Immunohematology - 09 Anti-Human Globulin Test

IMMUNOHEMATOLOGY BLOOD BANKING PROCEDURES: ANTI-HUMAN GLOBULIN TEST OUTLINE • Anti-Human Globulin Test o Stages of Antigen-Antibody Reaction o AHG Reagents (Commercially Prepared) o Direct Antiglobulin Test o Indirect Antiglobulin Test o Automated AHG Technique o Sources of Error in the AHG Technique o Factors Affecting AHG o ANTI-HUMAN GLOBULIN TEST • Principle: A technique for detecting cell-bound immunoglobulin. It is used to detect incomplete antibodies or non-agglutinating antibody (IgG) o IgG is incomplete, needs AHG Ig to be tested • Role of AHG: Links or connects cell-bound IgG thereby promoting lattice formation • (+) Lattice Formation → with agglutination • Thereby non-agglutinating antibody with AHG can now form agglutination • IgM o Natural o Complete o Large size o Agglutinating (readily available) o Most efficient antibody o Has coating which requires AHG medium to promote lattice formation o Cold-reacting (optimum temp: 4C) o Saline-reactive (immediate spin) o Eg. ABO antibody o Complement binding (most potent) • IgG o Immune o Incomplete (do not promote lattice formation) o Smallest antibody o Coating/sensitizing with the antigen o Warm reacting ▪ Require incubation at 37C o Albumin/AHG reactive (thermophase/AHG phase) o Eg. RH antibody, Kell, Kidd, Duffy o Complement binding (IgG3 > IgG1 > IgG2) ▪ IgG3 fastest STAGES OF ANTIGEN-ANTIBODY REACTION • Hypervariable portion of Fab region attaches to the epitope of antigen • Strength of Interaction: affinity • Affinity - measure binding strength only at single portion • Avidity - measures total binding strength of ab to ag • First stage: Sensitization o Sensitization occurs when antibodies recognize/eact with antigens on the cells and coat the cells o IgM and IgG • Second Stage: Agglutination o Aka lattice formation o Agglutination occurs when antibodies on coated cells form cross-linkages between cells resulting in visible clumping o IgM only (cross-linking ability because of its size) o For IgG to proceed to 2 nd stage, AHG must be added (anti-antibody directed against AHG (same with RF), FC portion of human IgG serves as the epitope AHG REAGENTS (COMMERCIALLY PREPARED) • Polyspecific AHG Reagents o Consists of a pool of rabbit anti-human IgG and mous monoclonal anti-C3b anti anti-C3d o Contains anti-antibody (anti-IgG), anti-complement o Also referred to as Broad Spectrum Coombs Reagent. • Monospecific AHG Reagents o Contains only one antibody specificity o Either: ▪ Anti-IgG ▪ Anti-C3b or C3d ANTIHUMAN GLOBULIN REAGENTS • Polyspecific o Rabbit polyclonal - contains anti-IgG and anti-C3d (may contain other anticomplement and other immunoglobulin antibodies) o Rabbit/murine monoclonal blend - contains a blend of rabbit polyclonal antihuman IgG and murine monoclonal anti-C3b and anti-C3d. o Murine monoclonal - contains murine monoclonal anti-IgG, anti-C3b, and anti-C3d. • Monospecific Anti-IgG o Rabbit polyclonal - contains anti-IgG with no anticomplement activity (not necessarily gamma-chain specific) o IgG heavy-chain specific - contains only antibodies reactive against human gamma chains o Monoclonal IgG - contains murine monoclonal anti- IgG • Anticomplement o Rabbit polyclonal ▪ Anti-C3d and anti-C3b - contains only antibodies reactive against the designated complement ▪ Anti-C3d, anti-C4b, anti-C4d - component(s), with no anti-immunoglobulin activity o Murine Monoclonal ▪ Anti-C3d - contains only antibodies reactive against the designated complement ▪ Anti-C3b, anti-C3d - component, with no anti- immunoglobulin activity DIRECT ANTIGLOBULIN TEST (DAT) • Detects for in vivo sensitization • Specimen : RBCs, EDTA-anticoagulated blood samples are preferred • Conditions associated : o Hemolytic Transfusion Reaction (HTR) o Hemolytic Disease of the Newborn (HDN) ▪ Main test for diagnosing HDN o Autoimmune Hemolytic Anemia (AHA)

• Samples : o Anti-complementary because it can inactivate C1 trimolecular complex (Calcium is chelated and qrs are dissociated) o Post- transfusion RBC of patient’s sample is collected and tested for DAT o EDTA o Citrate INDIRECT ANTIGLOBULIN TEST (IAT) • Detects for in vitro sensitization • Specimen : serum • Applications o Antibody detection ▪ Crossmatching ▪ Antibody screen ▪ Antibody panel o Antibody Titration o Determination of RBC phenotype using known antisera (weak D) • Requires incubation phase • 2 Steps o Incubation → in crossmatching (thermophase - 37C) o AHG → agglutination ▪ AHG must be added immediately ▪ Delayed AHG addition may cause elution rxn • Delay AHG Reagent Addition o Undergo spontaneous elution process (dissociation of Ab on the epitope of antigen (False Negative)) AUTOMATED AHG TECHNIQUE • Low Ionic Polybrene Technique o Relies on low ionic condition to promote rapid sensitization of IgG RBC o Polybrene → potent rouleaux forming agent; promotes the active lattice formation and agglutination of sensitized RBC • Enzyme Linked Anti-globulin Test o RBC suspension with sensitized IgG reacts with AHG labeled with enzyme and substrate SOURCES OF ERROR IN THE AHG TECHNIQUE • False-Positive Results o Improper specimen (refrigerated, clotted) may cause in vitro complement attachment o Overcentrifugation and overreading o Centrifugation after the incubation phase when PEG or other positively charged polymers are used as an enhancement medium o Bacterial contamination of cells or saline used in washing o Dirty glassware o Presence of fibrin in test tube may mimic agglutination. o Cells with a positive DAT will yield a positive IAT. o Polyagglutinable cells/Autoagglutinable cells o Saline contaminated by heavy metals or colloidal silica o Using a serum sample for a DAT (use EDTA, ACD, or CPD anticoagulated blood) o Samples collected in gel separator tubes may have unauthentic complement attachment. o Complement attachment when specimens are collected from infusion lines infusing dextrose solutions o Preservative-dependent antibody directed against reagents • False-Negative Results o Inadequate or improper washing of cells o Failure to wash additional times when increased serum volumes are used o Contamination of AHG by extraneous protein (i.e., glove, wrong dropper) o High concentration of IgG paraproteins in test serum o Early dissociation of bound IgG from RBCs due to interruption in testing o Early dissociation of bound IgG from RBCs due to improper testing temperature (i.e., saline or AHG too cold or hot) o AHG reagent nonreactive because of deterioration or neutralization (improper reagent storage) o Excessive heat or repeated freezing and thawing of test serum o Serum nonreactive because of deterioration of complement o AHG reagent, test serum, or enhancement medium not added o Undercentrifuged or overcentrifuged o Cell suspension either too weak or too heavy o Serum:cell ratios are not ideal o Rare antibodies are present that are only detectable with polyspecific AHG and when active complement is present. o Low pH of saline o Inadequate incubation conditions in the IAT o Poor reading technique o Serum not added in the indirect test FACTORS AFFECTING AHG • Temperature : 37C • Ratio of serum to cells : 2:1 (serum:RBC) o Prevent zonal phenomenon o Excess serum - prozone phenomenon o Excess RBC - postzone phenomenon

• Incubation time o Saline suspension: 30-120 minutes o LISS suspension: 1-15 minutes • Reaction medium : saline/ 22% albumin • Washing of cells : 3 times • Saline for washing : fresh and buffered to a pH (7.2 - 7.4) • Addition of AHG reagents: immediately after washing • Centrifugation : 1000 rcf for 15-20 seconds o Overcentrifugation - false positive