Blood Groups and Serologic Testing PDF

 Chapter 5 The Antiglobulin Test 105 Table 5–1 FDA Licensed Antihuman Globulin Reagents Reagent Definition Polyspecific 1. R...

 Chapter 5 The Antiglobulin Test 105 Table 5–1 FDA Licensed Antihuman Globulin Reagents Reagent Definition Polyspecific 1. Rabbit polyclonal Contains anti-lgG and anti-C3d (may contain other anticomplement and other anti-immunoglobulin antibodies) 2. Rabbit/murine monoclonal blend Contains a blend of rabbit polyclonal antihuman IgG and Anti-C3d is a murine monoclonal IgM antibody. Monospecific 1. Anti-IgG Contains anti-IgG with no anticomplement activity (Rabbit polyclonal) 2. Anti-IgG Murine monoclonal IgM antibody secreted by a hybridoma cell line (Gamma-clone AHG) 3. Anticomplement Anti-C3d The main component of this reagent is a murine monoclonal antibody to C3d. Anti-C3d will cause the aggluti- nation of red blood cells coated with human C3d and/or C3b complement components. Accessed From: https://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedProductsBLAs/BloodDonorScreening/BloodGroupingReagent/ default.htm polyspecific mixture may contain antibody activity to kappa human complement components injected into a rabbit result and lambda light chains common to all immunoglobulin in anticomplement. This type of response produces a poly- classes, thus reacting with IgA or IgM molecules.10 clonal antiglobulin serum. Polyclonal antibodies are a mixture of antibodies from different plasma cell clones. The Monospecific AHG resulting polyclonal antibodies recognize different antigenic determinants (epitopes), or the same portion of the antigen Monospecific AHG reagents contain only one antibody but with different affinities. Hybridoma technology can be specificity: either anti-IgG or antibody to specific components used to produce monoclonal antiglobulin serum. Mono- of complement, such as C3b or C3d (i.e., anticomplement). clonal antibodies are derived from one clone of plasma cells Licensed monospecific AHG reagents in common use are and recognize a single epitope. anti-IgG and anti-C3b,-C3d.10 Polyspecific AHG Anti-IgG Reagents labeled anti-IgG contain no anticomplement activ- Polyspecific AHG can be made using polyclonal or mono- ity. Anti-IgG reagents contain antibodies specific for the Fc clonal antibodies. The two types of antibody production fragment of the gamma heavy chain of the IgG molecule. If processes are very different from one another, yielding two not labeled “gamma heavy chain–specific,” anti-IgG may very different advantages and disadvantages in their usage. contain anti–light chain specificity and may therefore react with cells sensitized with IgM, IgA, and IgG.11 Polyclonal AHG Production Polyclonal AHG is usually prepared in rabbits. In contrast Anticomplement with the early production methods, in which a crude globu- Anticomplement reagents, such as anti-C3b,-C3d reagents, lin fraction of serum was used as the immunogen, modern are reactive against only the designated complement compo- production commences with the purification of the immuno- nents and contain no activity against human immunoglobu- gen from a large pool of normal sera. lins.11 Monospecific anticomplement reagents are often Conventional polyspecific antiglobulin reagents are pro- a blend of monoclonal anti-C3b and monoclonal anti-C3d duced by immunizing one colony of rabbits with human (see the following sections for descriptions of monoclonal immunoglobulin (IgG) antigen and another colony with and polyclonal antibody production). human C3 antigen. Because of the heterogeneity of IgG mol- ecules, using serum from many donors to prepare the pooled Preparation of AHG IgG antigen to immunize the rabbits and the pooling of anti- IgG from many immunized rabbits is essential in producing The classic method of AHG production involves injecting polyclonal reagents for routine use that are capable of de- human serum or purified globulin into laboratory animals tecting the many different IgG antibodies. This is an advan- such as rabbits. The human globulin behaves as foreign anti- tage of using anti-IgG of polyclonal origin for antiglobulin gen, the rabbit’s immune response is triggered, and an anti- serum.12 body to human globulin is produced. For example, human Both colonies of animals are hyperimmunized to produce IgG injected into a rabbit results in anti-IgG production; high-titer, high-avidity IgG antibodies. Blood specimens are 106 PART II Blood Groups and Serologic Testing drawn from the immunized animals, and if the antibody potency of anti-C3b and anti-C3d individually is one of the potency and specificity meet predetermined specifications, difficulties with polyclonal reagents that can be avoided with the animals are bled for a production batch of reagent. monoclonal products.12 Separate blends of the anti-IgG and anticomplement anti- bodies are made, and each pool is then absorbed with A1, B, Monoclonal AHG Production and O cells to remove heterospecific antibodies. The prepa- The monoclonal antibody technique devised by Kohler and ration of polyclonal AHG is diagrammed in Figure 5–1. The Milstein13 has been used to produce AHG and has proved par- total antibody content of each pool is determined, and the ticularly useful in producing high-titer antibodies with well- potency of the pools is analyzed to calculate the optimum defined specificities to IgG and to the fragments of C3.14–16 antibody dilution for use. Block titrations for anti-IgG pools Monoclonal antibody production begins with the immu- are performed by reacting dilutions of each antibody against nization of laboratory animals, usually mice, with purified cells sensitized with different amounts of IgG. This is a human globulin. After a suitable immune response, mouse critical step in the manufacturing process because excess spleen cells containing antibody-secreting lymphocytes antibody (especially anti-IgG) may lead to prozone, which are fused with myeloma cells. The resulting hybridomas leads to false-negative test results. (lymphocyte–myeloma hybrid cells) are screened for anti- Because it is not possible to coat cells with measured bodies with the required specificity and affinity. The antibody- amounts of components of complement, the potency of anti- secreting clones may then be propagated in tissue culture C3 pools is measured using at least two examples each of a or by inoculation into mice, in which case the antibody is C3b- and C3d-coated cell. Both anti-C3b and anti-C3d are collected as ascites (fluid accumulation in the abdomen of the present in the polyclonal anti-C3 pool. The level of anti-C3d mouse). Because the clonal line produces a single antibody, is critical in keeping false-positive tests to a minimum yet there is no need for absorption to remove heterospecific anti- also detecting clinically significant amounts of RBC-bound bodies. A hybridoma is a hybrid cell that is used as the basis C3d. Additionally, if the dilution of the anti-C3 pool is for the production of large amounts of antibodies for thera- determined on the basis of the amount of anti-C3d present, peutic, research, or diagnostic applications. the level of anti-C3b varies. The inability to determine the All antibody molecules produced by a clone of hybridoma cells are identical in terms of antibody structure and antigen specificity. This has advantages and disadvantages in AHG Conventional Method production. Once an antibody-secreting clone of cells has Polyclonal Antihuman Globulin been established, antibody with the same specificity and re- action characteristics will be available indefinitely. This allows Complement IgG the production of a consistently pure and uncontaminated Rabbit is injected with AHG reagent. The disadvantage is that all antibodies pro- pooled donor antigen duced by a clone of cells recognize a single epitope present on an antigen. For antigens composed of multiple epitopes such as IgG, several different monoclonal antibodies reacting with different epitopes may need to be blended, or a mono- clonal antibody specificity for an epitope on all variants of a particular antigen may need to be selected to ensure that all different expressions of the antigen are detected. Monoclonal antibodies to human complement compo- nents anti-C3b and anti-C3d may be blended with polyclonal anti-IgG from rabbits to achieve potent reagents that give fewer false-positive reactions as a result of anticomplement. Antibody collected from multiple rabbits Immucor, Inc., manufactures AHG reagents from an entirely and purified monoclonal source. The anti-IgG component is produced by Monospecific Monospecific exposing mice to RBCs coated with IgG. The resulting anti- Polyclonal Polyclonal IgG component is a murine monoclonal IgM antibody that Anti-C3b,d Anti-IgG reacts with the CH3 domain of the Fc region of human IgG subclasses 1, 2, and 3. The antibody does not react with human antibodies of the IgG4 subclass, but these are not con- sidered to be clinically significant. Blending the monoclonal AHG Polyspecific Polyclonal Blend anti-IgG with a monoclonal anti-C3b and monoclonal anti- C3d results in a polyspecific AHG reagent. The preparation Figure 5–1. Preparation of polyclonal AHG reagents. Pooled donor antigen of monoclonal AHG is diagrammed in Figure 5–2. allows for a broader spectrum of reactivity, but the source of antibody is limited to the life span of the inoculated animal. Polyspecific antihuman globulin may be Before the AHG is available for purchase, manufacturers manufactured by combining polyclonal anti-IgG with either polyclonal or monoclonal must subject their reagents to an evaluation procedure, and anticomplement components. the results must be submitted to the FDA for approval.  Chapter 5 The Antiglobulin Test 107 Hybridoma Technology Monoclonal Antihuman Globulin Complement IgG Mouse is injected with purified human antigen Antibody-producing lymphocytes collected from spleen Myeloma Myeloma cell cell Fusion with myeloma cell to form hybridoma Clones grown in Monoclonal tissue culture to Monoclonal Anti-C3b,d,g yield monoclonal Anti-IgG Figure 5–2. Preparation of monoclonal AHG reagents. The production of antibody is longer lasting than the polyclonal source, as the hybridoma antibodies can live indefinitely. A monoclonal blend may be manufactured by blending monoclonal anti-C3b, monoclonal anti-C3d, and monoclonal anti-IgG. Mono- specific antihuman globulin reagents can be manufactured by conventional AHG Polyspecific or hybridoma technology. Monoclonal Blend Whether produced by the polyclonal or monoclonal tech- a mixture of IgG1 and IgG3 subclasses. Rarely, nonaggluti- nique, the final polyspecific product is one that contains nating IgM antibodies may be found; however, they have both anti-IgG and anticomplement activity at the correct been shown to fix complement and may be detected by anti- potency for immediate use. The reagent also contains buffers, complement.17 While some antibodies to blood group anti- stabilizers, and bacteriostatic agents and may be dyed green gens can have IgA components, the only RBC alloantibodies for visual distinction in the laboratory reagent rack. that have been reported as being solely IgA have been exam- ples of anti-Pr,18 and those antibodies were agglutinating. Monospecific AHG IgA autoantibodies have been reported, although very rarely.19 Therefore, anti-IgG activity must be present in the Monospecific AHG production process is similar to that AHG reagent. Anti-IgM and anti-IgA activity may be present, described above for polyspecific AHG; however, it contains but neither is essential. The presence of anti–light chain only one antibody specificity. Monospecific anti-IgG is activity allows detection of all immunoglobulin classes. produced as a monoclonal, polyclonal, or blended formula. IgG1, IgG2, IgG3, and IgG4 are the human IgG subclasses; however, changes in the constant region has defined 29 isoal- Antibodies Required in AHG lotypes to date.20 Monospecific IgG AHG has the advantage of specificity, but there have been limitations/disadvantages The need to use both polyspecific and monospecific AHG noted based on the detection of IgG1–IgG4 and the reactivity reagents has already been discussed. However, the reader of isoallotypes. Gamma-clone anti-IgG (Immucor, Inc., should be aware of the specific requirements for the sub- Norcross, GA), is the only FDA-licensed monoclonal IgG classes and components of IgG and complement required AHG in the United States.21 Gamma-clone detects IgG1, in the reagent. IgG2, and IgG3, but as described in the package insert, it Anti-IgG fails to react with IgG4. This is not regarded as a significant limitation since IgG4-only antibody specificities are uncom- AHG must contain antibody activity to nonagglutinating mon, have not been associated with acute hemolytic reac- blood group antibodies. The majority of these antibodies are tions, and often have high-titer low-avidity-type features  Chapter 5 The Antiglobulin Test 111 nature and specificity of the recipient’s antibody. In the drug therapy, and recent transfusion history. A positive DAT investigation of HDFN, testing for complement proteins is may occur without clinical manifestations of immune- not necessary inasmuch as the protein sensitizing the new- mediated hemolysis. Table 5–5 outlines the in vivo phenom- born RBCs is presumed to be maternal IgG. Problems can ena that may be associated with a positive DAT. arise in accurate D typing in the case of a newborn with a The AABB Technical Manual states that “ a positive DAT positive DAT. If the DAT is positive due to IgG and the im- result alone is not diagnostic of hemolytic anemia. Under- mediate spin for D typing is negative, a test for weak D standing the significance of this positive result requires cannot be performed. The same is true for a patient with knowledge of the patient’s diagnosis; recent drug, pregnancy, AIHA due to a warm IgG antibody coating the patient transfusion, and hematopoietic transplantation history; cells. The antibody must be removed from the RBCs for and the presence of acquired or unexplained hemolytic accurate phenotyping. Other techniques can be used to anemia.”11 Answering the following questions before inves- remove antibody from the patient’s RBCs. These include tigating a positive DAT for patients other than neonates will chloroquine diphosphate, EDTA-glycine, and murine help determine what further testing is appropriate: monoclonal antibodies. Is there evidence of in vivo hemolysis? Evaluation of a Positive DAT Has the patient been transfused recently? If so, did the patient receive blood products or components containing Clinical consideration should dictate the extent to which a ABO-incompatible plasma? positive DAT is evaluated. Interpreting the significance of a Does the patient’s serum contain unexpected antibodies? positive DAT requires knowledge of the patient’s diagnosis, Is the patient receiving any drugs? Table 5–5 In Vivo Phenomena Associated With a Positive DAT Condition Cause Transfusion 1. Recipient alloantibody and Alloantibodies in the recipient of a recent transfusion that donor antigen react with antigen on donor RBCs 2. Donor antibody and recipient Antibodies present in donor plasma that react with antigen antigen on a transfusion recipient’s RBCs Drug Induced 1. Type I (hapten-dependent Ab) Drug binds covalently to membrane proteins and stimu- lates hapten-dependent Ab 2. Type II (autoantibody) Drug induces autoantibody specific for RBC membrane proteins through unknown mechanism; Ab reacts with normal RBCs in the absence of drug. 3. Type III (drug-dependent Ab) Drug induces Ab that binds to RBC only when drug is present in soluble form, unknown mechanism; Ab reacts with normal RBCs when soluble drug is present. Autoimmune Hemolytic Anemia 1. WAIHA (IgG and/or C3) Autoantibody reacts with patient’s RBCs in vivo. 2. CAS (C3) Cold-reactive IgM autoagglutinin binds to RBCs in periph- eral circulation (32°C). IgM binds complement as RBCs return to warmer parts of circulation; IgM dissociates, leaving RBCs coated only with complement. 3. PCH (IgG) The IgG autoantibody reacts with RBCs in colder parts of body, causes complement to be bound irreversibly to RBCs, and then elutes at warmer temperature. Hemolytic disease of fetus and newborn Maternal alloantibody crosses Maternal (IgG) alloantibody, specific for fetal antigen, placenta (IgG) coats fetal RBCs. DAT is reactive with anti-IgG. Miscellaneous 1. Absorbed proteins; administra- Heterophile antibodies that are present in ALG or ATG coat tion of equine preparations of recipient’s RBCs. High levels of protein causing RBCs to antilymphocyte globulin (ALG) spontaneously agglutinate. and antithymocyte globulin (ATG) 2. Administration of high-dose Nonantibody-mediated binding of immunoglobulin to IV gamma globulin and RBCs in patients with hypergammaglobulinemia hypergammaglobulinemia Modified from Fung MK, Grossman BJ, Hillyer CD and Westhoff CM (eds): Technical Manual, 18th ed. AABB, Bethesda, MD, 2014. AIHA = autoimmune hemolytic anemia; CAS = cold agglutinin syndrome; PCH = paroxysmal cold hemoglobinuria 112 PART II Blood Groups and Serologic Testing Is the patient receiving antilymphocyte globulin or Table 5–7 Tasks and Purposes of the antithymocyte globulin? Indirect Antiglobulin Test Is the patient receiving intravenous immune globulin (IVIG) or intravenous Rh immune globulin (IV RhIG)? Task Purpose Has the patient received a marrow or other organ transplant? Incubate RBCs with antisera Allows time for antibody molecule attachment to RBC antigen Indirect Antiglobulin Test Perform a minimum of three Removes free globulin molecules saline washes The IAT is performed to determine in vitro sensitization of RBCs and is used in the following situations: Add antiglobulin reagent Forms RBC agglutinates (RBC Ag + Ab + anti-IgG) Detection of incomplete (nonagglutinating) antibodies to potential donor RBCs (compatibility testing) or to screen- Centrifuge Accelerates agglutination by bring- ing cells (antibody screen) in serum ing cells closer together Determination of RBC phenotype using known antisera Examine for agglutination Interprets test as positive or (e.g., weak D, any other antigen testing that requires IAT) negative Titration of incomplete antibodies Grade agglutination reactions Determines the strength of reaction Table 5–6 lists the IATs and the in vitro sensitization Add antibody-coated RBCs to Checks for neutralization of anti- detected for each application. For in vitro antigen-antibody negative reactions sera by free globulin molecules reactions, the IAT tasks are listed and explained in Table 5–7. (Coombs’ control cells are The DAT does not require the incubation phase because D-positive RBCs coated with of the antigen-antibody complexes formed in vivo. anti-D) Factors Affecting the Antiglobulin Test The DAT can detect a level of 100 to 500 IgG molecules per Addition of AHG RBC and 400 to 1,100 molecules of C3d per RBC.34 Centrifugation for reading For the IAT, there must be between 100 and 200 IgG or C3 molecules on the cell to obtain a positive reaction. The Ratio of Serum to Cells number of IgG molecules that sensitize an RBC and the rate Increasing the ratio of serum to cells increases the sensitiv- at which sensitization occurs can be influenced by several ity of the test system. Generally, a minimum ratio can be factors, including the following: achieved by using 2 drops of serum and 1 drop of a 5% vol- Ratio of serum to cells ume of solute per volume of solution (v/v) suspension of Reaction medium cells.39 When using cells suspended in saline, it is often Temperature advantageous to increase the ratio of serum to cells in an Incubation time effort to detect weak antibodies (e.g., 4 drops of serum with Washing of RBCs 1 drop of a 3% [v/v] cell suspension will give a ratio of Saline for washing 133:1). Reaction Medium Table 5–6 Indirect Antiglobulin Test Reaction mediums include albumin, LISS, and polyethylene In Vitro glycol. Application Tests Sensitization Albumin Antibody detection Compatibility Recipient antibody react- testing ing with donor cells The macromolecules of albumin allow antibody-coated cells to come into closer contact with each other so that Antibody Antibody reacting with agglutination occurs. In 1965, Stroup and MacIlroy42 re- screening screening cells ported on the increased sensitivity of the IAT if albumin Antibody Antibody panel Antibody reacting with was incorporated into the reaction medium. Their reaction identification panel cells mixture, consisting of 2 drops of serum, 2 drops of 22% Antibody titration Rh antibody Antibody and selected (w/v) bovine albumin, and 1 drop of 3% to 5% (v/v) cells, titer Rh cells was shown to provide the same sensitivity at 30 minutes of incubation as a 60-minute saline-only test. The use of RBC phenotype RBC antigen Specific antisera + RBCs albumin does not seem to provide any advantage over LISS detection(ex: to detect antigen weak D, K, Fy) techniques and adds to the cost of the test.42 Petz and coworkers36 also showed that an albumin technique may  Chapter 5 The Antiglobulin Test 113 miss several clinically significant antibodies. Therefore, it antibody to elute from the RBCs, decreasing the sensitivity is rarely, if ever, used as an IAT media for routine tests. of the test.47 However, this could not be confirmed by Voak and coworkers.48 Low Ionic Strength Solutions Low ionic strength solutions (LISS) enhance antibody Washing of RBCs uptake and allow incubation times to be decreased—from 30 to 60 minutes to 10 to 15 minutes—by reducing the When both the DAT and IAT are performed, RBCs must be zeta potential surrounding the RBC. Some LISS also saline-washed a minimum of three times before adding AHG contain macromolecular substances. The LISS technique, reagent. Washing the RBCs removes free unbound serum introduced by Low and Messeter,43 has critical require- globulins. Inadequate washing may result in a false-negative ments with respect to the serum-to-cell ratio. Moore and reaction because of neutralization of the AHG reagent by Mollison44 showed that optimum reaction conditions were residual unbound serum globulins. The washing phase of a obtained using 2 drops of serum and 2 drops of a 3% (v/v) DAT and IAT is one of the most important steps in testing. suspension of cells in LISS. Increasing the serum-to-cell The wash phase can be controlled using check cells, or group ratio increased the ionic strength of the reaction mixture, O cells sensitized with IgG. leading to a decrease in sensitivity and counteracting the Washing should be performed immediately after being re- shortened incubation time of the test. A LISS medium moved from the incubator and in as short a time as possible may be achieved by either suspending RBCs in LISS or to minimize the elution of low-affinity antibodies. The cell using a LISS additive reagent, with the latter being the button should be completely resuspended before adding the more common practice. next saline wash. All saline should be discarded after the final wash because residual saline dilutes the AHG reagent Polyethylene Glycol and therefore decreases the sensitivity of the test. Centrifu- Polyethylene glycol (PEG) is a water-soluble linear gation at each wash should be sufficient to provide a firm polymer and is used as an additive to increase antibody cell button and therefore minimize the possible loss of cells uptake. Its action is to remove water molecules surround- with each discard of saline. ing the RBC (the water of hydration theory), thereby effectively concentrating antibody. Anti-IgG is the AHG Saline for Washing reagent of choice with PEG testing to avoid false-positive reactions.11 Because PEG may cause aggregation of RBCs, Ideally, the saline used for washing should be fresh (sug- reading for agglutination following 37°C incubation in the gested open expiration of 30 days) and buffered to a pH of IAT is omitted. Several investigators45 compared the per- 7.2 to 7.4. Saline stored for long periods in plastic contain- formance of PEG as an enhancement media with that of ers has been shown to decrease in pH, which may increase LISS. Findings indicated that PEG increases the detection the rate of antibody elution during the washing process, of clinically significant antibodies while decreasing detec- yielding a false-negative result.49 Changes in pH may have tion of clinically insignificant antibodies. Barrett and important implications when monoclonal AHG is used, associates46 reported that since PEG has been used for pre- inasmuch as monoclonal antibodies have been shown to transfusion antibody screening, 6,353 RBC components have narrow pH ranges for optimum reactivity. Significant have been transfused without any reported acute or levels of bacterial contamination in saline have been delayed HTRs. reported;50 this situation can contribute to false-positive results. Temperature Addition of AHG The rate of reaction for the majority of IgG antibodies is optimal at 37°C; therefore, this is the usual incubation tem- AHG should be added to the cells immediately after wash- perature for the IAT. This is also the optimum temperature ing to minimize the chance of antibody eluting from the for complement activation. cell and subsequently neutralizing the AHG reagent. The volume of AHG added should be as indicated by the man- Incubation Time ufacturers. However, Voak and associates51 have shown that adding two volumes of AHG may overcome washing For cells suspended in saline, incubation times may vary problems when low levels of serum contamination remain. between 30 and 120 minutes. The majority of clinically These authors indicated that the neutralization of AHG significant antibodies can be detected after 30 minutes of is a problem only with free IgG left in serum following incubation, and extended incubation times are usually not inadequate saline washings and not with residual serum necessary. If a LISS or PEG technique is being used,43,44 in- complement components. The complement fragments free cubation times may be shortened to 10 to 15 minutes. With in serum are not the same as the complement fragments these shortened times, it is essential that tubes be incubated bound to RBCs. Therefore, residual serum does not con- at a temperature of 37°C. Extended incubation (i.e., up to tain C3b and C3d to neutralize the anti-C3b and anti-C3d 40 minutes) in the LISS technique has been shown to cause in AHG reagent. 114 PART II Blood Groups and Serologic Testing Centrifugation for Reading hemagglutination of the RBCs with the anti-IgG in the AHG reagent. If no hemagglutination follows the addition Centrifugation of the cell button for reading of hemagglu- of IgG-coated check cells, the test result is invalid and the tination along with the method used for resuspending test must be repeated. The most common technical errors the cells is a crucial step in the technique. The CBER- that result in failure to demonstrate hemagglutination after recommended method for the evaluation of AHG uses the addition of IgG-coated RBCs are inadequate washing, 1,000 relative centrifugal forces (RCFs) for 20 seconds, nonreactive AHG reagent, and failure to add AHG reagent. although the technique described in this chapter suggests Monospecific anti-C3d reactivity may be verified with the 500 RCFs for 15 to 20 seconds. The use of higher RCFs addition of C3d-coated RBCs to negative reactions.52 yields more sensitive results; however, depending on how the button is resuspended, it may give weak false-positive Modified and Automated Antiglobulin results because of inadequate resuspension, or may give a Test Techniques negative result if resuspension is too vigorous. The opti- mum centrifugation conditions should be determined for Modifications to the antiglobulin test technique (LISS, PEG, each centrifuge. and albumin) have just been described; however, additional modifications have historically been used, including the low- Sources of Error ionic polybrene technique53-56 and the enzyme-linked antiglobulin test (ELAT). Solid-phase and gel technologies Some of the most common sources of error associated with are fully automated modifications now widely used in the performance of the AHG test have been outlined in the immunohematology laboratories. previous section. Box 5–1 lists reasons for false-positive and false-negative AHG reactions. An anticoagulant such as Solid-Phase Technology EDTA should be used to collect blood samples for the DAT to avoid the in vitro complement attachment associated with Solid-phase technology may be used for performing refrigerated clotted specimens.52 antiglobulin tests. Several different techniques have been re- All negative antiglobulin test reactions must be checked ported using either test tubes57 or microplates.58,59 With the by the addition of IgG-sensitized cells. Adding IgG-coated availability of microplate readers, this technology can be RBCs to negative test reactions should demonstrate fully automated. Direct and indirect tests can be performed BOX 5–1 Sources of Error in Antihuman Globulin Testing False-Positive Results False-Negative Results Improper specimen (refrigerated, clotted) may cause in vitro Inadequate or improper washing of cells complement attachment Failure to wash additional times when increased serum volumes are Overcentrifugation and overreading used Centrifugation after the incubation phase when PEG or other Contamination of AHG by extraneous protein (i.e., glove, wrong positively charged polymers are used as an enhancement medium dropper) Bacterial contamination of cells or saline used in washing High concentration of IgG paraproteins in test serum Dirty glassware Early dissociation of bound IgG from RBCs due to interruption in testing Presence of fibrin in the test tube may mimic agglutination. Early dissociation of bound IgG from RBCs due to improper testing Cells with a positive DAT will yield a positive IAT. temperature (i.e., saline or AHG too cold or hot) Polyagglutinable cells AHG reagent nonreactive because of deterioration or neutralization Saline contaminated by heavy metals or colloidal silica (improper reagent storage) Using a serum sample for a DAT (use EDTA, ACD, or CPD anticoagu- Excessive heat or repeated freezing and thawing of test serum lated blood) Serum nonreactive because of deterioration of complement Samples collected in gel separator tubes may have unauthentic com- AHG reagent, test serum, or enhancement medium not added plement attachment. Undercentrifuged or overcentrifuged Complement attachment when specimens are collected from infusion Cell suspension either too weak or too heavy lines infusing dextrose solutions Serum-to-cell ratios are not ideal. Preservative-dependent antibody directed against reagents Rare antibodies are present that are only detectable with polyspecific AHG and when active complement is present. Low pH of saline Inadequate incubation conditions in the IAT Poor reading technique Modified from Fung MK, et al. Technical Manual, 19th ed. AABB, Bethesda, MD, 2017.  Chapter 5 The Antiglobulin Test 115 using solid-phase methodology. In the former, antibody Comparison of AHG Methodologies is attached to a microplate well, and RBCs are added. If antibody is specific for antigen on RBCs, the bottom of the Transfusion service departments typically work to detect all well will be covered with suspension; if no such specificity clinically significant antibodies, both DAT and IAT types, and occurs, RBCs will settle to the bottom of the well. In the as few clinically insignificant antibodies such as warm and latter, known RBCs are bound to a well that has been treated cold-reacting autoantibodies. A common question that arises with glutaraldehyde or poly L-lysine. Test serum is added to in these departments is which detection method should be RBC-coated wells, and if antibody in serum is specific for employed to reach such goals. Chapter 12 describes various antigen on fixed RBCs, a positive reaction occurs as previ- AHG methods for DAT and IAT testing. Table 5–8 outlines ously described. For a detailed description of this technology, some of the advantages and disadvantages in various AHG see Chapter 12, “Blood Bank Testing Technologies and testing methodologies. Automation.” Gel Test CASE STUDIES The gel test is a process that detects RBC antigen-antibody Case 5-1 reactions by means of a chamber filled with polyacrylamide gel. The gel acts as a trap; free unagglutinated RBCs form You are working second shift at a suburban hospital. Your buttons in the bottom of the tube, whereas agglutinated laboratory utilizes the conventional tube testing method RBCs are trapped in the tube for hours. Therefore, negative using LISS as an enhancement medium. About an hour reactions appear as buttons in the bottom of the microtube, before the end of your shift, the phlebotomist brings you and positive reactions are fixed in the gel. For a detailed a routine type and screen for a patient just admitted to description of this technology, see Chapter 12. Table 5–8 Comparison of AHG Methodologies Testing Methodology Advantages Disadvantages Saline-tube testing No additives Long incubation Reduced cost Least sensitive Avoids reactivity with auto Abs Requires highly trained staff Ability to assess multiple phases of reactivity Most procedural steps Fewer method-dependent Abs detected LISS-tube testing Reduced cost Inability to be automated Avoids reactivity with auto Abs Requires highly trained staff Shortest incubation time Many procedural steps Increased Ab uptake Fewer method-dependent Abs detected Most common tube method Ability to assess multiple phases of reactivity PEG-tube testing Reduced cost Requires highly trained staff Decreased incubation time Many procedural steps Increased Ab uptake Detects more unwanted Abs Enhances most Abs Inability to be automated Ability to assess multiple phases of reactivity Fewer method-dependent Abs detected (not 37°C) Gel More sensitive DAT method Warm auto Abs enhanced No washing steps Mixed-cell agglutination with cold Abs No need for check cells Increased costs Stable endpoints Increased need for additional instrumentation Small test volume Increased chances of detected unwanted Abs Enhanced anti-D detection Ability to be automated Solid phase No need for check cells Increased sensitivity for all Abs Stable endpoints Detects unwanted Abs Small test volume Warm auto Abs enhanced Enhanced anti-D Increased costs Increased sensitivity for all Abs Increased need for additional instrumentation Ability to be automated Ab = antibody; LISS = low ionic strength saline; PEG = polyethylene glycol