EDUCATIONAL COMMENTARY ANTIBODY TITRATIONS

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1 EDUCATIONAL COMMENTARY ANTIBODY TITRATIONS Educational commentary is provided through our affiliation with the American Society for Clinical Pathology (ASCP). To obtain FREE CME/CMLE credits click on Earn CE Credits under Continuing Education on the left side of the screen. LEARNING OUTCOMES On completion of this exercise, the participant will be able to explain clinical reasons to perform antibody titration. explain the testing phases for antibody titration. discuss causes for discrepant antibody titration results. describe ways to standardize titration studies. Educational Commentary Blood banks routinely detect and identify antibodies; in some clinical situations, however, it is important to semiquantitate the concentration of an antibody. Antibodies are titrated by making serial, two-fold dilutions of the patient s plasma and grading the strength of reactivity with selected red blood cells (RBCs) that possess the corresponding antigen. The results of a titer are reported as the reciprocal of the highest dilution of plasma demonstrating macroscopic agglutination. In blood banking, standardization of both qualitative and quantitative testing has ensured that laboratories report similar results for many tests. Antibody titrations, however, are among the few tests in blood banking that have not been standardized. Indications for Antibody Titration Antibody titration is clinically indicated in prenatal studies, separating multiple antibodies, antibody identification, and performing ABO isohemagglutinin titers on apheresis donor platelet units or organs that are to be transplanted across ABO boundaries. When a clinically significant antibody capable of causing hemolytic disease of the newborn or fetus is detected in a pregnant woman, it is important to determine the antibody titer, particularly if the father has the corresponding RBC antigen. Throughout pregnancy, the mother s antibody titer is repeated to determine if there is a rise in titer or concentration of the antibody. A rise in antibody titer by two dilutions is considered clinically significant and suggests the fetal RBCs possess the antigen to the corresponding antibody. Once the titer reaches a critical threshold and the fetus is at least eighteen weeks gestational age, additional studies, such as ultrasound, Doppler sonography, amniocentesis, or cordiocentesis, will be performed. These procedures monitor the level of anemia in the fetus and indicate the necessity of intrauterine transfusion or other interventions.

2 Antibody titration can also be used to identify antibodies. Some antibodies react weakly in the antihuman globulin (AHG) phase, but when titers are performed, these antibodies have remarkably high titers. Known as high-titer, low-avidity (HTLA) antibodies, they are directed against high frequency RBC antigen systems that include Chido, Rogers, York, Knops, McCoy, John Milton Hagen, and others. These antibodies are not clinically significant for transfusion, but they mask underlying alloantibodies capable of causing hemolytic transfusion reactions. Since these antibodies are directed against high-frequency antigens, all donor RBC products will be crossmatch incompatible; physicians should administer transfusions conservatively to such patients. In some cases, multiple antibodies can be detected and identified by performing antibody titers. The specimen can be diluted before testing with reagent RBCs, thereby removing one antibody and allowing identification of the second, underlying antibody with the higher titer. Some institutions perform titers of anti-a and anti-b antibodies, also known as isohemagglutinin titers, on group O apheresis donor platelets before dispensing to non group O patients. Apheresis platelets from group O donors contain large plasma volumes and may contain unusually high titers of anti-a or anti-b antibodies. There are multiple reports of patients who have suffered from passively mediated hemolytic transfusion reactions due to minor ABO incompatibilities. Medical centers have implemented policies to minimize this risk for hemolytic transfusion reactions in patients who receive ABO-incompatible platelet transfusions. Some have focused on performing isohemagglutinin titers on all apheresis group O platelets. The challenges in this practice include a lack of standardization for performing isohemagglutinin titers; an agreed-upon critical titer to restrict these products for transfusion to group O patients; and variable reports that document a wide range of titers implicated in causing hemolytic transfusion reactions. To avoid this risk completely, some institutions will not transfuse group O apheresis platelets to frequently transfused, non group O patients. Recently, immunosuppression medication combined with apheresis has allowed for successful ABO-incompatible renal and liver transplant. Performing ABO titers is critical for determining both the effectiveness of pretreatment regimens and when titers are low enough to permit transplant, but there is no uniform method for determining a safe anti-a or anti-b titer. In some cases, patients may be undertreated or overtreated for their ABO-incompatible transplant, owing to the inconsistency in performing and monitoring ABO titers during the conditioning regimen. Even after transplant has taken place, ABO titers must be monitored and compared with previous levels to determine if antibody-mediated rejection is a threat to the graft. Antibody Titration Methodology When performing an antibody titration for the first time for a patient, some laboratories will require that two technologists perform the titration. It is also a common practice to freeze an aliquot of the sample so the titration can be repeated alongside a subsequent sample. The conventional tube titration is performed by labeling tubes according to the specimen dilution. The first tube will contain a volume of undiluted sample and the second and subsequent tubes will contain an equal volume of saline. An equal volume of sample is added to the second tube, which is mixed, and one volume of the mixture is transferred to the next tube. The same process is

3 continued for all dilutions with a clean pipette tip to mix and transfer each dilution. After the dilutions, two drops of the RBC suspension possessing the antigen are added to the tubes. Some laboratories add an enhancement medium, such as low ionic saline solution, and some do not. The tubes are incubated for times and temperatures appropriate for the antibody. The tubes may simply be centrifuged and read for reactivity or washed and AHG or anti-immunoglobulin G (IgG) added before centrifugation and interpretation of results (Table I). Gel cards may also be used for antibody titration. The dilutions are performed in a test tube and then the sample is transferred to the gel cards. The type of gel card used, temperature, and time of incubation are dependent on the type of antibody titrated (Table II). Table I. Conventional Tube Methods for Antibody Titers Clinically Significant Antibodies,such as: Rh, Kell, Kidd, Duffy Enhancement None or LISS Reagent RBC concentration 2%-5% Incubation 37 C For m 4 Times with saline Anti-A/ Anti-B Titers Enhancement None or LISS Reagent RBC concentration 2%-5% First incubation 30 m at room temperature, centrifuge and read Second incubation 37 C for 30 m 4 Times with saline AHG indicates antihuman globulin; IgG, immunoglobulin G; LISS, low ionic saline solution; RBC, red blood cell; and m, minute(s). Table II. Gel Card Antibody Titration Clinically Significant Antibodies, such as: Rh, Kell, Kidd, and Duffy Gel card type Anti IgG Reagent RBC concentration 0.8% Incubation 37 C for 15 m Anti-A/Anti-B Titers, 2 Separate Cards Gel only card Room temperature incubation, 15 m, centrifuge and read Anti IgG card 37 C for 15 m Reagent RBC concentration 0.8% 4 times with saline AHG indicates antihuman globulin; IgG, immunoglobulin G; LISS, low ionic saline solution; RBC, red blood cell; m, minute(s).

4 Testing Inconsistencies Antibody titrations have been difficult to standardize in the laboratory. It has been a longstanding practice to repeat an antibody titration on a subsequent sample alongside the previously tested sample. This practice was established to reduce inconsistencies of technique and interpretation among technologists performing titrations in the same laboratory. Because technical variables can significantly affect results, uniform practices within the same laboratory should be emphasized. Some variables include pipetting technique and changing tips between dilutions to prevent carryover from one dilution to the next. The zygosity, phenotype, age, and concentration of the reagent RBCs can also influence results; therefore, the same reagent RBCs, or RBCs of the same phenotype, should be used for subsequent samples. The optimal incubation time, temperature, and centrifugation speed and time should be consistent when performing titrations. In addition, interlaboratory titration methods vary significantly in methodology, incubation time, temperature, testing phase, and endpoint to interpret the results. The College of American Pathologists (CAP) has demonstrated significant disparity in titration results in proficiency testing. Even when results are grouped by methodology, the results can vary between laboratories by five or more dilutions. In an effort/attempt to combat this variation, the Transfusion Medicine Resource Committee for CAP created a detailed and uniform procedure for performing antibody titrations and provided proficiency samples to 36 participating laboratories. This investigation found that, even when providing a detailed procedure, there was wide variation in the results from each laboratory. The detailed procedure instructed laboratories to use a grade of 1+ as the uniform endpoint; however titration results still varied significantly. The committee then asked for laboratories to review their results and provide titers on samples using a weak positive endpoint. Applying this weak positive endpoint reduced the variance of results. The investigation also found that the gel-card technique generated less variance between laboratories than tube testing, although fewer laboratories used the gel technique than tube method. (1,2) Anti-A and anti-b titers can be performed in conventional test tubes both at room temperature and at the AHG phase, and both of these titers may be reported to the clinical team. A recent study at Johns Hopkins Hospital reported that room-temperature ABO titers performed in tube are not necessary and do not offer any more data pertinent to clinical decision making than AHG titers. Researchers compared AHG tube titers to IgG gel-card titers and found 86% of the titer results were identical. They concluded that the AHG tube method and IgG gelcard ABO titers were comparable, but the gel card method reduced turnaround times in half by decreased incubations and by performing titrations in batches. (3) Summary Antibody titrations are important in antenatal evaluations, antibody identifications, transfusing ABO-incompatible plasma products, and performing and monitoring ABO-incompatible organ transplants. Unfortunately, standardization of antibody titers has continued to elude laboratorians, and proficiency testing performed by CAP has demonstrated up to a 5-fold disparity in titration results. By using a detailed procedure and a weak positive

5 endpoint, the CAP Transfusion Medicine Resource Committee has been able to reduce the interlaboratory variation. The committee also concluded that using detailed and consistent laboratory methods for antibody titers may render the current practice of repeating titers from a previous sample with the current sample unnecessary, because the variation of titers reduces over time in each laboratory. SUGGESTED READING 1. AuBuchon JP, de Wildt-Eggen J, Dumont LJ. Reducing the variation in performance of antibody titrations. Vox Sang. 2008;95: AuBuchon JP, de Wildt-Eggen J, Dumont LJ. Reducing the variation in performance of antibody titrations. Arch Pathol Lab Med. 2008;132: Shirey RS, Cai W, Montgomery RA, et al. Streamlining ABO antibody titrations for monitoring ABOincompatible kidney transplants. Transfusion. 2010;50: Walker PS. Identification of antibodies to red cell antigens. In: Roback J, Combs MR, Grossman BJ, et al, eds. Technical Manual. 17th ed. Bethesda, MD: American Association of Blood Banks; Judd WJ, Johnson ST, Storry JR, eds. Judd s Methods in Immunohematology. 3rd ed. Bethesda, MD: American Association of Blood Banks; Cooling L, Downs T. Immunohematology. In: McPherson RA, Pincus MR, eds. Henry s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia, PA: Elsevier Saunders; ASCP 2012