MONOCLONAL ANTIBODIES TO HUMAN CELL SURFACE ANTIGENS. Mouse Anti CD19 R-Phycoerythrin (R-PE)-labeled

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1 MONOCLONAL ANTIBODIES TO HUMAN CELL SURFACE ANTIGENS Mouse Anti CD19 R-Phycoerythrin (R-PE)-labeled CATALOG No. MHCD tests 0.5 ml CATALOG No. MHCD tests 2.0 ml Mouse Anti CD19 TRI-COLOR (TC)-labeled CATALOG No. MHCD tests 0.5 ml CATALOG No. MHCD tests 2.5 ml FOR IN-VITRO DIAGNOSTIC USE

2 I. INTENDED USE LIFE TECHNOLOGIES anti-human CD19 R-PE and CD19 TRI-COLOR are fluorochrome-conjugated monoclonal antibody reagents that may be used to enumerate CD19 + lymphocytes in human peripheral blood by flow cytometric methods. (4) II. SUMMARY AND EXPLANATION The major cellular or cell derived elements of human peripheral blood include lymphocytes, monocytes, granulocytes, red blood cells and platelets. The mature lymphocyte population contains functionally distinct components. The two main components are referred to as T (thymus-derived) and B lymphocytes. Two major subsets of T lymphocytes have distinctly different functional properties, and can be identified by correspondingly different cell surface glycoprotein antigens (1). The CD3 + mature T lymphocytes expressing the CD4 surface antigen are responsible for helper-inducer activity, while CD3 + cells that express the CD8 surface antigen have suppressor and cytotoxic activity (2). The antigen which is recognized by the CD19 monoclonal antibody is expressed on all mature B lymphocytes in human peripheral blood. This molecule is also found on B cells at all stages of maturation in bone marrow, but is lost during the terminal differentiation of B cells to plasma cells. CD19 is thus a lineage marker for B cells (3). CD19 is detectable in the earliest stages of B cell differentiation, and prior to the rearrangement and expression of immunoglobulin receptor genes and has a molecular weight of approximately 90 kda. Although the function of the molecule is uncertain, it may have roles in the regulation of immunoglobulin secretion, the control of B cell proliferation, or in the terminal differentiation of B cells to plasma cells (5). III. CLINICAL RELEVANCE LIFE TECHNOLOGIES anti-human CD19 monoclonal antibody recognizes the CD19 antigen (6). The monoclonal antibody may be used to enumerate mature B lymphocytes in human peripheral blood. Monoclonal antibodies recognizing the CD19 antigen may also be used, in combination with other indicators, for the diagnosis or prognosis of some immunodeficiency diseases. These include X-linked or Bruton's agammaglobulinemia (7,8), common variable immunodeficiency disease (CVID) (9), severe combined immunodeficiency disease (SCID) (10), as well as autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (11). In all of these diseases, the number of peripheral blood B lymphocytes is reported to be substantially reduced. In general, diseases that exhibit decreased cellular immunocompetence may exhibit reduced numbers of CD19 + mature B lymphocytes as well. IV. PRINCIPLES OF THE TEST LIFE TECHNOLOGIES anti-human CD19 R-PE- and TRI-COLOR -conjugated monoclonal antibodies bind to the surfaces of viable blood cells that express the CD19 antigen. LIFE TECHNOLOGIES produces R-PEand TRI-COLOR -conjugated isotype controls as well. To identify cells that express the CD19 antigen, peripheral blood leukocytes are incubated with the monoclonal antibody, and washed to remove unbound antibody. Prior to removal of unbound antibody, lysis solution is added to lyse red blood cells. An appropriate fixative solution is added to the lysed and washed cells. Stained and fixed cells are subsequently analyzed by flow cytometry. An appropriate isotype control should be used in sample analysis to determine the amount of any background or nonspecific fluorescence. These R-PE- or TRI-COLOR -conjugated isotype controls should be of the same immunoglobulin isotype as the specific antibody. The amount of these antibodies used should be the same as the CD19 conjugated monoclonal antibodies. An appropriate cell gating control should be used in sample analysis. The CD45 pan-lymphocyte and CD14 pan-monocyte reagents are intended to optimize the resolution of lymphocyte and monocyte populations (12).

3 V. REAGENTS A. LIFE TECHNOLOGIES MONOCLONAL ANTIBODY REAGENTS Cluster Designation: CD19 WHO Classification: Leukocyte Workshop II (6) Clone: SJ25-C1 Isotype: IgG1 Species: Mouse Composition: IgG1 heavy chain Kappa light chain Source: Mouse ascites fluid or tissue culture supernatant Method of Purification: Column chromatography Fluorochrome: R-Phycoerythrin (R-PE)* Excitation wavelength 488 nm Emission wavelength 575 nm TRI-COLOR Excitation wavelength 488 nm Emission wavelength 667 nm Reagents contents: 0.5 ml vial containing monoclonal antibody for 100 tests, in PBS buffer with BSA and sucrose, 0.1% sodium azide. 2.0 ml vial containing monoclonal antibody for 400 tests, in PBS buffer with BSA and sucrose, 0.1% sodium azide. 2.5 ml vial containing monoclonal antibody for 500 tests, in PBS buffer with BSA and sucrose, 0.1% sodium azide. B. ADDITIONAL LIFE TECHNOLOGIES MATERIALS REQUIRED Isotype Control Reagent: Mouse IgG1 R-PE, 0.5 ml vial containing antibody for 50 tests, Catalog No. MG104 Mouse IgG1 TRI-COLOR, 0.5 ml vial containing antibody for 50 tests, Catalog No. MG106 Lysing Reagent: Cal-Lyse TM Lysing Solution, 25 ml vial sufficient for 250 tests, Catalog No. GAS-010 * U.S. Patent No. 4,520,110, Canadian Patent No. 1,179,942, European Patent No. 76,695

4 VI. STATEMENT OF WARNINGS Reagents contain sodium azide. Under acidic conditions sodium azide yields hydrazoic acid, an extremely toxic compound. Solutions containing sodium azide should be diluted with running water before being discarded. This is recommended to avoid deposits in plumbing where explosive conditions may develop. Do not expose reagents to a direct light source during incubation with cells or prior to analysis. Do not pipette by mouth. Samples should be handled as if capable of transmitting infection. Appropriate disposal methods should be used. The sample preparation procedure employs a fixative (formaldehyde). Avoid contact with skin or mucous membranes. Do not use antibodies beyond the stated expiration dates of the products. Deviations from the recommended procedure enclosed within this product insert may invalidate the results of testing. VII. APPROPRIATE STORAGE CONDITIONS Store reagents at 2-8 C. Freezing is not recommended. Reagents should be brought to room temperature (22 ± 3 C) before use. Protect cells from light sources during incubation with antibodies and prior to analysis. VIII. EVIDENCE OF DETERIORATION Reagents should not be used if any evidence of deterioration, such as increase in compensation, or substantial loss of reactivity is observed. The normal appearance of the R-PE-conjugated monoclonal antibody is a clear, pink-red liquid. The normal appearance of the TRI-COLOR -conjugated monoclonal antibody is a purple liquid. IX. SPECIMEN COLLECTION Collect venous blood samples into blood collection tubes using an appropriate anticoagulant. For optimal results the sample should be processed within 6 hours of venipuncture. EDTA, ACD or heparin may be used if the blood sample is processed for analysis within 30 hours of venipuncture. ACD or heparin, but not EDTA, may be used if the sample is not processed within 30 hours of venipuncture. Samples that cannot be processed within 48 hours should not be used (13,14). Unstained anticoagulated blood should be retained at C prior to sample processing. Blood samples that are hemolyzed, clotted or appear to be lipemic, discolored or to contain interfering substances should not be used. Refer to"standard Procedures For The Collection Of Diagnostic Blood Specimens", published by the National Committee For Clinical Laboratory Standards (NCCLS) for additional information on the collection of blood specimens.

5 X. SAMPLE PREPARATION 1. Collect blood into an appropriate anticoagulant. 2. Determine leukocyte count and, if necessary, dilute blood in an isotonic solution to a concentration of approximately 5 x 10 6 leukocytes per ml. Determine cell viability using Trypan Blue or propidium iodide. If the cell viability is not at least 85%, the blood sample should not be used. 3. Pipette 100 µl of well mixed blood into 12 x 75 mm polypropylene centrifuge tubes marked CD19 and Control, and add 5 µl of LIFE TECHNOLOGIES CD19 R-PE or CD19 TRI-COLOR to the tube marked CD Add 10 µl of corresponding LIFE TECHNOLOGIES IgG1 R-PE-or IgG1 TRI-COLO R-conjugated isotypic control reagent to tubes marked Control. Mix gently. 5. Incubate all tubes for 15 minutes at room temperature (22 ± 3 C) in the dark. 6. Add 100 µl of Cal-Lyse solution to all tubes. 7. Incubate all tubes containing Cal-Lyse solution for 10 minutes at room temperature (22 ± 3 C). 8. Add 2-4 ml of deionized water to all tubes containing Cal-Lyse solution. 9. Centrifuge all tubes at 400 x g for 3 minutes at room temperature and remove supernate. 10. Add fixing solution to all tubes according to the manufacturer's directions. Retain cells in fixing solution for not less than 30 minutes at room temperature (22 ± 3 C). 11. Centrifuge all tubes at 400 x g for 3 minutes at room temperature and remove supernate. 12. Resuspend the cells in 1 ml of PBS or sheath fluid, and store tubes at 2-8 C in the dark until flow cytometric analysis is performed. It is recommended that samples be analyzed within 18 hours of fixation. 13. Analyze on a flow cytometer according to the manufacturer's instructions. XI. MATERIALS REQUIRED BUT NOT SUPPLIED Centrifuge capable of at least 400 x g Vortex mixer 12 x 75 mm polypropylene centrifuge tubes Micropipette capable of dispensing 5 µl, 10 µl, 20 µl, 100 µl, and 500 µl volumes Blood collection tubes with anticoagulant Phosphate buffered saline (PBS) Trypan Blue or propidium iodide, 0.25% (w/v) in PBS for the determination of cell viability (15) Lysing solution Fixing solution Flow cytometer: Becton Dickinson FACScan TM, Coulter Profile or equivalent

6 XII. INTERPRETATION OF RESULTS FLOW CYTOMETRY Analyze antibody-stained cells on an appropriate flow cytometer according to the manufacturer's instructions. The right angle light scatter, or side scatter (SSC), versus forward angle light scatter (FSC) is collected to reveal the lymphocyte cell cluster. A gate is drawn for the lymphocyte cluster (lymphocyte bitmap). The fluorescence attributable to the R-PE- or TRI-COLOR -conjugated monoclonal antibody is collected, and the percentage of antibody-stained lymphocytes is determined. An appropriate R-PE- or TRI-COLOR -conjugated isotypic control of the same heavy chain immunoglobulin class and antibody concentration must be used to estimate and correct for nonspecific binding to lymphocytes. An analysis region is set to exclude background fluorescence and to include positively stained cells. The following histograms are representative of cells stained and gated on the lymphocyte region from a normal donor: LIFE TECHNOLOGIES IgG1 R-PE ISOTYPE CONTROL Cell Number Neg Pos Fluorescence Intensity LIFE TECHNOLOGIES CD19 R-PE MONOCLONAL ANTIBODY Cell Number Neg Pos Fluorescence Intensity

7 LIFE TECHNOLOGIES IgG1 TRI-COLOR ISOTYPE CONTROL Cell Number Neg Pos Fluorescence Intensity LIFE TECHNOLOGIES CD19 TRI-COLOR MONOCLONAL ANTIBODY Cell Number Neg Pos ABSOLUTE COUNTS Fluorescence Intensity Use the following formula for the calculation of Absolute Counts: Absolute Counts = Total White Blood Cell Count x % Positive Lymphocytes x % Positively Stained Cells/10 4. Due to an unacceptable variance among the different laboratory methods for determining absolute counts, an assessment of the accuracy of the method is necessary (16).

8 XIII. QUALITY CONTROL PROCEDURES Nonspecific fluorescence identified by the R-PE- and TRI-COLOR -conjugated isotypic controls is usually not greater than 4% in normal individuals. If the background level exceeds these values, test results may be in error. Increased nonspecific fluorescence may be seen in some disease states. A blood sample from each normal and abnormal donor should be stained with the CD45 panlymphocyte and CD14 panmonocyte monoclonal antibodies (12). When used in combination, these reagents assist in identifying the lymphocyte analysis region, and distinguish lymphocytes from monocytes, granulocytes and unlysed or nucleated red cells and cellular debris. A blood sample from a healthy normal donor should be analyzed as a positive control on a daily basis or as frequently as needed to ensure proper laboratory working conditions. Each laboratory should establish their own normal ranges, since values obtained from normals may vary from laboratory to laboratory. An appropriate isotype control should be used as a negative control with each patient sample to identify nonspecific Fc binding to lymphocytes. An analysis region should be set to exclude the nonspecific fluorescence identified by the isotypic control, and to include the brighter fluorescence of the lymphocyte population that is identified by the specific antibody. Refer to the appropriate flow cytometer instrument manual and other available references for recommended instrument calibration procedures (17,18). XIV. LIMITATIONS OF THE PROCEDURE 1. Incubation of antibody with cells for other than the recommended time and temperature may result in capping or loss of antigenic determinants from the cell surface. 2. The values obtained from normal individuals may vary from laboratory to laboratory; therefore, it is recommended that each laboratory establish its own normal range. 3. Abnormal cells or cell lines may have a higher or lower antigen density than normal cells. This could, in some cases, require the use of a larger quantity of monoclonal antibody than is indicated in the procedures for Sample Preparation. 4. Blood samples from abnormal donors may not always show abnormal values for the percentage of lymphocytes stained with a given monoclonal antibody. Results obtained by flow cytometric analysis should be considered in combination with results from other diagnostic procedures. 5. When using the whole blood method, red blood cells found in some abnormal donors, as well as nucleated red cells found in normal and abnormal donors may be resistant to lysis by lysis solutions. Longer red cell lysis periods may be needed to avoid the inclusion of unlysed red cells in the lymphocyte gated region. 6. Blood samples should not be refrigerated or retained at ambient temperature for an extensive period (longer than hours) prior to incubating with monoclonal antibodies. 7. Accurate results with flow cytometric procedures depend on correct alignment and calibration of the laser, as well as proper gate settings. 8. Due to an unacceptable variance among the different laboratory methods for determining absolute lymphocyte counts, an assessment of the accuracy of the method used is necessary (16).

9 XV. EXPECTED VALUES Blood samples were collected from a total of 155 apparently healthy, normal donors in an age range of 16 to 72 years with a mean age of 41 years. Samples were collected and analyzed in three independent laboratories. Roughly equal numbers of males and females were collected and analyzed in each laboratory. The normal donor population included members of differing ethnic origins. Including: adult Caucasians, Asians and Hispanics. Donors from geographically diverse areas of the United States participated in this study. These areas included the Western, Eastern and South Central regions. Blood samples collected from each donor were stained with the LIFE TECHNOLOGIES CD19 R-PE- and CD19 TRI-COLOR -conjugated monoclonal antibodies. Summary of expected values for LIFE TECHNOLOGIES anti-human CD19-conjugated monoclonal antibodies for all normal donors: Procedure Mean S.D. Range n CD19 R-PE CD19 TRI-COLOR Expected values for pediatrics and adolescents have not been established. The values obtained from normal individuals may vary from laboratory to laboratory; therefore, it is recommended that each laboratory establish its own normal range. XVI. PERFORMANCE CHARACTERISTICS SPECIFICITY Blood samples were obtained from healthy, normal donors of Caucasian, Black, Hispanic and Asian ethnic origins. Samples of each donor were stained with LIFE TECHNOLOGIES anti-human CD19 R-PE- and CD19 TRI-COLOR -conjugated monoclonal antibodies. Cells contained in the lymphocyte (Lymph.), monocyte (Mono.) and granulocyte (Gran.) regions were selected for analysis. Separate samples from the same donors were prepared for analysis of red blood cells (RBC) and platelets (Plt.) and stained with each of the LIFE TECHNOLOGIES monoclonal antibodies. CD19 PE Percent of Stained Cells Ethnic Origin Lymph. Mono. Gran. Plt. RBC Caucasian Caucasian Hispanic Asian Black Mean ±1 S.D

10 CD19 TRI-COLOR Percent of Stained Cells Ethnic Origin Lymph. Mono. Gran. Plt. RBC Caucasian Caucasian Hispanic Asian Black Mean ±1 S.D Specific and/or nonspecific antibody binding to Fc receptors on monocytes in a patient sample can be excluded by proper gating on lymphocytes on the flow cytometer. CORRELATION The correlation study was performed on 175 donors, including 155 normal and 20 abnormal donors. Comparison of the LIFE TECHNOLOGIES CD19 R-PE-conjugated monoclonal antibody with the Coulter CD19 RD1- conjugated monoclonal antibody Procedure Mean % r 2 Slope Y Positive Value Intercept n CD19 R-PE CD19 RD CD19 R-PE Linear regression y = 0.92x Comparison of the LIFE TECHNOLOGIES CD19 R-PE-conjugated monoclonal antibody with the Coulter CD19 FITC-conjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive Value Intercept n CD19 R-PE CD19 FITC 16.7 CD19 R-PE Linear regression y = 0.93x

11 Comparison of the LIFE TECHNOLOGIES CD19 TRI-COLOR -conjugated monoclonal antibody with the Coulter CD19 RD1-conjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive Value Intercept n CD19 TRI-COLOR CD19 RD CD19 TRI-COLOR Linear regression y = 0.92x Comparison of the LIFE TECHNOLOGIES CD19 TRI-COLOR -conjugated monoclonal antibody with the Coulter CD19 FITC-conjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive Value Intercept n CD19 TRI-COLOR CD19 FITC 16.7 CD19 TRI-COLOR Linear regression y = 0.94x Comparison of the LIFE TECHNOLOGIES CD19 TRI-COLOR -conjugated monoclonal antibody with the Life Technologies CD19 R-PE-conjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive Value Intercept n CD19 TRI-COLOR CD19 R-PE 16.4 CD19 TRI-COLOR Linear regression y = 0.99x REPRODUCIBILITY, INTRA-LAB Intra-lab reproducibility for LIFE TECHNOLOGIESanti-human CD19 R-PE- and CD19 TRI-COLOR - conjugated monoclonal antibodies was assessed by performing 10 replicate determinations for each antibody in each of three ranges, high, medium and low. Thus, a total of 30 determinations were performed for each conjugate of CD19. In this manner, reproducibility was demonstrated throughout the entire measuring range. The 10 determinations for each range were performed by the staining, processing and analysis of 10 separate samples. Lymphocytes were selected for the analysis of percent cells stained in each of the three ranges. To perform this study, anticoagulated blood was obtained from an abnormal donor expressing a high percentage of CD19 + cells. Mid range and low range samples were obtained by adding known CD19 - cells in appropriate ratios, while maintaining approximately the same total cell concentrations for the three ranges. The study was performed in each of three independent laboratories, in the manner that each laboratory obtained, stained and analyzed separate blood samples. The following data are representative:

12 Procedure Level Mean % S.D. % CV n Positive CD19 R-PE high mid low Procedure Level Mean % S.D. % CV n Positive CD19 high TRI-COLOR mid low REPRODUCIBILITY, INTER-LAB Inter-lab reproducibility for LIFE TECHNOLOGIES anti-human CD19 R-PE- and CD19 TRI-COLOR - conjugated monoclonal antibodies was assessed by performing 10 replicated determinations for each antibody in each of three ranges; high, medium and low. Thus, a total of 30 determinations were performed for each form of CD19. In this manner, reproducibility was demonstrated throughout the entire measuring range. The 10 determinations for each range were performed by the staining, processing and analysis of 10 separate samples. Lymphocytes were selected for the analysis of percent cells stained in each of the three ranges. The study was performed in each of three laboratories. All laboratories stained and analyzed blood samples from the same blood donors. Lysed, unstained samples containing cells in the appropriate ranges were prepared by one of the participating laboratories for staining and analysis in each of the other laboratories. The following data were obtained: SITE 1 Procedure Level Mean % S.D. % CV n Positive CD19 R-PE high mid low Procedure Level Mean % S.D. % CV n Positive CD19 high TRI-COLOR mid low SITE 2 Procedure Level Mean % S.D. % CV n Positive CD19 R-PE high mid low

13 Procedure Level Mean % S.D. % CV n Positive CD19 high TRI-COLOR mid low SITE 3 Procedure Level Mean % S.D. % CV n Positive CD19 R-PE high mid low Procedure Level Mean % S.D. % CV n Positive CD19 high TRI-COLOR mid low SENSITIVITY STUDY The measurement of sensitivity for LIFE TECHNOLOGIES monoclonal antibodies was determined by staining a series of blood samples from normal donors with decreasing concentrations of each of the antibodies to affect a titration curve relating the antibody concentration to percent of lymphocytes stained and the mean channel fluorescence. The concentration ranges of antibodies were selected to include one or more concentrations expected to saturate all epitopes on appropriate target cells (saturation point), and one or more lower antibody concentrations expected to represent the least amount of antibody (detection threshold) that may be used to detect the same percentage of cells as was detected at the higher antibody concentrations. Thus, a titration curve was generated for each of the monoclonal antibodies to identify the saturation point and detection threshold. The study provides a measurement of the potency of the antibodies for the intended target cells, and indicates the minimal concentrations of CD19 R-PE and CD19 TRI-COLOR that are required to detect all target cells. In practice, samples containing 10 6 leukocytes/ml were stained with not less than 6 concentrations of each of the antibodies, in the range of 1.0 µg/test to µg/test. Measurement sensitivity for LIFE TECHNOLOGIES CD19 R-PE- and CD19 TRI-COLOR -conjugated monoclonal antibodies in samples obtained from a representative normal donor are as follows:

14 CD19 R-PE: ANTIBODY CONCENTRATION VS PERCENT + CELLS % Positive Cells in Whole Blood CD19 R-PE (ng/ml) CD19 R-PE: ANTIBODY CONCENTRATION VS MEAN FLUORESCENCE % Maximal MFI CD19 R-PE (ng/ml)

15 CD19 TRI-COLOR : ANTIBODY CONCENTRATION VS PERCENT + CELLS % Positive Cells in Whole Blood CD19 TRI-COLOR (ng/ml) CD19 TRI-COLOR : ANTIBODY CONCENTRATION VS MEAN FLUORESCENCE % Maximal MFI CD19 TRI-COLOR (ng/ml)

16 XVII. BIBLIOGRAPHY 1. Hsu S., Cossman J., Jaffe E. Lymphocyte subsets in normal human lymphoid tissues. Am. J. Clin. Path. 80: 21-30, Morimoto C., Letvin N.L., Distaso J.A., et al.: The cellular basis for the induction of antigen-specific T8-suppressor cells. Eur. J. Immunol. 16: , Dorken B., Moller P., Pezzutto A., Schwartz-Albiez R., Moldenhauer G. B-cell antigens: CD19, In Fourth International Workshop and Conference On Human Leukocyte Differentiation Antigens, pp 34-36, Vienna, Pirruccello S.J., Johnson D.R. Reagents for flow cytometry: monoclonal antibodies and hematopoietic cell antigens, in Flow Cytometry And Clinical Diagnosis, pp 56-78, Pub. American Society of Clinical Pathologists, Shields J.G., Rigley K.P., Callard R.E. Regulation of human B cell proliferation by the CD19 cellsurface glycoprotein, in Fourth International Workshop and Conference On Human Leucocyte Differentiation Antigens, pp 40-43, Vienna, Nadler L.M., in Leukocyte Typing II, Volume 2, B cell/leukemia Panel Workshop: Summary and Comments, Chapter 1: 2-20, Springer Verlag, New York, Wedgwood R.J. X-linked agammaglobulinemia, in CRC Handbook Series In Clinical Laboratory Science, CRC Press, West Palm Beach Florida, pp 41-50, Pearl E.R., Vogler L.B., Okos A.J., et al.: B lymphocyte precursors in bone marrow. An analysis of normal individuals and patients with antibody-deficiency states. J. Immunol. 120: , Spickett G.P., Webster A.D., Farrant J. Cellular abnormalities in common variable immunodeficiency. Immunodefic. Rev. 2: , Small T.N., Keever C., Collins N., et al.: Characterization of B cells in severe combined immunodeficiency disease. Hum. Immunol. 25: , Goldstein R., Izaguirre L.C., Douglas J., et al.: Systemic lupus erythematosus and common variable panhypogammaglobulinemia. Theoretical and practical considerations, Fed. Proc. 29: , Loken M.L., Brosnan N.N., Back B.A., Ault K.A. Establishing optimal lymphocyte gates for immunophenotyping for flow cytometry. Cytometry 11: , Guidelines for the performance of CD4 + T-cell determinations in persons with human immunodeficiency virus infection. Morbidity And Mortality Weekly Report (MMWR), Volume 41/No. RR-8, May 8, Nicholson J.K.A., Green T.A. and Collaborative Laboratories. Selection of anticoagulants for lymphocyte immunophenotyping. J. Immunol. Methods 165: 31-35, Tennant J.R. Evaluation of the Trypan Blue technique for determination of cell viability, Transplantation. 2: , Koepke J.A., Landay A.L. Precision and accuracy of absolute lymphocyte counts. Clin. Immunol. and Immunopath. 52: 19-27, Brown M.C., Hoffman R.A., Kirchanski S. Controls for flow cytometers in hematology and cellular immunology. Ann. N.Y. Acad. Sci. 468: , Durrand R.E. Calibration of flow cytometer detector systems. Cytometry 2: , 1981.

17 Explanation of symbols Symbol Description Symbol Description Symbol Description Manufacturer Catalog number Batch code Use by Consult instructions for use Temperature limitation Caution, consult accompanying documents Life Technologies Corporation 7335 Executive Way Frederick, MD Life Technologies Limited European Regulatory Affairs 3 Fountain Drive, Inchinnan Business Park Paisley PA49RF, Scotland, UK Tel: +44 (0) DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. For support visit or techsupport@lifetech.com