BD CellFIX IVD. 10X concentrate Catalog No BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company.

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1 BD CellFIX X concentrate Catalog No INTENDED USE BD CellFIX is intended for the fixation of human peripheral blood cell suspensions after immunofluorescence staining with monoclonal antibodies and prior to flow cytometric analysis. 2. SUMMARY AND EXPLANATION BD CellFIX is a premixed fixing concentrate, especially formulated for the treatment of samples prior to analysis with (automated) flow cytometers such as the BD FACS brand instruments. 3/ IVD BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. 15 BD Becton Dickinson Benelux N.V. Becton, Dickinson and Company Erembodegem-Dorp 86 B-93 Erembodegem Belgium Tel Fax Benex Limited Pottery Road, Dun Laoghaire, Co. Dublin, Ireland Tel Fax BD Biosciences European Customer Support Tel Fax help.biosciences@europe.bd.com bdbiosciences.com ClinicalApplications@bd.com 3. PRINCIPLES OF THE PROCEDURE The interaction of BD CellFIX with human peripheral blood cells results in cross-linking of membrane-bound proteins. BD CellFIX effectively preserves stained human lymphocytes for at least 24 hours without significant alteration of lymphocyte subset enumeration values and fluorescence intensity results. The fixative may be used in conjunction with monoclonal antibodies conjugated to FITC, PE, PerCP, PerCP-Cy 5.5 *, PE- Cy 7, APC and APC-Cy7. Prior to flow cytometric analysis, whole blood is stained with monoclonal antibodies, treated with BD FACS lysing solution to lyse erythrocytes, then washed and fixed with BD CellFIX. * Cy is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and is made and sold under license from GE Healthcare. This product is licensed for sale only for in vitro diagnostics. It is not licensed for any other use. If you require any additional license to use this product and do not have one, return this material, unopened, to BD Biosciences, 235 Qume Drive, San Jose, CA 95131, and any money paid for the material will be refunded. APC-Cy7: US patent 5,714,386 1

2 4. REAGENT Reagent Provided BD CellFIX X concentrate, 5 ml (sufficient for fixations) of a proprietary buffered solution. Precautions Solution contains.% formaldehyde, CAS number 5--, 3.55% methanol, CAS number , and.93% sodium azide, CAS number Danger H311 Toxic in contact with skin. H331 Toxic if inhaled. H341 Suspected of causing genetic defects. H35 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. H318 Causes serious eye damage. H2 Harmful if swallowed. H315 Causes skin irritation. H317 May cause an allergic skin reaction. H413 May cause long lasting harmful effects to aquatic life. Wear protective gloves / eye protection. Wear protective clothing. 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: Call a POISON CENTER/doctor if you feel unwell. WARNING All biological specimens and materials coming into contact with them are considered biohazards. Handle as if capable of transmitting infection 1,2 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. Dilution Instructions For use, dilute BD CellFIX 1: with reagent-grade (distilled and deionized) water. Store in a glass container at room temperature ( C 25 C) for up to one month. Storage and Handling When stored at C 25 C, the reagent concentrate is stable until the expiration date shown on the label. Do not use after the expiration date. Alteration in the appearance of the reagent, such as precipitation or discoloration, indicates instability or deterioration. In such cases, the reagent should not be used. 5. SPECIMEN COLLECTION AND PREPARATION Collect blood aseptically by venipuncture into a sterile BD Vacutainer EDTA blood collection tube or equivalent. Store anticoagulated blood at room temperature ( C 25 C) for up to 6 hours until ready for staining. CAUTION Use standard precautions when obtaining, handling, and disposing of all human blood samples and/or potentially carcinogenic reagents. 6. PROCEDURE Reagent provided BD CellFIX, X concentrate (Catalog No. 3181) 2

3 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. Micropipettor with tips. 5- or -ml serological pipet. BD monoclonal antibodies to human leucocyte antigens. Vortex mixer. Low-speed centrifuge (minimum speed g) with swinging bucket rotor with 12 x 75-mm tube carriers. Vacuum aspirator with trap. Reagent-grade (both distilled and deionized) water. BD FACS lysing solution (X) (Catalog No. 3492) BD CellWASH (Catalog No ) or a wash buffer of phosphate buffered saline (PBS) with.1% sodium azide. 1X PBS (Dulbecco s modified PBS, ph 7.2 ±.2,.1 mol/l PO 4 and.15 mol/ L NaCl) 3. This reagent does not contain calcium, magnesium, phenol red, or sodium azide. Filter PBS through a.2- µm filter before use. Store at 2 C 8 C. Preparing Samples 1. Stain whole blood or peripheral blood mononuclear cells (PBMCs) with monoclonal antibodies using the amounts, incubation times, and temperatures specified in the appropriate reagent instructions for Falcon is a registered trademark of Corning Incorporated. use (IFU). Use care to protect tubes from direct light. 2. In the case of PBMCs, proceed to step 3. In the case of whole blood samples, lyse red blood cells following staining, using diluted BD FACS lysing solution (1X). Use care to protect tubes from direct light. Perform the procedure (incubation and centrifugation) as directed in the specific reagent data sheet or IFU or the BD FACS lysing solution IFU. Discard or aspirate the supernatant, leaving approximately 5 µl of residual fluid in the tube to avoid disturbing the pellet. 3. Vortex thoroughly at low speed to resuspend the cell pellet. Add 2 ml of BD CellWASH solution or 1X PBS with.1% sodium azide to the cell suspensions. Vortex thoroughly at low speed for 3 seconds. Centrifuge at g for 5 minutes at room temperature ( C 25 C). 4. Discard or aspirate the supernatant, leaving approximately 5 µl of residual fluid in the tube to avoid disturbing the pellet. 5. Vortex thoroughly at low speed to resuspend the cell pellet in the residual fluid. 6. Dilute room-temperature ( C 25 C) X BD CellFIX to 1X, following the instructions in Reagent, Section 4. Add.5 ml of 1X BD CellFIX solution to each tube except if the monoclonal antibody instructions for use specify other volumes. Vortex thoroughly at low speed for 3 seconds. Make sure that the cell suspension is well mixed with BD CellFIX. 3

4 7. Tubes 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. Vortex the cells thoroughly at low speed to help reduce aggregation before running them on the flow cytometer. CAUTION Some PE-Cy7 conjugates show changes in their emission spectra with prolonged exposure to paraformaldehyde. For overnight storage of stained cells, wash and resuspend in buffer without paraformaldehyde after 1 hour of fixation. 7. EXPECTED RESULTS Aliquots of blood specimens from normal and abnormal donors were stained with BD Multitest reagents CD3/CD8/CD45/ CD4 and CD3/CD16+CD56/CD45/ CD19, then lysed with BD FACS lysing solution, washed, and fixed with BD CellFIX (see Section 6, Preparing Samples). Samples were analyzed on a BD FACSCalibur flow cytometer with BD Multiset software, and lymphocyte subset populations (in %) were determined. The reference ranges for the parameters studied are presented in the following table. Refer to Section 9 for limitations about reference ranges. Lymphocyte subset BD Multiset software (n=29) Reichert et al 4 (n=271) Mean range Mean range %CD (6) a %CD16 + CD (6) a 6 29 %CD (4) a PERFORMANCE CHARACTERISTICS Performance Results obtained for cell preparations fixed with BD CellFIX were compared to non-fixed cell preparations (PBS) and to cell preparations fixed with a freshly prepared fixative solution (1X PBS, 1% paraformaldehyde,.1% sodium azide). Blood samples of one donor were prepared for analysis with BD Multitest reagents CD3/CD8/CD45/CD4 and CD3/ CD16+CD56/CD45/CD19. Lymphocyte subsets (in %) were determined in nonfixed samples, in samples treated with the reference fixative solution, and in samples treated with 6 different BD s identified as a through f. Cells were analyzed immediately after preparation (T= h) and after 24 hours (T=24 h). Figure 1 % T lymphocytes in non-fixed blood cell preparations (--), in cell preparations fixed with 1% paraformaldehyde solution/.1% sodium azide (reference fixative), and in blood cell preparations fixed with BD a, b, c, d, e or f. % NK lymphocytes in non-fixed blood cell preparations (--),in cell preparations fixed with 1% paraformaldehyde solution/.1% sodium azide (reference fixative), and in blood cell preparations fixed with BD a, b, c, d, e, or f. % B lymphocytes in non-fixed blood cell preparations (--), in cell preparations fixed with 1% paraformaldehyde solution/.1% sodium azide a. Values given in parentheses as standard deviations (SD). 4

5 (reference fixative), and in blood cell preparations fixed with BD a, b, c, d, e, or f Fixative a % T Lymphocytes f Fixative a % NK Lymphocytes f a Fixative c e f % B Lymphocytes T=h T=24h % T Lymphs, no fixation b T=h T=24h b Precision Repeatability was assessed using the BD Multitest reagents CD3/CD8/CD45/ CD4 and CD3/CD16+CD56/CD45/ CD19. Thirty aliquots of the same blood sample were stained, lysed, and treated c c d d % NK Lymphs, no fixation b T=h T=24h d e e % B Lymphs, no fixation with BD CellFIX, and lymphocyte subsets were determined. Samples were run on the BD FACSCalibur instrument immediately after treatment (T = h) and after 24 hours (T = 24 h). Lymphocyte subset at T=h n Mean SD %CV a %CD %CD16 + CD %CD a. CV = coefficient of variation Lymphocyte subset at T=24h n Mean SD %CV a %CD %CD16 + CD %CD a. CV = coefficient of variation Accuracy To determine accuracy of BD CellFIX, flow analysis of BD CellFIX treated and non-bd CellFIX treated 4-color stained cell preparations was performed, and resulting lymphocyte subsets were compared. Aliquots of blood specimens from 29 normal or abnormal donors were stained, lysed, washed, and fixed with BD CellFIX and immediately run on a BD FACSCalibur flow cytometer. Aliquots of same blood specimens were prepared in a similar way for 4-color analysis but were not fixed with BD CellFIX. Figure 2 % T lymphocytes in non-fixed blood cell preparations vs % T lymphocytes in cell preparations fixed with BD CellFIX; % NK lymphocytes in non-fixed blood cell preparations vs % NK lymphocytes in cell preparations fixed with BD CellFIX; % B lymphocytes in 5

6 non-fixed blood cell preparations vs % B lymphocytes in cell preparations fixed with BD CellFIX. % T Lymphs in blood cell preparations fixed with CellFIX % NK Lymphs in blood cell preparations fixed with CellFIX % B Lymphs in blood cell preparations fixed with CellFIX % T Lymphocytes Expected values y =,9774x + 1,1417 R 2 =, % T Lymphs in non-fixed blood cell preparations % NK Lymphocytes Expected values y = 1,22x -,1176 R 2 =,9818 % NK Lymphs in non-fixed blood cell preparations % B Lymphocytes Expected values y =,9774x +,3194 R 2 =, % B Lymphs in non-fixed blood cell preparations 9. LIMITATIONS Each laboratory should establish normal ranges for leucocyte subsets using its own test conditions. EDTA is the anticoagulant of choice. BD Biosciences has limited information concerning use of other anticoagulants. Samples should be processed within 6 hours of collection. Store blood samples in BD Vacutainer blood collection tubes or equivalent at room temperature prior to staining, lysing, and fixing. Incubation times or temperatures other than those specified may be a source of error. The volumes of reagent recommended are based on studies of normal human blood. WARRANTY Unless otherwise indicated in any applicable BD general conditions of sale for non-us customers, the following warranty applies to the purchase of these products. THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT. REFERENCES 1. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 5. M29-A3. 2. Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37: 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. 3rd ed. Washington, DC: American Society for Microbiology; 1986:

7 4. Reichert TA, DeBruyere M, Deneys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopathol. 1991; 6: