activated T lymphocytes


Q4CD3+Anti–HLA-DR–

T lymphocytes

Q1CD3–CD16+CD56+ NK lymphocytes

Q2CD3+CD16+CD56+ T lymphocyte subset


Q4CD3+CD16–CD56– T lymphocytes

Tube C (CD3/CD19) Tube D(CD4/CD8)

Tube E (CD3/Anti–HLA-DR) Tube F (CD3/CD16+CD56)

27

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.

28

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

29

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

30

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).

31

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)

Helper/Suppressor

Ratio‡

C (CD3/CD19) Total T lymphocytes Q4 82

Total B Lymphocytes Q1 9

D (CD4/CD8) Helper/Inducer lymphocytes; monocytes

Q2, Q4 50

Suppressor/cytotoxic and NK lymphocytes

Q1, Q2 41 1.2

E (CD3/Anti–HLA-DR)

Total T lymphocytes Q4 81

Activated T lymphocytes

Q2 18

F (CD3/CD16+CD56)

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

32

N = total number of events in the lymphocyte gate defined by BD Leucogate = 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 gate.

Equation 1. Percent gated lymphocytes (%L) = L/N x 100

Equation 2a. Percent gated, corrected lymphocytes in Q1, Q2, or Q4 =

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.

Number of gated events in Q1, Q2, or Q4N (%L/100)×

---------------------------------------------------------------------------------------------- 100×

33

Equation 2b. Percent gated, corrected, unstained lymphocytes in Q3 (Corr %L) =

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 =

(Number gated events in Q3 – Number gated nonlymphocytes) 100×

N x (%L/100)----------------------------------------------------------------------------------------------------------------------------------------------------------------------

%CD4+

100----------------------- %L

100---------- (WBCs/µL)××

34

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.

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.

Table 2. Three-part differential data from Figure 2

Three-Part Differentiala

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.

% Leucocytes

Lymphocytes 25

Monocytes 5

Granulocytes 70

35

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 103 WBC/µL and greater than 9.8 x 103 WBC/µL will require special handling to obtain correct results. Samples with counts of greater than 9.8 x 103 WBC/µL will need to be diluted with PBS containing 0.1% sodium azide. Samples with counts of less than 3.5 x 103 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

36

the BD Simultest IMK Plus software report to compute absolute counts. The differential count provided by BD Simultest IMK Plus 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 nor entered into BD Simultest IMK Plus software to obtain absolute counts.

• If the BD Simultest IMK Plus lymphocyte differential count differs from the independent differential white count by more than ±10%, there is a significant discrepancy and care should be taken in interpreting absolute count results. Either the BD Simultest IMK Plus differential count or the laboratory differential white count is in error. See Quality Control under Section 7, Procedure.

• Variation in either automatic or manual lymphocyte acquisition gate settings will change the relative amounts of subsets assayed. BD Simultest IMK Plus software uses the BD Leucogate tube (A) to include at least 95% of the total lymphocytes in the sample to set the lymphocyte acquisition gate and requires a visual inspection of the gate setting for validation.

• Abnormal states of health are not always represented by abnormal percentages of T or B lymphocytes or their subsets. That is, an individual who can be in an abnormal state of health can exhibit the same T- or B-lymphocyte percentages as a healthy individual. Results from BD Simultest IMK Plus must be used in conjunction with other information available from the clinical evaluation and additional independent diagnostic procedures.

• The information obtained from this kit must be combined with other information and interpreted by a medically qualified diagnostician to establish presence or absence of specific disease states.

37

• Note that normal reference ranges established by BD for the CD4+/CD8+ (helper/suppressor) lymphocyte ratio extend below a ratio of 1.0 (see Table 3 under Leucocyte Subsets in Section 10, Expected Values).

• The BD Simultest IMK Plus software Quadrant Correction option assumes that all nonlymphocytes are unstained. Therefore, CD4-positive monocytes that appear in Q4 will be included as CD4-positive lymphocytes. The limit of monocyte contamination allowed is 4% before the sample is flagged. See the BD Simultest IMK Plus User’s Guide for more information on quadrant correction.

• Note that some BD Simultest IMK Plus antibodies react with other non-lymphocyte-formed elements in blood. See Cross-Reactivity in Section 11, Performance Characteristics.

• The helper/suppressor T-lymphocyte ratio as determined by BD CONSORT 30 or BD CONSORT 32 with BD Simultest IMK Plus software can vary because of changes in various subsets of CD8+ lymphocytes that are also positive for CD16, CD56, or both. Dual-fluorescence studies employing each of these antibodies with CD8 can determine which populations are responsible for variations.

• Samples with nucleated RBCs can show incomplete lysis because Reagent G, BD FACS lysing solution, 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, for example, myelofibrosis, spherocytosis. Nucleated erythrocytes will be counted as debris and, if debris is greater than 20%, the software will flag the sample as “too many nonlymphs in gate.” We recommend that such sample results be rejected. When the Quadrant Correction software option has been selected, nonlymphocytes such as nucleated or unlysed red cells determined using BD Leucogate are assumed to be unstained and are subtracted from Q3 by the software.

38

• BD Simultest IMK Plus software checks for nonantigen-specific antibody binding in Control tube B only. The operator should examine the printout for any evidence of nonantigen-specific antibody binding in subsequent tubes (C through F). For example, on the dot plot display of tube C, if excessive events occur in Q2, nonantigen-specific antibody binding should be suspected. The presence of such nonantigen-specific antibody binding can reflect a change in the reagents or an error in the preparation of the samples. Such samples should be repeated.

• The presence of abnormal (blast) cells might not allow BD Leucogate to set an adequate lymphocyte acquisition gate. The software will flag the sample and results will not be printed.

• For whole blood samples, prolonged exposure of cells to Reagent G, BD FACS lysing solution, beyond 12 minutes can cause white cell destruction (see item 4 under Staining and Fixing the Cells in Section 7, Procedure).

• The ability of the flow cytometer to select lymphocytes and to eliminate platelets, red cells, debris, granulocytes, and monocytes from the lymphocyte count depends on the existence of a clear demarcation between these formed elements and lymphocytes on a display of FSC vs SSC. For some patients, this demarcation is not clear and lymphocyte gating will be less effective.

• Performance characteristics have been determined with BD FACScan flow cytometers. Performance characteristics with any other instruments have not been established.

10. EXPECTED VALUES

Leucocyte Subsets

Four sets of counts are collected for each tube, including the Control tube B. Each count is represented in a different quadrant on the BD FACScan display (Figure 2).

39

The cell populations present in each quadrant, by tube, are shown in Figure 3 in this IFU and in the BD Simultest IMK Plus User’s Guide. The clinically significant results are found in the Physician’s Report.71 BD has reported a mean helper/suppressor lymphocyte ratio of 1.4 and a 95% range of the ratio between 0.6 to 2.8 in the peripheral blood of 304 healthy male and female subjects whose ages ranged from 20 to 70 years.71

BD has investigated the normal reference ranges for BD Simultest IMK Plus parameters in normal male and female subjects using the BD FACScan flow cytometer at six sites (five European clinical centers and one US site). The expected normal reference ranges for LWB are shown in Table 3.71 Ranges are presented as corrected percentages of lymphocytes.

The ranges obtained were tested for differences by clinical site, by sex, and by age of subject. For each BD Simultest IMK Plus parameter, nonparametric comparisons were made between values obtained at each study center, between values obtained for males and females, and between values obtained for subjects age 18 to 40 and age 41 to 70. If 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 fitted distribution.71 The ability to pool reference ranges for the BD Simultest IMK Plus parameters (Table 3) is an indication of between-laboratory reproducibility.

There are small but significant differences between sexes for ranges of total T lymphocytes determined by Reagent F and differences between sexes and subjects 40 or below versus subjects over 40 years old for T-helper/inducer lymphocytes, the CD4+/CD8+ lymphocyte ratio, activated T lymphocytes, and NK lymphocytes.

40

Table 3. Representative reference rangesa for BD Simultest IMK Plus parameters in hematologically normal adults as percentages of total lymphocytes (corrected) (data pooled from six clinical centers)

a. This data was collected using BD Simultest IMK Plus reagents, BD Simultest IMK Plus software, and the BD FACScan flow cytometer.

Parameter Sex Age n Mean (%) 95% Rangeb

b. The 95% range is obtained from fitting an appropriate distribution to the data and then estimating the central 95% area of the fitted distribution.

Total T lymphocytes MaleFemale

18–7018–70

161143

71.373.9

58.2–84.362.8–85.0

Total B lymphocytes Both 18–70 319 14.0 7.1–23.3

Helper/inducer lymphocytes MaleMaleFemaleFemale

≤40>40≤40>40

77848558

39.943.944.048.9

27.3–52.528.5–59.231.4–56.734.0–63.8

Suppressor/cytotoxic lymphocytes Both 18–70 304 33.4 18.9–47.9

Helper/suppressor ratio MaleMaleFemaleFemale

≤40>40≤40>40

77848558

1.21.51.31.7

0.6–2.20.6–3.00.7–2.30.8–3.3

Activated T lymphocytes MaleMaleFemaleFemale

≤40>40≤40>40

58676444

9.411.07.89.1

3.5–20.63.6–25.92.8–17.34.2–17.2

NK lymphocytes MaleMaleFemaleFemale

≤40>40≤40>40

80889160

13.216.312.313.6

5.4–27.16.7–33.54.8–26.16.0–26.7

41

Adult reference ranges should not be used with pediatric blood samples (neonate to 13 years of age). Preliminary results on 50 pediatric samples (infants and children) suggest that percentage values relative to adult values can be slightly higher for total B lymphocytes. Children less than one year old tended to have significantly lower total T-lymphocyte values.

Race can also be a variable in the establishment of normal reference ranges,72 although insufficient data was collected by BD to determine this. BD presents more than one upper and lower limit for the ranges for most BD Simultest IMK Plus parameters (Table 3 on page 40) because of the age and sex differences that have been observed.

NOTE Expected normal values can vary depending upon age, sex, or race of patient. Because of these differences, each laboratory should establish its own normal reference range for each parameter.

Absolute Counts

The BD FACScan system with BD Simultest IMK Plus software automatically calculates the absolute count if a WBC and a differential percentage of lymphocytes or an absolute lymphocyte count is obtained and entered into the software.

NOTE Do not use the BD Simultest IMK Plus three-part differential values for absolute count determinations. 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.

Absolute counts will exhibit significant inter-laboratory variation depending upon the procedure employed for obtaining the WBC and differential white counts.

42

Representative absolute counts are shown in Table 4. The table is generated from data acquired at Laboratoire d’Hématologie, Ecole de Santé Publique, Université Catholique de Louvain, Belgium, the site with the largest single population of subjects between the ages of 20 and 70. These subjects represent a selected population between the ages of 20 to 70 to which the criterion T + B + NK = 100 ±5% was applied. BD Simultest IMK Plus parameters were expressed as a percentage of lymphocytes in the lymphocyte acquisition gate after subtracting nonlymphocytes (granulocytes, monocytes, and debris) from quadrant 3.

Table 4. Representative reference rangesa for BD Simultest IMK Plus parameters in hematologically normal adults (absolute count datab from one clinical site)

Parameter Sex Age n Mean 95% Rangec

WBC Both 20–70 98 6.08d 3.45–9.76

% Lymphs Both 20–70 98 30.96 20.7–44.6

Total T lymphocytes MaleFemale

20–7020–70

4055

1.311.39

0.594–1.9920.772–2.201

Total B lymphocytes Both 20–70 98 0.27 0.109–0.532

Helper/inducer lymphocytes MaleMaleFemaleFemale

≤40>40≤40>40

11292728

0.770.820.860.86

0.439–1.1120.356–1.3510.309–1.5710.496–1.354

Suppressor/cytotoxic lymphocytes

Both 20–70 95 0.58 0.282–0.999

Helper/suppressor ratio MaleMaleFemaleFemale

≤40>40≤40>40

11292728

1.281.571.371.84

0.865–1.8850.784–3.1000.775–2.3750.846–3.500

43

11. PERFORMANCE CHARACTERISTICS

Performance of BD Simultest IMK Plus was established by testing at either one US clinical center or at BD laboratories in San Jose, California, or both.

Within-Sample Reproducibility

Blood samples from each of five normal and five abnormal donors were obtained, aliquoted (four times for normals, three for abnormals), stained with the BD Simultest IMK Plus reagents, lysed, and fixed within 2 hours of staining. Flow cytometric analysis was performed within 24 hours on a BD FACScan flow cytometer in the same laboratory. Table 5 shows the average within-sample reproducibility obtained for both normal and abnormal subjects.

Activated T lymphocytes MaleMaleFemaleFemale

≤40>40≤40>40

11302928

0.140.170.120.15

0.050–0.2680.042–0.4510.034–0.3210.054–0.496

NK lymphocytes MaleMaleFemaleFemale

≤40>40≤40>40

11302928

0.190.260.210.23

0.078–0.3450.102–0.5110.072–0.5430.106–0.605

a. This data was collected using BD Simultest IMK Plus reagents, BD Simultest IMK Plus software, and the BD FACScan flow cytometer.

b. Absolute counts are expressed as 1 x 103 cells/µLc. The 95% range is estimated directly from the data using the 2.5 and 97.5 percentiles.d. Data was obtained from a Technicon™ H6000 automated cell counter. (Technicon is a trademark of Miles

Instruments.)

Table 4. Representative reference rangesa for BD Simultest IMK Plus parameters in hematologically normal adults (absolute count datab from one clinical site) (continued)

Parameter Sex Age n Mean 95% Rangec

44

Between-Instrument Reproducibility

Using a protocol similar to that above, samples of whole blood from ten normal subjects were aliquoted, stained with the BD Simultest IMK Plus reagents, and lysed. Single samples from each donor were then analyzed on three different BD FACScan flow cytometers. Table 6 shows these results.

Between-Laboratory Reproducibility

Between-laboratory reproducibility is indicated by the ability to pool normal reference ranges for BD Simultest IMK Plus parameters (Table 3 on page 40).

BD Simultest IMK Plus vs Comparative Methods

Results of aliquots from the same blood samples from normal and abnormal subjects were analyzed with the BD Simultest IMK Plus reagents, the BD Simultest™ Immune Monitoring Kit, CD3 FITC, and CD19 PE, using LWB and the BD FACScan. Results were compared using linear regression. A summary of the results is presented in Table 7.Table 5. Within-sample reproducibility for BD Simultest IMK Plus parameters (five normal subjects and five abnormal subjects) as percentages of lymphocytes (corrected)

Parameter Subjects nMeana

(%) SDb CVc dfd

Total T lymphocytes NormalAbnormal

55

68.480.4

1.61.9

2.22.0

14e

15e

Total B lymphocytes NormalAbnormal

55

9.59.3

2.41.0

21.59.0

1415

Helper/inducer lymphocytes NormalAbnormal

55

46.318.8

1.52.1

3.212.2

1515

Suppressor/cytotoxic lymphocytes NormalAbnormal

55

34.865.5

1.32.6

3.63.7

1515

45

Helper/suppressor ratio NormalAbnormal

55

1.40.3

0.10.04

5.213.4

1515

Activated T lymphocytes NormalAbnormal

55

6.438.9

1.51.6

24.54.3

1515

NK lymphocytes NormalAbnormal

55

24.712.6

2.00.9

7.06.7

14e

15

a. Mean is the pooled mean, Y = the mean of the individual means.b. SD = standard deviation, the pooled standard deviation.

si2 = variance of the ith sample for 1 ≤ i ≤ k.

k = number of samples.n = number of observations in a sample.

c. CV = coefficient of variation

d. df = degree of freedom = (number of replicates – number of donor means) x number of subjects; 4 aliquots – 1 mean = 3 degrees of freedom. Three degrees of freedom for 5 subjects = 15 degrees of freedom, or 1 aliquot x 3 instruments – 1 mean = 2 degrees of freedom. Two degrees of freedom for 10 subjects = 20 degrees of freedom.

e. One of the replicates was discarded by the clinician.

Table 6. Between-instrument reproducibility for BD Simultest IMK Plus parameters (ten normal subjects) as percentages of lymphocytes (corrected)

Parameter Meana (%) SDb CVc dfd

Total T lymphocytes 72.1 2.13 2.29 20

Total B lymphocytes 12.7 1.26 10.59 20

Helper/inducer lymphocytes 45.8 1.26 2.47 20

Suppressor/cytotoxic lymphocytes 39.0 1.37 3.28 20

Table 5. Within-sample reproducibility for BD Simultest IMK Plus parameters (five normal subjects and five abnormal subjects) as percentages of lymphocytes (corrected)

Parameter Subjects nMeana

(%) SDb CVc dfd

SDn1 1–( ) s1

2× n2 1–( ) s2

2× ... nk 1–( ) sk

2×+ + +

n1 n2 ... nk k–+ + +------------------------------------------------------------------------------------------------------------------------------=

46

Stability of Stained Cell Preparations

Cell preparations from five normal subjects were acquired and analyzed on the BD FACScan. The preparations were stored at 2°C– 8°C in the dark for 24 hours and flow cytometric analysis repeated. Studies indicated no significant difference between day 0 and day 1

Helper/suppressor ratio 1.2 0.06 4.80 20

Activated T lymphocytes 11.8 1.56 14.09 20

NK lymphocytes 16.8 1.28 6.93 20

a. Mean is the pooled mean, Y = the mean of the individual means.b. SD = standard deviation, the pooled standard deviation.

si2 = variance of the ith sample for 1 ≤ i ≤ k. k = number of samples.n = number of observations in a sample.

c. CV = coefficient of variation =

d. df = degree of freedom = (number of replicates – number of donor means) x number of subjects; 4 aliquots – 1 mean = 3 degrees of freedom. Three degrees of freedom for 5 subjects = 15 degrees of freedom, or 1 aliquot x 3 instruments – 1 mean = 2 degrees of freedom. Two degrees of freedom for 10 subjects = 20 degrees of freedom.

Table 6. Between-instrument reproducibility for BD Simultest IMK Plus parameters (ten normal subjects) as percentages of lymphocytes (corrected)

Parameter Meana (%) SDb CVc dfd

SDn1 1–( ) s1

2× n2 1–( ) s2

2× ... nk 1–( ) sk

2×+ + +

n1 n2 ... nk k–+ + +------------------------------------------------------------------------------------------------------------------------------=

SD Y

x 100

47

readings in any parameter. We recommend analyzing stained samples within 24 hours.

Cross-Reactivity

The specificity of monoclonal antibodies with a CD number has been established by blind testing at a number of laboratories by the International Leucocyte Workshop group.25,27,34,38,42,64,65

The CD4 antibody reacts with monocytes as well as lymphocytes.47

The Anti–HLA-DR antibody reacts with monocytes/macrophages as well as B lymphocytes.14,19,57–60 The CD14 antibody reacts with granulocytes as well as monocytes/macrophages.33 The CD16 antibody reacts weakly with neutrophils.20 Potentially interfering granulocytes should be eliminated from the lymphocyte count by their light-scatter properties.

Although Reagent B is designed to detect nonantigen-specific antibody binding, nonspecific binding due to poor condition of cells can be observed with any antibody reagent. Nonantigen-specific antibody

Table 7. BD Simultest IMK Plus vs comparative method using LWB and the BD FACScan

Parameter Slope Intercept r na

a. Composed of normal and abnormal samples.

Range of Data (%)

Total T lymphocytesb

b. Defined by CD3 FITC.

0.98 4.06 0.98 58 10–90

Total B lymphocytesc

c. Defined by CD19 PE.

0.97 1.47 0.99 65 0–80

Helper/inducer lymphocytes 1.16 –1.81 0.96 31 10–60

Suppressor/cytotoxic lymphocytesd

d. As compared to the BD Simultest Immune Monitoring Kit.

0.96 7.05 0.93 31 25–80

Helper/suppressor ratiod 1.02 –0.02 0.98 31 0.125–2.0

Activated T lymphocytes 1.06 0.34 0.92 27 5–50

48

binding can be inferred if no clear demarcation between negative and positive cells is seen on the fluorescence plot or if there are more than 5% of counts from the negative Control outside quadrant 3. Consult Troubleshooting, Section 12, at the end of this IFU if nonantigen-specific antibody binding is seen.

Linearity-Recovery

All lots of BD Simultest IMK Plus reagents are titered against actual cell suspensions to provide optimal performance at 1.0 x 106 WBC/sample. For an LWB sample, the normal range is 3.5 x 103 to 9.8 x 103 WBC/µL. Results are expected to be linear from 3.5 x 103 to 9.8 x 103 WBC/µL.

49

12. TROUBLESHOOTING

Problem Possible Cause Solution

Insufficient separation between positive and negative fluorescent populations.

• Decreased fluorescence due to incorrect PMT voltage or amplifier gain.

• Insufficient antibody reagent.

1. Run BD Autocomp software with BD Calibrite 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.

No demarcation found between debris and lymphocyte clusters on light-scatter dot plot.

• Presence of excess platelets or nucleated red blood cells or unlysed red blood cells.

• Amplifier gain improperly set.• Sample not stained within 6

hours of collection.• FSC threshold set too high or

too low.

1. Restain a fresh sample. Wash cells at lower speed, for example 100g for 15 minutes at room temperature (20°C–25°C).

2. Adjust 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 BD Autocomp to whole blood determinations. See 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

Suboptimal antibody-staining performance.

• Sample not stained within 6 hours of collection.

• Analysis gate selected by software included too many nonlymphocyte events.

• Fluorescence markers set incorrectly.


2. Reprocess data using BD CONSORT 30, BD LYSYS II, or BD Simulset 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. See the appropriate software user's guide.

3. Reprocess data using BD CONSORT 30, BD LYSYS II, or BD Simulset software and set manual fluorescence markers.


51

Software unable to set an acceptable lymphocyte gate.

• Sample is lymphopenic.• FSC threshold set too high or

too low.• Amplifier gain improperly set.• Sample not stained within 6

hours of collection.• Software marker setting not

appropriate.

1. Concentrate cells using a density-gradient method or reacquire a larger number of events using BD CONSORT 30 or BD LYSYS II.

2. Adjust FSC threshold so that there are approximately 10 channels of debris (256-channel scale).

3. Adjust 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 BD Autocomp to whole blood determinations. See the appropriate user's guide.


5. Reacquire data using BD CONSORT 30 or BD LYSYS II.

Unstained cells in the stained sample move over marker set with Control tube B into Q4 or Q1.

• Nonantigen-specific antibody binding due to sample containing dead or damaged cells.

• Nonantigen-specific antibody binding due to Fc receptors.


2. Check for excessive monocyte, granulocyte, or debris contamination in the lymphocyte acquisition gate.

Staining is dim or inconsistent.

• Cell concentration too high at staining step.

• Insufficient reagent.• Cells not analyzed within 24

hours of staining.• Improper medium

preparation (sodium azide omitted).

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.


52

REFERENCES1 Wolde-Mariam W, Peter J. Recent diagnostic advances in cellular immunology. Diagnost

Med. 1984;7:25-32.

2 Henry C. Hemolytic plaque assays. In: Mishell B, Shiigi S, eds. Selected Methods in Cellular Immunology. New York: WH Freeman and Co; 1980:69-123.

3 Grabstein K, Chen Y. Cell-mediated cytolytic responses. In: Mishell B, Shiigi S, eds. Selected Methods in Cellular Immunology. New York: WH Freeman and Co; 1980:124-137.

4 Wofsy L, Henry C, Kimura J, North J. Modification and use of antibodies to label cell surface antigens. In: Mishell B, Shiigi S, eds. Selected Methods in Cellular Immunology. New York: WH Freeman and Co; 1980:287-304.

5 Jackson A, Warner N. Preparation, staining, and analysis by flow cytometry of peripheral blood leukocytes. In: Rose N, Friedman H, Fahey J, eds. Manual of Clinical Laboratory Immunology. Washington, DC: American Society for Microbiology; 1986:226-235.

6 Jackson A. Basic phenotyping of lymphocytes: selection and testing of reagents and interpretation of data. Clin Immunol Newsletter. 1990;10:43-55.

7 Nicholson J, Jones B. Immunophenotyping by flow cytometry: its use in HIV infection. Labmedica. 1989;6:21-26.

8 Landay A, Muirhead K. Procedural guidelines for performing immunophenotyping by flow cytometry. Clin Immunol Immunopathol. 1989;52:48-60.

Few or no cells. • Cell concentration too low.• Flow cytometer

malfunctioning.

1. Resuspend fresh sample at a higher concentration. Repeat staining and analysis.

2. Troubleshoot instrument.

Increased autofluorescence.

• Possible bacterial contamination of PBS or fixative.

• Poor sample preparation.

1. Prepare fresh 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.

Weak positive fluorescence.

Possible bacterial contamination of PBS or fixative. Buffer or fixative at improper pH.

Prepare fresh PBS with reagent-grade water and fresh fixative with PBS with sodium azide. Adjust to the proper pH. Filter through 0.2-µm filter.


53

9 Schmidt R. Monoclonal antibodies for diagnosis of immunodeficiencies. Blut. 1989;59:200-206.

10 Ohno T, Kanoh T, Suzuki T, et al. Comparative analysis of lymphocyte phenotypes between carriers of human immunodeficiency virus (HIV) and adult patients with primary immunodeficiency using two-color immunofluorescence flow cytometry. Tohoku J Exp Med. 1988;154:157-172.

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58

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THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.

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