WHOLE BLOOD LYSING SOLUTION FOR FLOW CYTOMETRIC APPLICATIONS
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1 FOR IN-VITRO DIAGNOSTIC USE INVITROGEN CAL-LYSE TM Lysing Solution WHOLE BLOOD LYSING SOLUTION FOR FLOW CYTOMETRIC APPLICATIONS CAL-LYSE TM CATALOG No. GAS tests 25 ml CATALOG No. GAS-010S tests 100 ml Explanation of symbols Symbol Description Symbol Description Catalogue Number Research Use Only Use by Batch code In vitro diagnostic medical device Temperature limitation Manufacturer European Community authorised representative [-] Without, does not contain [+] With, contains Protect from light Consult accompanying documents Directs the user to consult instructions for use (IFU), accompanying the product.
2 I. INTENDED USE Invitrogen Cal-Lyse TM is a lysing solution formulated to enable the lysis of erythrocytes in samples of anticoagulated human peripheral blood. Invitrogen Cal-Lyse TM lysing solution is intended to be used as an aid in the enumeration of leukocytes that have been stained with Invitrogen monoclonal antibodies for analysis using flow cytometric methods. II. SUMMARY AND EXPLANATION Invitrogen Cal-Lyse TM is a premixed solution for use in whole blood staining procedures. Lysis of erythrocytes is performed immediately following staining of the blood samples with Invitrogen fluorochrome-conjugated monoclonal antibodies. Blood samples may then be washed by centrifugation to remove cellular debris resulting from the lysis procedure. The remaining erythrocyte-free intact antibodystained leukocytes are suitable for flow cytometric analysis. Failure to completely remove erythrocytes from blood samples may interfere with the subsequent flow cytometric enumeration of antibody-stained leukocytes. III. PRINCIPLES OF THE TEST Invitrogen monoclonal antibodies bind to the surfaces of viable blood cells that express the corresponding antigens. To identify cells bearing these antigenic determinants, peripheral blood samples are incubated with fluorochrome-conjugated monoclonal antibodies. Prior to the removal of unbound antibody, Invitrogen Cal-Lyse TM lysing solution is added to lyse red blood cells. Cells may subsequently be washed, resulting in the elimination of red cell debris as well as unbound antibody. Invitrogen Cal-Lyse TM lysing solution contains formaldehyde as fixative, and no additional fixation is required. Antibody-stained and fixed leukocytes are subsequently analyzed using flow cytometric methods. IV. REAGENT PROVIDED Invitrogen Cal-Lyse TM Lysing Solution is sufficient for the lyse of 250 samples (GAS-010) of or 1000 samples (GAS-010S-100) of anticoagulated whole blood. The ingredients of the lysing solution are as follows: Polyvinylpyrrolidone Ethylene-bis (oxyrthylenenitruol) - tetra acetic acid Sodium phosphate Formaldehyde Deionized water Invitrogen Cal-Lyse TM solution is not a concentrate, and should not be diluted prior to use. For optimal results, use only as directed.
3 V. STATEMENTS OF WARNING Do not pipet by mouth. Samples should be handled as if capable of transmitting infection. Appropriate disposal methods should be used. Do not use reagent beyond the stated expiration date of the product. Invitrogen Cal-Lyse TM solution is not a concentrate, and should not be diluted prior to use. For optimal results, use only as directed. Invitrogen Cal-Lyse TM solution contains formaldehyde as a fixative. Formaldehyde is toxic, allergenic and a suspected carcinogen. Avoid ingestion, inhalation and contact with eyes, skin and clothing. Deviations from the recommended procedure enclosed within this product insert may invalidate the results of testing FOR IN VITRO DIAGNOSTIC USE VI. APPROPRIATE STORAGE CONDITIONS Store at Room Temperature. Do not freeze. VII. EVIDENCE OF DETERIORATION Reagent should not be used if any evidence of deterioration such as cloudiness or substantial loss of reactivity is observed. We recommend that you check this mixture against your standard periodically. The normal appearance of Invitrogen Cal-Lyse TM lysing solution is a clear, particulate-free liquid. Do not use this reagent if discoloration occurs or a visible precipitate forms. VIII. SAMPLE PREPARATION Invitrogen Antibody Staining and Wash Lysis Procedure: 1. Collect blood into an appropriate anticoagulant. 2. Pipette 100 µl of thoroughly mixed blood into 12 x 75 mm polypropylene centrifuge. 3. Add antibody as indicated in the manufacturer s package insert to appropriately labeled tubes from step 2. Mix gently. 4. Incubate all tubes for 15 minutes at room temperature (22 ± 3 C) in the dark. 5. Add 100 µl of Invitrogen Cal-Lyse TM lysing solution to all tubes. 6. Incubate all tubes for 10 minutes at room temperature (22 ± 3 C) in the dark. 7. Fill all tubes by adding approximately 1.0 ml of room temperature deionized water. Immediately vortex tubes individually. 8. Serofuge all tubes for 10 minutes at room temperature and remove supernatant. 9. Resuspend the cells in all tubes in 1 ml of phosphate buffered saline (PBS) or Sheath Fluid. 10. Analyze on a flow cytometer according to the manufacturer's instructions. Samples should be refrigerated if analysis is delayed.
4 Alternatively, the Invitrogen Antibody Staining and No-Wash Lysis Procedure may be employed, with a minor modification to the procedure above: Invitrogen Antibody Staining and No-Wash Lysis Procedure: 1. Collect blood into an appropriate anticoagulant. 2. Pipette 100 µl of well mixed blood into 12 x 75 mm polypropylene centrifuge tubes. 3. Add antibody as indicated in the manufacturer s package insert to appropriately labeled tube from step 2. Mix gently. 4. Incubate all tubes for 15 minutes at room temperature (22 ± 3 C) in the dark. 5. Add 100 µl of Invitrogen Cal-Lyse TM lysing solution to all tubes. 6. Incubate all tubes for 10 minutes at room temperature (22 ± 3 C) in the dark. 7. Add 1 ml of room temperature deionized water. Retain cells at room temperature for approximately 10 minutes. 8. Analyze on a flow cytometer according to the manufacturer's instructions. Samples should be refrigerated if analysis is delayed. IX. MATERIALS REQUIRED BUT NOT SUPPLIED: Centrifuge with swinging bucket rotor, at least 1000 x g Vortex mixer 12 x 75 mm polypropylene centrifuge tubes Micropipette capable of dispensing 100 µl volumes Blood collection tubes with anticoagulant Phosphate Buffered Saline (PBS) Flow cytometer
5 X. 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 fluorochrome-conjugated monoclonal antibody is collected, and the percentage of antibody-stained cells is determined. A fluorochrome-conjugated isotypic control may be used to estimate and correct for nonspecific binding to lymphocytes. The isotypic control must be of the same heavy chain immunoglobulin class as the specific antibody. The istotypic control should be used at approximately the same antibody concentration as the specific antibody. An analysis region is set to exclude background fluorescence and to include positively stained cells. The following histograms are representative of cell staining from a normal donor, gated on the lymphocyte region and stained with a representative Invitrogen T cell-specific monoclonal antibody, CD3 FITC, and a B cell-specific monoclonal antibody, CD19 R-PE. Cells were processed with the Invitrogen whole blood sample preparation method. Red blood cells were lysed with the Invitrogen Cal-Lyse TM lysing solution. LYSED WHOLE BLOOD 10 µl anti-cd19 R-PE 10 µl anti-cd3 FITC
6 INVITROGEN CD3 FITC MONOCLONAL ANTIBODY Cell Number Anti-CD3 FITC INVITROGEN CD19 R-PE MONOCLONAL ANTIBODY Cell Number Anti-CD19 R-PE
7 XI. LIMITATIONS OF THE PROCEDURE 1. The values obtained from normal individuals may vary from laboratory to laboratory; therefore, it is recommended that each laboratory establish its own normal range. 2. 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. Longer red cell lysis periods may be needed to avoid the inclusion of unlysed red cells. 3. Blood samples should not be refrigerated or retained at ambient temperature for an extensive period (longer than hours) prior to incubation with monoclonal antibodies. 4. Invitrogen Cal-Lyse TM lysing solution has been formulated for use with flow cytometers commonly used in a clinical laboratory environment, including the Becton Dickinson FACScan TM and Coulter EPICS Profile flow cytometers. Only limited information is available on the use of the lysing solution with other flow cytometers. 5. Invitrogen Cal-Lyse TM lysing solution has been specifically formulated for use with Invitrogen monoclonal antibodies. Only limited information is available on the use of the lysis solution with monoclonal antibodies from other sources. XII. EXPECTED VALUES Blood samples were collected from a total of 155 apparently healthy adult normal donors in an age range of 16 to 72, with a mean age of 41. Samples were stained with Invitrogen monoclonal antibodies and red blood cells were lysed with Invitrogen Cal-Lyse TM lysing solution. Samples were collected and analyzed in each of three independent laboratories. An approximately equal number of males and females were collected and analyzed in each laboratory. The normal donor population included members of differing ethnic origins, including adult Caucasians, Blacks, Asians and Hispanics. Donors in geographically diverse areas of the United States, including the Western, Eastern and SouthCentral regions, participated in this study. Summary of expected values for the Invitrogen T and B cell monoclonal antibodies, CD3 FITC and CD19 R-PE, for all normal donors: Procedure Mean % S.D. Range n Positive ± 2 S.D. CD3 FITC CD19 R-PE 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.
8 XII. 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 Invitrogen monoclonal antibodies and red blood cells were lysed with Invitrogen Cal-Lyse TM lysing solution. 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 (Pit.). All were stained with Invitrogen monoclonal antibodies. The following specificity data were obtained with the Invitrogen T and B cell monoclonal antibodies, CD3 FITC and CD19 R-PE, following the lysis of red blood cells with Invitrogen Cal-Lyse TM lysing solution: CD3 FITC Ethnic Percent of Stained Cells Origin Lymph. Mono. Gran. Plt. RBC Caucasian Caucasian Hispanic Asian Black Mean ± 1 S.D CD19 R-PE Ethnic Percent of Stained Cells Origin Lymph. Mono. Gran. Plt. RBC Caucasian Caucasian Hispanic Asian Black Mean 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.
9 CORRELATION The correlation between the Invitrogen and Coulter B cell monoclonal antibodies using the Invitrogen Cal- Lyse TM lysing solution was based on the performance of the B cell monoclonal antibodies CD19 R-PE and CD19 TRI-COLOR. The percentage, as well as the mean fluorescence, of B lymphocytes is substantially lower than is observed for T lymphocytes in normal peripheral blood. The resulting analysis of B lymphocytes by flow cytometric methods may be more susceptible to uncontrolled variations in the staining and lysis methods employed. A total of 155 normal donor samples were collected and analyzed in each of three independent laboratories. The samples were analyzed on either the FACScan TM, FACSCalibur TM, or Profile flow cytometer. The normal donor population included members of differing ethnic origins, including adult Caucasians, Blacks, Asians and Hispanics. Donors in geographically diverse areas of the United States, including the Western, Eastern and SouthCentral regions, participated in this study. Males and females were represented in approximately equal numbers. A total of 20 abnormal donors were analyzed on both the FACScan TM and Profile flow cytometers at a single site. Correlations were obtained between the Invitrogen and Coulter B cell monoclonal antibodies for all normal and abnormal donors (n = 175). Red blood cells from all samples were lysed with Invitrogen Cal-Lyse TM lysing solution. In this correlation study samples obtained from normal and abnormal donors were stained with Invitrogen and a comparable Coulter B cell monoclonal antibodies. Red blood cells were lysed with Invitrogen Cal- Lyse TM lysing solution in samples that had been stained with both Invitrogen and Coulter monoclonal antibodies. Comparison of the Invitrogen CD19 R-PE-conjugated monoclonal antibody with the Coulter CD19 RD1 conjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive value intercept CD19 R-PE CD19 RD CD19 R-PE Linear regression y = 0.92x Comparison of the Invitrogen CD19 R-PE-conjugated monoclonal antibody with the Coulter CD19 FITCconjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive value intercept CD19 R-PE CD19 FITC 16.7 CD19 Linear regression y = 0.93x
10 Comparison of the Invitrogen CD19 TRI-COLOR -conjugated monoclonal antibody with the Coulter CD19 RD1-conjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive value intercept CD19 TRI-COLOR CD19 RD CD19 TRI-COLOR Linear regression y = 0.92x Comparison of the Invitrogen CD19 TRI-COLOR -conjugated monoclonal antibody with the Coulter CD19 FITC-conjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive value intercept CD19 TRI-COLOR CD19 FITC 16.7 CD19 TRI-COLOR Linear regression y = 0.94x Comparison of the Invitrogen CD19 TRI-COLOR-conjugated monoclonal antibody with the Invitrogen CD19 R-PE-conjugated monoclonal antibody: Procedure Mean % r 2 Slope Y Positive value intercept CD19 TRI-COLOR CD19 R-PE 16.4 CD19 TRI-COLOR Linear regression y = 0.99x LEUKOCYTE RECOVERY This study was conducted on blood samples obtained from 5 normal donors consisting of representatives from Caucasian, Black, Hispanic and Asian ethnic origins. An appropriate hematology analyzer was used to enumerate leukocytes prior to, and immediately following the lysis of red blood cells with Invitrogen Cal-Lyse TM lysing solution as well as with the Ortho-mune TM Lysing Reagent. Leukocyte recovery from members of differing ethnic origins was comparable. Leukocyte recovery following lysis with Invitrogen Cal-Lyse TM and Ortho-mune TM lysing reagents was comparable. It should be noted that the washing of cells alone, in the absence of a lysis procedure may result in a modest loss of cells.
11 In the following table describing leukocyte recovery, leukocyte counts are expressed as cells per cu. mm. Leukocyte Count Donor Prior Following Percent No. Race to Lysis Lysis Recovered 1 Caucasian Caucasian Black Hispanic Asian Mean ± 1 SD RED BLOOD CELL LYSIS This study was conducted on blood samples obtained from 5 normal donors, to determine whether essentially all red cells were lysed by the lysing solution. Red cells were lysed in a sample of blood from each donor with Invitrogen Cal-Lyse TM lysing solution. An appropriate hematology analyzer was used to enumerate red blood cells prior to and immediately following lysis. The differing orders of magnitude of red cells counted prior to and following lysis should be noted in the following table. Red Blood Cell Count Donor Prior to Lysis Following Lysis Percent No. cells/cu. mm. x 10 6 cells/cu. mm x 10 5 Lysed Mean S.D XIII. BIBLIOGRAPHY 1. Mishell B.B., and A.M. Shilgi Selected methods in cellular immunology. W.H. Freeman and Company. 2. Whittam R. editor Transport and diffusion of red blood cells. Williams and Wilkins, Baltimore. 3. Gorgi, J.V., H. Cheng, J. Margolick et al Quality control in the flow cytometric measurement of T-lymphocyte subsets: the multicenter AIDS cohort study experience. Clin. Immunol. Immunopathol. 55: NCCLS Document H42-T, Clinical applications of flow cytometry: Quality Assurance and immunophenotyping of peripheral blood lymphocytes. Tentative Guideline, May, 1992.
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