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Albumin-indirect antiglobulin test

J.R. Hamilton

Albumin was the first widely used additive solution for Reagents/Supplies hemagglutination tests. Its major effect is to decrease the repulsive forces that keep red cells (RBCs) apart. This Reagents Supplies effect may enable some RBC , particularly those in the • Reagent RBCs for detection • Test tubes (10 × 75 or Rh blood group system, to directly agglutinate antigen-positive and/or antibody identification 12 × 75 mm) RBCs after 37°C incubation. The impact of albumin on antibody • 22% bovine albumin binding before detection by an indirect antiglobulin test (IAT) is • Pipettes minimal. Use of albumin in antibody identification may help with • 0.9% saline or PBS pH 6.5–7.5 • Calibrated serofuge separation of RBC antibody specificities in a mixture when one • AHG (polyspecific or anti-IgG) and/or cell washer or more antibodies demonstrate reactivity after 37°C incubation. • IgG-coated reagent RBCs • Calibrated timer Warm autoantibodies can show decreased reactivity in albumin RBCs = red blood cells; PBS = phosphate-buffered saline; IATs, allowing recognition of underlying alloantibodies. AHG = antihuman . REVIEW METHOD SEROLOGIC Immunohematology 2019;35:63–64. Procedural Steps Key Words: albumin, IAT, antibody identification • Add two drops plasma or to a labeled tube. Principle • Add one drop reagent RBC suspension. • Centrifuge, and perform immediate spin reading (if desired). • Add two drops albumin (per MI). Albumin is added to serologic tests to overcome forces • Incubate 30–60 minutes at 37°C (per MI). keeping red blood cells (RBCs) apart and, in doing so, makes • Centrifuge, and perform 37°C reading. hemagglutination reactions more likely to occur. Use of • Wash three to four times in isotonic saline. albumin in antibody detection tests began as early as the 1940s • Add one to two drops AHG reagent (per MI). • Centrifuge, and perform AHG reading. before the advent of the antihuman globulin (AHG) phase of Compiled from Fung et al.6 testing, when direct agglutination tests were the only available RBC = ; MI = manufacturer’s instructions; method for visualizing the antigen-antibody reaction. Initially, AHG = antihuman globulin. the reagent was used in albumin layering1,2 or albumin replacement methods.3 These methods were very sensitive when supporting hemagglutination but were not adopted particular, can show increased reactivity after 37°C incubation widely in the United States. The albumin method used for in albumin tests. It has also been postulated, however, that routine tube testing in the 1970s and still in use today involves the final concentration of albumin in the test environment the simple addition of albumin to a RBC suspension/plasma (considering dilution by plasma and RBC diluent) is too low mixture. Two theories as to the mechanism of albumin’s effect to significantly affect direct hemagglutination.4 Historically, exist; one being that the albumin raises the dielectric constant some albumin preparations were formulated in a low-ionic of the test environment. The RBCs’ negative charge (zeta environment, leading to a belief that albumin increased the potential) is dispersed, causing the RBCs to come into close speed of antibody binding.5 However, albumin alone does enough contact that an antibody molecule can bridge between not increase the speed or amount of antibody that is bound two RBCs. Alternatively, the albumin molecule disrupts the to RBCs. As stated earlier, it only allows for closer proximity water molecules associated with the RBC membrane, again of the RBCs to occur, resulting in observable agglutination. allowing RBCs to be in closer proximity to allow for antibody This finding makes it necessary to incubate a test long enough crosslinking. In reality, each mechanism likely plays a role. to allow maximum antibody binding equilibrium. A 30- to The practical effect of albumin’s impact on RBC 60-minute incubation in a normal ionic strength environment crosslinking is that certain antibodies may show direct is required for adequate antibody binding. The reading after agglutination after a 37°C incubation using albumin that they the 37°C incubation is followed by the AHG test. would not show without its use. Rh system antibodies, in

IMMUNOHEMATOLOGY, Volume 35, Number 2, 2019 63 J.R. Hamilton

Indications Quality Control

The albumin-indirect antiglobulin test (IAT) is a method The albumin reagent should be tested for reactivity on used in antibody identification studies. The increased tendency each day of use, utilizing an antibody of known specificity and for 37°C agglutination when using albumin may allow antigen-positive and -negative RBCs. The tests should give recognition of antibody specificity before the AHG phase. This expected results at the AHG phase. If the antibody is known step is especially helpful when multiple alloantibodies are to show reactivity after 37°C incubation, this reactivity should present; Rh system antibodies are most likely to react at this be observed with the antigen-positive RBCs. Quality control test phase. Newly developing antibodies may show increased of other components of the test system should be performed reactivity in 37°C tests when compared with AHG tests using according to laboratory protocols. anti-IgG. Conversely, warm autoantibodies generally have decreased reactivity in albumin-IAT tests. This method may Limitations be used to avoid their reactivity while retaining the ability to detect clinically significant alloantibodies when proper No single test method, including those using incubation time is used. albumin enhancement, will detect all clinically significant alloantibodies. Insufficient incubation time is expected to Procedure decrease the sensitivity of the albumin-IAT. The centrifuge used for the direct agglutination Place two drops of patient or donor plasma or serum reading after 37°C incubation must have been calibrated into a properly labeled tube, followed by one drop of the 2–5 for centrifugation of a high- test. The centrifugation percent RBC suspension to be tested. If an immediate spin time for this test is generally longer than the spin time for a reading is desired, centrifuge at the calibrated time for a low- low-protein test. Improper centrifugation may result in false protein test, and gently resuspend while examining the tube results. for RBC agglutination. Add two drops of 22 percent bovine albumin to the tube. Incubate the tube at 37°C for 30–60 References minutes or the incubation time specified in the manufacturer’s 1. Diamond LK, Denton RL. Rh agglutination in various media instructions. Centrifuge at the calibrated time for a high- with particular reference to the value of albumin. J Lab Clin protein test. If serum is being tested, the tube can be examined Invest 1945;30:821–30. for before beginning resuspension of the RBCs. 2. Dunsford I, Bowley CC. Techniques in blood grouping. Springfield, IL: Charles C. Thomas, 1955. Gently resuspend the RBC button while examining the tube 3. Case J. The albumin layering method for D typing. Vox Sang for RBC agglutination. This step is the albumin 37°C reading. 1959;4:403–5. Wash the RBCs three to four times with large quantities of 4. Case J. Potentiators of agglutination. In: Bell CA, Ed. A isotonic saline. Add one to two drops of antiglobulin reagent seminar on antigen-antibody reactions revisited. Arlington, VA: American Association of Blood Banks, 1982. as specified by the manufacturer’s insert. Two drops of 5. Rohil SD. Enhancement techniques for antigen-antibody antiglobulin reagent generally produces a more sensitive test. interactions. Lab Med 1986;17:203–6. Centrifuge at the calibrated time for an AHG reading. Gently 6. Fung MK, Eder AF, Spitalnik SL, Westhoff CM, Eds. Method resuspend the RBC button while examining the tube for RBC 3-3: Albumin or LISS additive indirect antiglobulin test. In: agglutination. Add IgG-coated reagent RBCs to all negative Technical manual. 19th ed. Bethesda, MD: AABB, 2017. tests. Centrifuge, and read for agglutination. Tests that do not show agglutination or show agglutination that is weaker than Janis R. Hamilton, MS, MT(ASCP)SBB, Manager, Immunohema- tology Reference Laboratory, American Red Cross, Southeastern expected per the manufacturer’s instructions indicate that Michigan Region, 1415 Trumbull Avenue, Detroit, MI 48216, jan. inadequate washing has occurred and the test is invalid and [email protected]. must be repeated.

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