Resolve Panel A A Qualitative Test for the Identification of Unexpected Blood Group Antibodies
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1 Revised October 2015 Reagent Red Blood Cells Resolve Panel A A Qualitative Test for the Identification of Unexpected Blood Group Antibodies Rx ONLY SUMMARY AND EXPLANATION When an unexpected antibody has been detected in serum, it must be identified to determine its clinical significance. Blood group antibodies are not all equally dangerous in transfusion therapy or in pregnancy. Once the identity of the antibody (or antibodies) has been established, standard texts may be consulted for guidance in specific situations. Antibody identification is accomplished by testing the serum against a panel of red cells having different antigen characteristics, observing the presence or absence of hemolysis or agglutination and comparing the pattern of reactivity with the antigen profile of the cells. RESOLVE Panel A consists of red blood cells from 11 individual adult donors, a vial of diluent and an ANTIGRAM Antigen Profile. A separate product, RESOLVE Panel B, is available should additional cells be required for the resolution of complex mixtures of antibodies. PRINCIPLE OF PROCEDURE Using the conditions under which the antibody was originally detected, the serum is combined with each cell sample of the panel. Most often these conditions will include an incubation temperature of 37 C and the addition of an antibody enhancement solution during the incubation phase. If, on initial testing, reactivity of the antibody was observed to occur at room temperature and decrease in strength after incubation at 37 C, identification tests should be done by the cold panel procedure. Occasionally it may be necessary to use both the warm panel and cold panel procedures. Antibody identification may be facilitated by recording results at each phase of testing and grading the strength of positive reactions. REAGENT RESOLVE Panel A is a series of red blood cells in 3% suspensions from 11 group O individuals. One or more of the red blood cells used in RESOLVE Panel A may have been held in frozen storage. The cells are suspended in a preservative developed by Ortho-Clinical Diagnostics, Inc. a phosphate-citrate buffered diluent to which a purine, a steroid and nucleosides have been added to maintain reactivity and/or retard hemolysis during the dating period. Chloramphenicol (1:3,000), neomycin sulfate (1:10,000) and gentamicin (1:20,000) have been added to retard bacterial contamination. The individual cells have been carefully selected to include most of the blood group factors of clinical significance. RESOLVE Panel A Reagent Red Blood Cells should be used directly from the vial. As with all reagent red blood cells, the reactivity of the cells may decrease during the dating period. The rate at which antigen reactivity (e.g., agglutinability) is lost is partially dependent upon individual donor characteristics that are neither controlled nor predicted by the manufacturer. Do not use if marked hemolysis is observed. Reagent Red Blood Cell Diluent is the same diluent in which the cells of RESOLVE Panel A are suspended. For in vitro diagnostic use. No U.S. Standard of Potency. Do not freeze. Do not use beyond expiration date. The expiration date of each lot is no longer than 70 days, excluding the days in frozen storage, from the date of collection of red blood cells from any donor in the lot. Store at 2 to 8 C. All donors have been tested with a wide variety of reagents for antigens of low incidence and high incidence. Antigens for which donors have been tested and cells positive for an antigen of low incidence or negative for an antigen of high incidence are noted in the accompanying ANTIGRAM Antigen Profile. CAUTION: All blood products should be treated as potentially infectious. Source material from which this product was derived was found negative when tested in accordance with current FDA required tests. No known test methods can offer assurance that products derived from human blood will not transmit infectious agents. SPECIMEN COLLECTION AND PREPARATION Either serum or plasma may be used. Specimen collection should be accomplished by acceptable medical procedures. No special preparation of the patient is required prior to specimen collection. Bacterial contamination may interfere with the results and interpretation of the test. Specimen storage should be within applicable regulating agencies requirements. If specimens are stored before testing, they should be stored at 2 to 8 C. PROCEDURE Material Provided Reagent Red Blood Cells RESOLVE Panel A Required Supplementary Materials 1. Test tubes, 10 x 75 mm or 12 x 75 mm 2. Pasteur pipettes 3. Centrifuge 4. Isotonic saline 5. Antibody enhancement solution, such as a low ionic strength solution or bovine albumin (optional) 6. Anti-human globulin [such as ORTHO Anti-Human Globulin, Anti-lgG, -C3d, polyspecific; Anti-Human Globulin (Rabbit and Murine Monoclonal) BioClone, Anti-IgG, -C3d, polyspecific or ORTHO Anti-IgG] ORTHO 1
2 7. Coombs control cells (such as ORTHO Coombs Control) 8. Incubator, 37 C (warm panel) 9. Incubator, 15 to 18 C (cold panel) 10. Optical aid Directions for Use An auto control, i.e., a test of the individual s serum with his own red blood cells, should be set up with every panel and carried through the same procedures. Use the Reagent Red Blood Cell Diluent provided, or isotonic saline, to resuspend the individual s red cells for the auto control as follows: Place one drop of the Reagent Red Blood Cell Diluent (or isotonic saline) in a small test tube. Add the individual s cells to make an approximate 3% to 5% suspension. or If the individual s red cells were resuspended to 3% to 5% in saline, transfer one drop of this suspension to a test tube, centrifuge, decant the saline and replace with one drop of Reagent Red Blood Cell Diluent. The most clinically significant unexpected antibodies generally fall into the warm reactive category and will persist through antiglobulin testing. Consequently, most sera will be tested with the warm panel procedure. NOTE: If the antibody was originally detected without an antibody enhancement solution, two warm panels should be set up: one omitting Step 7 and increasing incubation time at 37 C to one hour, and the other including Step 7. Warm Panel Saline Wash Method 1. Label a series of 12 small test tubes #1 through #12 and place in a rack. 2. To each tube, with a Pasteur pipette, add at least two drops of the serum or plasma under test. 3. To tube #1, add one drop of thoroughly mixed RESOLVE Panel A Cell #1, to tube #2 add one drop of thoroughly mixed RESOLVE Panel A Cell #2, etc. 4. To auto control tube, with a Pasteur pipette, add one drop of a 3% to 5% suspension of the patient s cells in the Reagent Red Blood Cell Diluent or in isotonic saline. 5. If a room temperature test is desired, mix well, centrifuge and read as in Step 6. Otherwise proceed to Step 7. Suggested centrifugation: approximately 15 seconds at 3400 rpm ( rcf) or 1 minute at 1000 rpm ( rcf). 6. Resuspend the cells in all test tubes by gentle agitation and examine macroscopically* for hemolysis and/or agglutination. Complete hemolysis precludes further testing and must be interpreted as a positive result. If partial hemolysis or agglutination is observed, record and proceed to Step 7. Grade agglutination from 4+ to 1+ at each stage of reading. 7. To each test tube, add antibody enhancement solution according to the manufacturer s directions. 8. Mix well and incubate all tests at 37 C for 15 to 60 minutes. 9. Centrifuge (see Step 5 for suggested centrifugation). 10. Resuspend the cells by gentle agitation and examine macroscopically* for hemolysis and/or agglutination. 11. Wash cells in each tube three times with tubes full of isotonic saline. Decant and drain completely after the last washing. 12. To each test tube, add two drops of anti-human globulin or follow directions accompanying anti-human globulin being used. 13. Mix the contents of each tube well and centrifuge (see Step 5 for suggested centrifugation). 14. Resuspend the cells by gentle agitation and promptly examine macroscopically* for agglutination. 15. It is recommended that all negative antiglobulin tests be controlled by adding red blood cells sensitized with lgg antibody, e.g., ORTHO Coombs Control (see package insert for procedure). Increasing the serum-to-cell ratio by adding three to four drops of serum is an accepted means of enhancing antibody identification in other than low ionic strength systems. * An optical aid, such as a hand lens or magnifying mirror, may enhance visibility of weak reactions. Caution must be exercised when reading microscopically. Interpretation Identification of the antibody present in the serum may be made by matching the reactions obtained with the ANTIGRAM Antigen Profile furnished with the reagent. For example, if the serum reacts with the D(Rh o ) positive cells but not the D(Rh o ) negative cells, the antibody is probably anti-d (anti-rh o ). If the antibody specificity is not evident, additional cells may be required. Such cells may be selected from RESOLVE Panel B or obtained locally. Stability of Final Reaction Mixture All results should be interpreted upon test completion. Cold Panel RESOLVE Panel A Reagent Red Blood Cells may also be used in the cold panel procedure. AFFIRMAGEN Reagent Red Blood Cells and ORTHO A 2 Cells Reagent Red Blood Cells are also helpful in the identification of cold agglutinins. In the cold panel procedure, the patient s serum and reagent cells are incubated without an antibody enhancement solution for one to two hours at 15 to 18 C and read without centrifugation. 1. A series of 12 small test tubes, labeled #1 through #12, is placed in a rack. 2. To each tube, with a Pasteur pipette, add two drops of the serum or plasma containing the antibody. 3. To tube #1, add one drop of thoroughly mixed RESOLVE Panel A Cell #1, to tube #2 add one drop of thoroughly mixed RESOLVE Panel A Cell #2, etc. 4. To tube #12, with a Pasteur pipette, add one drop of a 3% to 5% suspension of the patient s cells in the Reagent Red Blood Cell Diluent or in isotonic saline. 5. Incubate all tubes at 15 to 18 C for one to two hours. (This temperature can be maintained by placing the rack of test tubes in a large flat pan of cold water to which a few ice cubes have been added.) 6. Remove tubes from the cold bath and read quickly. Do Not Centrifuge. Examine for agglutination and record reactions, grading positives from 4+ to 1+. 2
3 Interpretation Identification of specific cold antibodies present in the serum may be made by matching the reactions obtained with the ANTIGRAM Antigen Profile furnished with RESOLVE Panel A. Stability of Final Reaction Mixture All results should be interpreted upon test completion. CONTROL OF ERROR 1. A control consisting of the serum and autologous red blood cells should be tested in parallel with RESOLVE Panel A through all phases of the identification procedure. Positive reactions in any phase of the control tube indicate a patient abnormality which must be resolved before the test results can be interpreted. It is recommended that the auto control be resuspended in the Reagent Red Blood Cell Diluent provided in order to more closely duplicate the conditions of RESOLVE Panel A Reagent Red Blood Cells. (Exact duplication may not be possible due to the length of time RESOLVE Panel A Reagent Red Blood Cells are exposed to the Reagent Red Blood Cell Diluent.) 2. Negative antiglobulin tests should be controlled with group O red blood cells weakly sensitized with lgg antibody, such as ORTHO Coombs Control. Agglutination of the cells indicates: The anti-human globulin was capable of reacting in the test. The anti-human globulin had not been neutralized by improper washing of the cell/serum mixture. The anti-human globulin had, in fact, been added to the test. 3. For quality assurance, RESOLVE Panel A should be tested periodically with weak antibodies. LIMITATIONS OF PROCEDURE 1. Contaminated supplementary materials used in the procedures described may interfere with the test results. 2. Improper technique may invalidate the results obtained with this reagent. 3. If multiple antibodies are present in the serum, additional cells may be required for identification. 4. False-positive results may occur if antibodies to components of the preservative solution are present in the serum tested. 5. Variations in cell concentration can affect the sensitivity of test results. When the cell concentration of reagent red blood cells is modified for use in certain applications, such as the gel test or column agglutination methods, the accuracy of the cell concentration becomes more critical. When cells are too concentrated, weaker results may be obtained due to the increase in the antigen/antibody ratio. In addition, cells may fail to completely migrate to the bottom of the column or microtube and could cause a false-positive interpretation. When the cell concentration is too low, the red cells may be difficult to see, potentially causing a weak reaction to be missed. Care should be taken to follow the manufacturer s recommendations when altering the concentration of the Reagent Red Blood Cells. 6. Complement-dependent antibodies may not be detected if a plasma specimen is used. 7. Optimal reaction conditions vary across antibody specificities. No single test method will detect all antibodies. SPECIFIC PERFORMANCE CHARACTERISTICS When properly stored and used according to the procedures described under Directions for Use, these reagent red blood cells will aid in the identification of antibodies directed against the antigens present on them. The antigen structure will vary with each individual lot. The presence or absence of each antigen listed on the accompanying ANTIGRAM Antigen Profile has been demonstrated by testing with at least two sources of antiserum unless rarity of the antiserum precludes it. Each of these tests have been conducted and interpreted independently. Each cell sample is shown to have a negative direct antiglobulin test, indicating that no human lgg or human complement components are detectable on the cell surface. Meets requirements of the FDA. Technical questions concerning this reagent should be directed to Customer Technical Support at The centrifugal force applied to cell/serum mixtures should be the minimum required to produce a button of red cells and a clear supernate. Overcentrifugation, i.e., the application of forces in excess of the minimum, causes the cells to adhere to the bottom of the test tube so that vigorous agitation is necessary before they can be resuspended. During such agitation, weak agglutination may be dispersed causing a positive reaction to be missed. Undercentrifugation, i.e., the failure to apply forces necessary to cause the celis to form a button and a clear supernate, may result in a weak or negative reaction. No one speed and time of centrifugation can be recommended which will cover the wide variety of centrifuges available; each laboratory must calibrate its own equipment and determine the time required at a given speed to achieve the desired result. SUMMARY OF REVISIONS Section Front Page SPECIFIC PERFORMANCE CHARACTERISTICS Back Page Revision Added Rx ONLY. Updated section to align with regulations for testing of source material. Updated corporate logo. Updated copyright information. 3
4 BIBLIOGRAPHY 1. Beattie K. Control of the antigen-antibody ratio in antibody detection/compatibility tests. Transfusion 1980;20: Technical manual. 14th ed. Bethesda, MD: American Association of Blood Banks, Issitt PD. From kill to overkill: 100 years of (perhaps too much) progress. Immunohematology 2000;16:18. 4
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