Chapter 1: Serology Chapter 3: Flow Cytometry/Luminex Section: Technical Modules Module: (a) Serum Preparation Author: Stephanie Skibby, HLA Laboratory, Inland Northwest Blood Center, Spokane, WA Date prepared/revised: December 23, 2009 1
Objectives Ø Understand the clinical rationale for using different types of serum preparations for antibody screening or crossmatch testing. Ø Understand the limitations of and quality control necessary for different types of serum preparations. Ø Understand the pros and cons of different types of serum preparations. Ø Understand the ASHI Standards that are applicable to serum preparation. 2
Introduction A critical component of testing in the HLA laboratory is the ability to detect antibodies in the patient serum. This can be accomplished by antibody screening and identification or crossmatch techniques. IgG antibodies directed against foreign HLA molecules are considered to be the most clinically relevant class of antibody. Whatever the method used, it is important for laboratories to be able to process patient serum samples in such a way to minimize background and/or non-specific reactivity. It is common for patient serum to contain non-clinically relevant antibodies or complexes that interfere with the detection of clinically relevant antibodies. Patient serum may also contain contaminating substances from the patient s immunosuppressive regimen, such as OKT3. In some instances, plasma may have been collected from the patient or donor. If a large volume is required to be collected, it may be collected in the form of a standard blood bank donation or plasmapheresis. For most assays, serum is recommended by the test manufacturer as the anticoagulant present in plasma may interfere with testing. It is possible to convert the plasma into serum by recalcifying the plasma and allowing coagulation to occur. Calcium is added to the plasma to form a complex with the citrate present in the anti-coagulant. This restores a normal ionized calcium level allowing clotting to occur. In a typical serum collection, patient peripheral blood is collected without anticoagulant and allowed to clot. The tube is spun to allow the serum to be collected and transferred to another tube. Samples that are hemolyzed or do not clot are unacceptable. Serum can be stored at - 70 o C indefinitely. Prior to the advent of single antigen identification beads for solid phase assays, it was often challenging to determine specificities in patients with multiple antibodies. Polyspecific antiserum can be absorbed with lymphocytes to determine if there is a single antibody or multiple antibodies. For example, a serum that reacts with A1 and B8 can be absorbed with lymphocytes expressing A1 but not B8. The absorbed serum will be retested against B8 to see if there is still reactivity or if the reactivity is only directed against A1. Another application of lymphocyte absorption is to use the patient s own cells to remove autoantibody. Absorption can be performed at cold or warm temperatures depending on the type of antibody. Autoantibodies are typically cold reacting (4 o C) and B cell antibodies are typically warm reacting (22-37 o C). Patient serum may contain autoantibodies and/or IgM antibodies that may interfere the binding of anti-hla IgG antibodies to the antigen. In complement dependent assays (CDC), IgM antibodies can activate complement; potentially leading to false positive reactivity. DTT (dithiothreitol, also 3
known as Cleland s Reagent) treatment and heat inactivation are commonly used to remove IgM antibodies from serum. DTT inactivates IgM molecules by cleaving the disulphide bonds in the pentamer structure. Heat inactivation works in the same manner, but is less specific than DTT. An additional contaminating substance in patient serum may be introduced by the immunosuppressive regimen the patient is receiving. Many patients receive therapeutic antilymphocyte antibodies such as Thymoglobulin (purified, pasteurized, gamma immune globulin, obtained by immunization of rabbits with human thymocytes) or OKT3 (a murine IgG monoclonal antibody directed against CD3) as part of their post transplant therapy. If these therapeutic antilymphocyte antibodies are present in the patient serum, they can cause false positive reactivity in T cell cytotoxic assays. Therefore, such interfering substances must be removed from the serum prior to testing, allowing any clinically significant patient antibodies to be detectable. This removal is accomplished by absorption using a magnetic beads coated with anti-rabbit or anti-mouse immunoglobulin for Thymoblobulin or OKT3, respectively. Methodologies and protocols are provided for the following types of serum preparations: 1. Recalcificiation of plasma 2. Adsorption with lymphocytes 3. Inactivation of IgM antibodies a. DTT treatment b. Heat Inactivation 4. Depletion of therapeutic anti-lymphocyte antibodies from serum These instructions are intended to be a guideline. All reagents and procedures must be validated prior to clinical use. When using commercial kits, refer to the manufacturer s instructions regarding serum preparation. Any deviation from the manufacturer s instructions must be validated by the laboratory. 4
Methodologies 1. Recalcification of Plasma Reagent Purpose 2 M Calcium Chloride solution Restore normal calcium level and allow clotting to occur Topical thrombin Used if Calcium Chloride solution does not work Equipment: Centrifuge Refrigerator QA/QC Information: N/A Vendor Products Available: Sigma Parke Davis (Thrombin) Procedure: Recalcification of Plasma Purpose: To convert plasma to serum for use in clinical testing. Materials: Citrated plasma from whole blood or plasmapheresis product 2M Calcium Chloride solution Topical thrombin (1000 units/ml) Procedure: 1. Determine plasma volume. For large volumes, use the weight and assume that 1 gram 1 ml. 2. Add 1 ml of 2M Calcium Chloride for each 100 ml of plasma. Invert several times to mix. 3. Incubate at room temperature overnight. 4. Remove the clot from the wall of the container. Incubate overnight at 1-6 o C. 5. Centrifuge for 10 minutes at 4000g at 1-6 o C. If there is still a precipitate, recentrifuge at 10000g or higher at 1-6 o C. 5
6. Extract the serum and store in new container. 7. If no clot has formed, add 0.1 ml topical thrombin and mix well. Incubate overnight. Repeat steps 5-6. Limitations: N/A Precautions: Any human blood specimen should be considered as potentially infectious and handled with universal precautions. Quality Control/Assurance Procedure: Quality Control for Recalcification of Plasma Purpose: To describe the appropriate quality control measures that must be in place to perform recalcification of plasma. Materials: Centrifuge, refrigerator Standards: 1. Verify that all micropipettes used have been calibrated. Calibration is preformed twice a year. 2. All reagents should be used prior to the manufacturer s expiration dates. Reagents should also be visually inspected for contamination and discarded if any contamination is detected. 3. Verify that all equipment used, including centrifuges and refrigerators are calibrated and maintained according to the manufacturer s instructions. Corrective Action: Any equipment or reagent that falls outside of normal testing parameters must be resolved immediately and before clinical results can be reported. 6
2. Absorption with Lymphocytes No reagents required Equipment: Centrifuge Refrigerator and/or 37 o C water bath QA/QC Information: Negative control, Positive control, B cell control, Monocyte control Vendor Products Available: N/A Procedure: Absorption with Lymphocytes Purpose: To determine the specificity of antibody in patient serum that is highly reactive with multiple antibody specificities present. Materials: Patient serum Autologous lymphocytes or reference lymphocytes Procedure: 1. Isolate desired lymphocyte preparation T or B lymphocytes. 2. Label 4 tubes as #1, #2, #3 and #4. To tubes 1-3, add 5 x 10 6 lymphocytes. To tube 4, add 0.5 x 10 6 lymphocytes. Refrigerate tubes 2-4. 3. Centrifuge tube 1 to pellet lymphocytes. Remove supernatant. 4. Resuspend lymphocyte pellet in 200 ml of patient serum. 5. For cold antibody removal, incubate for 1 hour a 4 o C. For warm antibody removal, incubate for 1 hour at 37 o C. 6. Centrifuge tubes 1 and 2. Discard supernatant in tube 2 only. 7. Transfer absorbed serum in tube 1 to tube 2. Mix thoroughly to resuspend lymphocytes. 8. Repeat incubation step 5. 9. Centrifuge tube 2 to pellet lymphocytes. Transfer 2X absorbed supernatant to a clean tube. 10. Prepare serial dilutions of the 2X absorbed serum (typically neat, 1:2 and 1:4 dilutions are used). 11. Add 1 ul of the following controls and sera for testing to the microtiter tray: a. Negative control 7
b. Positive control c. Untreated serum (1:1, 1:2 and 1:4) d. 2X absorbed serum (1:1, 1:2 and 1:4) 12. Perform testing with lymphocytes saved in tube 4 using original test method. 13. If necessary, another absorption step can be performed as follows: a. Centrifuge tube 3 to pellet lymphocytes. Remove supernatant. b. Collect the 2X absorbed serum and add it to the lymphocyte pellet in tube 3. c. Repeat the incubation step 5. d. Centrifuge tube 3 to pellet lymphocytes. Transfer 3X absorbed supernatant to a clean tube. Limitations: 1. Several controls are necessary when testing absorbed serum. a. Negative control used to show that the extent of non-specific cell death b. Positive control used to show that a positive reaction will occur with lymphocytotoxic antibodies. c. Cell purity positive control used as a positive control to demonstrate the percent of a particular cell type in the cell population i. B cell should be positive with B cells, negative with T cells ii. Monocyte should be positive with Monocytes, negative with T and B cells 2. Lymphocyte absorptions performed to remove antibody, there should be a positive reaction with untreated serum and a negative reaction with treated serum. If this is not the case, another absorption step may need to be performed. If reactivity persists, the clinician should be notified as there are high tittered autoantibodies present in the patient serum. Precautions: Any human blood specimen should be considered as potentially infectious and handled with universal precautions. Quality Control/Assurance Procedure: Quality Control for Absorption with Lymphocytes Purpose: To describe the appropriate quality control measures that must be in place to perform lymphocyte absorption of plasma. 8
Materials: Centrifuge Refrigerator and/or 37 o C water bath Standards: 1. Verify that all micropipettes used have been calibrated. Calibration is preformed twice a year. 2. All reagents should be used prior to the manufacturer s expiration dates. Reagents should also be visually inspected for contamination and discarded if any contamination is detected. 3. Verify that all equipment used, including centrifuges, refrigerators and water baths are calibrated and maintained according to the manufacturer s instructions. Corrective Action: Any equipment or reagent that falls outside of normal testing parameters must be resolved immediately and before clinical results can be reported. 9
3. Inactivation of IgM Antibodies a. DTT Treatment Reagent DTT Purpose Inactivate IgM antibodies in patient serum Equipment: 37 o C heat block, incubator or water bath QA/QC Information: Dilution control, IgG Positive Control, IgM Control, Negative Control Vendor Products Available: Sigma - DL-Dithiothreitol ThermoScientific - DTT (Dithiothreitol) J.T.Baker - DITHIOTHREITOL Procedure: DTT treatment of Serum Purpose: To inactivate autoantibodies in patient serum by disrupting the disulfide bond present in IgM class antibodies. Materials: 0.05M working solution of DTT (stock solution diluted 1/20 in PBS) Patient Serum PBS Procedure: 1. Mix 10 ul 0.05M DTT working solution with 90 ul patient serum. 2. Mix well and incubate at 37 o C for 30-45 minutes. Limitations: 3. Several controls are necessary when using a DTT treated serum. a. Dilution Control used to show that the reactivity was not changed by dilution i. 90 ul patient serum + 10 ul PBS ii. Should be positive if untreated serum is positive b. IgG Positive Control to show that DTT has only a minimal effect on IgG class antibodies and does not inhibit complement 10
i. High PRA human serum that is known to be IgG, tested with and without DTT treatment ii. Should be positive with and without DTT treatment c. IgM Control to show that DTT inactivates IgM i. High PRA human serum that is known to be IgM, tested with and without DTT ii. Should be negative with DTT treatment, positive without DTT treatment d. Negative Control to show that DTT is not cytotoxic i. Normal human serum (may be pooled) known to be 0% PRA, tested with and without DTT treatment ii. Should be negative with and without DTT treatment Precautions: Any human blood specimen should be considered as potentially infectious and handled with universal precautions. Avoid skin contact or inhalation of DTT. In the case of skin contact, wash with soap and copious amounts of water. If inhaled, remove to fresh air and contact a physician if breathing becomes difficult. Quality Control/Assurance Procedure: Quality Control for DTT Treatment of Serum Purpose: To describe the appropriate quality control measures that must be in place to DTT treatment of serum. Materials: 37 o C heat block, incubator or water bath Standards: 1. Verify that all micropipettes used have been calibrated. Calibration is preformed twice a year. 2. All reagents should be used prior to the manufacturer s expiration dates. Reagents should also be visually inspected for contamination and discarded if any contamination is detected. 3. Verify that the 37 o C incubator used has been calibrated. 4. Each lot/batch of DTT must meet quality control specifications before being used for clinical testing. A known serum sample with established reactivity must be tested along with the 4 DTT controls described in the procedure. 11
Corrective Action: Any equipment or reagent that falls outside of normal testing parameters must be resolved immediately and before clinical results can be reported. b. Heat Inactivation No reagents required Equipment: 63 o C heat block, incubator or water bath Centrifuge QA/QC Information: IgG Positive Control, IgM Control, Negative Control Vendor Products Available: N/A Procedure: Heat Inactivate of Serum Purpose: To inactivate autoantibodies in patient serum by disrupting the disulfide bond present in IgM class antibodies. Materials: Patient Serum PBS Procedure: 1. Aliquot patient serum into microcentrifuge tube 2. Incubate serum in 63 o C heat block for 13 minutes. 3. Remove from heat and centrifuge for 1 minute. 4. Transfer supernatant to clean tube without disrupting the pellet or gel in the spun tube. Limitations: 1. Several controls are used when using a heat treated serum a. IgG Positive Control to show that heat inactivation has only a minimal effect on IgG class antibodies and does not inhibit complement 12
i. High PRA human serum that is known to be IgG, tested with and without DTT treatment ii. Should be positive with and without heat inactivation b. IgM Control to show that heat inactivation inactivates IgM i. High PRA human serum that is known to be IgM, tested with and without heat inactivation ii. Should be negative with heat inactivation, positive without heat inactivation c. Negative Control to show that heat inactivation is not cytotoxic i. Normal human serum (may be pooled) known to be 0% PRA, tested with and without heat inactivation ii. Should be negative with and without heat inactivation Precautions: Any human blood specimen should be considered as potentially infectious and handled with universal precautions. Quality Control/Assurance Procedure: Quality Control for Heat Treatment of Serum Purpose: To describe the appropriate quality control measures that must be in place to heat treatment of serum. Materials: 63 o C heat block, incubator or water bath Standards: 1. Verify that all micropipettes used have been calibrated. Calibration is preformed twice a year. 2. All reagents should be used prior to the manufacturer s expiration dates. Reagents should also be visually inspected for contamination and discarded if any contamination is detected. 3. Verify that the 63 o C incubator used has been calibrated. Corrective Action: Any equipment or reagent that falls outside of normal testing parameters must be resolved immediately and before clinical results can be reported. 13
4. Depletion of Therapeutic Anti-Lymphocyte Antibodies from Serum Reagent* PBS Dynabead M-280 Sheep anti-rabbit IgG Dynabead M-280 Sheep anti-mouse IgG Purpose To absorb Thymoglobulin present in patient serum To absorb OKT3 present in patient serum *If other therapeutic anti-lymphocyte antibodies are used, the same basic procedure may be used by substituting an appropriate antibody conjugated to magnetic beads. Refer to the manufacturer s product list. Equipment: Dynal Magnet 37 o C incubator QA/QC Information: Thymoglobulin or OKT3 Standard, Positive control, Polyvalent antibody control, Negative control Vendor Products Available: Dynabeads - Invitrogen Procedure: Depletion of therapeutic anti-lymphocyte antibodies from serum Purpose: To remove therapeutic anti-lymphocyte antibodies from serum in order to eliminate false positive reactions in histocompatibility assays... Materials: Dynabeads M-280 Sheep anti-mouse IgG or Dynabeads M-280 Sheep anti-rabbit IgGt Serum PBS Procedure: 1. Make 4-200ul aliquots of Sheep anti-mouse IgG or Sheep anti-rabbit IgG beads in a 1.5ml plastic tube. Add approximately 1ml PBS to each. 2. Place tubes on magnet for 1 min (or centrifuge if using a non-magnetic substrate) 3. Remove and discard supernatant. 4. Repeat wash steps to ensure that beads are adequately washed. 5. Add 0.2ml of the following to the 4 aliquots. 14
a. tube 1 Serum (unknown) b. tube 2 Negative control c. tube 3 Known serum with multi-specific antibodies d. tube 4 Thymoblobulin/OKT3 standard, approximately 1000 ng/ml (positive control) 6. Incubate all tubes at 37oC for 45 minutes, mixing occasionally. 7. Place tubes on a magnet for 1 minute. 8. Collect supernatant and transfer to new, labeled tubes. 9. Continue with crossmatch testing or antibody testing with collected serum. 10. Control reactivity should be as follows: a. tube 1 serum (unknown) reactive in untreated serum to less reactive or non-reactive dependent on the specificity of HLA antibodies present in the serum. b. tube 2 Negative control should be non-reactive untreated and treated. c. tube 3 Known serum with multi-specific antibody should have the same reactivity treated and untreated. d. tube 4 Positive control should be highly reactive untreated and non-reactive when treated. Limitations: If the serum is still reactive following treatment, the serum may contain high levels of OKT3 or Thymoglobulin. Repeat the procedure to ensure complete removal. If the serum is still reactive after a second treatment and correlates with HLA specificity identified, it is highly likely this reactivity is due to HLA antibodies and should be considered a true positive reaction. Precautions: Any human blood specimen should be considered as potentially infectious and handled with universal precautions. Quality Control/Assurance Procedure: Quality Control for Depletion of therapeutic anti-lymphocyte antibodies from Serum Purpose: To describe the appropriate quality control measures that must be in place to perform the depletion of therapeutic anti-lymphocyte antibodies from serum. Materials: 37 o C incubator 15
Standards: 1. Verify that all micropipettes used have been calibrated. Calibration is preformed twice a year. 2. All reagents should be used prior to the manufacturer s expiration dates. Reagents should also be visually inspected for contamination and discarded if any contamination is detected. 3. Verify that the 37 o C incubator used has been calibrated. Corrective Action: Any equipment or reagent that falls outside of normal testing parameters must be resolved immediately and before clinical results can be reported. 16
Frequently Asked Questions Question: Why is calcium added to plasma to convert it to serum? Answer: Calcium is added to the plasma to combine with the citrate present in the anticoagulant. This restores a normal ionized calcium level and allows clotting to occur. Once a clot has formed, the serum can be collected and used for testing. Question: At what temperature should serum absorption with lymphocytes be performed? Answer: Typically, auto-antibodies are cold reacting and should be absorbed at 4 o C while alloantibodies are warm reacting and should be absorbed at 37 o C. Question: Should we use DTT treatment or heat inactivation to remove IgM antibodies? Answer: The specific type of serum treatment used depends on the agreement between your program s clinicians and your laboratory. DTT treatment is more specific than heat treatment in removing IgM antibodies. However, DTT can also affect the disulfide bonds in IgG antibodies. It is important to run all of the controls described to ensure that either treatment has only a minimal effect on IgG and does not inhibit complement. Question: Why do we need to use so many controls when treating serum with DTT? Answer: The controls are necessary when using treated serum to ensure that treatment is denaturing the IgM antibody and not affecting the IgG antibody. Additionally, the dilution control used with DTT treatment ensures that any diminished reactivity is not due to the dilution of serum with DTT. Question: What happens if we don t remove OKT3 prior to cytotoxicity assays? Answer: OKT3 is a monoclonal antibody directed against the CD3 molecule. Therefore, the binding of OKT3 to any CD3+ cell will activate complement and lyse the target cell. This results in falsely positive reactivity. 17
Standards D.5.2.7 Laboratories performing Antibody Analysis and/or Crossmatch testing must: D.5.2.7.1 Test each patient serum undiluted or at a dilution that has been established to be optimal for the method used, and document the dilution(s) in the test records. D.5.2.7.2 Have written criteria or protocols for: D.5.2.7.2.1 Selecting appropriate patient serum samples. D.5.2.7.2.2 Preparation of donor cells or cellular component isolations (for example, solubilized antigens) as applicable to the technique(s) performed. D.5.2.7.3 Use a negative control of human serum documented to be non-reactive against the antigenic target. D.5.2.7.4 Use a positive control of an appropriate isotype and specificity, known to react with the specific cell types or antigens being tested, as applicable. D.5.2.7.5 Use the positive control at a dilution appropriate for the assay (i.e., a titer at which moderate changes in assay sensitivity are likely to be detected). D.5.2.7.12 Have a process for distinguishing HLA class I and class II antibodies from non-hla antibodies as appropriate for clinical applications. D.5.2.7.13 Use appropriate methods and/or controls to assess the impact of xenogeneic, chimeric, monoclonal or other therapeutic antibodies in the assay. D.5.2.7.14 Ensure that there is a procedure to monitor and adjust for non-specific binding of antibody. 18
Literature Cited and Recommended Reading Teresi, Gary A and Fuller, Anne: Absorption with Lymphocytes. ASHI Laboratory Manual 4 th Edition. 2000. Arnold ML, Zacher T, Dechant M, Kalden JR, Doxiadis II and Spriewald BM: Detection and specification of noncomplement binding anti-hla alloantibodies. Human Immunology 65(11):1288-96, 2004. Barocci S, Valente U, Gusmano R, Perfumo F, Cantarella S, Leprini A, Icardi A and Nocera A: Autoreactive lymphocytotoxic IgM antibodies in highly sensitized dialysis patients waiting for a kidney transplant: identification and clinical relevance. Clinical Nephrology 36(1): 12-20, 1991. Hahn, Amy B: Inactivation of IgM Antibodies: A. DTT Treatment and B. Heat Inactivation. ASHI Laboratory Manual 4 th Edition. 2000. Perkins, Herbert A, Sakahara, Nancy and Ganton, Zenaida P: Recalcification of Plasma. ASHI Laboratory Manual 4 th Edition. 2000. Zachary AA, and Hart JM: Relevance of antibody screening and crossmatching in solid organ transplantation. In: Handbook of Human Immunology, MS Leffell, NR Rose and AD Donnenberg, eds., CRC Press, Boca Raton; pp. 477-519, 1997. 19