Immunohematology methods used in evaluating patients with warm and cold autoantibodies
| Method . | Description/purpose . | Comments/pitfalls . |
|---|---|---|
| Routine: | ||
| ABO group/RhD type | Determine patient's ABO group and RhD type | • Presence of high titer CAA may cause autoagglutination at room temperature. Steps to resolve this may include: o Warm/wash patient RBCs at 37 °C o Perform testing at 37 °C o Treat RBCs with thiol reagent (DTT/2-ME) to remove IgM coating patient RBCs |
| DAT • Polyspecific AHG | Detects IgG and/or C3 (indirectly detects IgM) on patient's RBCs | • Positive in >90% of cases of AIHA • Screening assay; when positive must repeat testing with anti-IgG and anti-C3d to define protein coating patient RBCs |
| DAT • Monospecific o Anti-IgG o Anti-C3d | Individual reagents to detect whether IgG, C3d, or both on patient's RBCs | • Both reagents plus control must be performed • False positives can occur due to autoagglutination (most common with IgM) |
| Antibody detection (screen) | Use of 2-3 reagent RBCs of known phenotype to screen for auto and/or alloantibody in patient's serum • Detects IgM CAA when performed at IS/RT (direct agglutination) using a test tube method • Detects IgG WAA at IAT with any method. | • Detection depends on test method used (test tubes, CAT, SP) • May be negative if all autoantibody bound to patient’s RBCs |
| Antibody identification | Use of panel reagent RBCs (10-16) of known phenotype to determine specificity of antibody. • Reactivity at IS/RT (direct agglutination) confirms CAA. Some show autoanti-I or -i specificity. • Confirms broad reactivity or specificity of WAA at IAT. | • Subsequent testing when antibody is detected to identify antibodies of clinical significance • Identifies specificity of any underlying alloantibody once autoantibody removed from patient serum |
| Crossmatch | Confirms ABO compatibility by either serologic (IS) or electronic/computer. | • If known clinically significant alloantibody, must perform serologic crossmatch at IAT (also known as AHG or full crossmatch) |
| Phenotype | Serologically type patient RBCs using known antisera. | • Performed when patient has not been transfused in last 3 months • Strong positive DAT may cause false positive; not all antisera able to give accurate results when DAT is positive • Partial antigens or variant antigens may not be detected |
| Advanced: | ||
| Elution | • Elution removes IgG bound to the patient's RBCs using a dilute acid solution. Resulting eluate is then tested against a panel of reagent RBCs to check for specificity of the RBC bound IgG. o Positive with all cells confirms WAA. o If negative, suspect drug-dependent antibody. | • Once diagnosis of AIHA, no need to perform with each subsequent pretransfusion test • Also used in suspected delayed transfusion reaction to detect binding of alloantibody to circulating transfused RBCs |
| Adsorption—autologous | • Removes IgG autoantibody (when incubated at 37 °C) from patient plasma using patient RBCs; then adsorbed plasma tested to identify underlying alloantibody. • Removes IgM autoantibody (when incubated at 4C) from patient plasma using patient RBCs; then adsorbed plasma tested to identify underlying alloantibody. | • Performed when patient has not been transfused in last 3 months • Patient RBCs are first treated to remove bound autoantibody to allow sites for autoantibody in plasma to bind • Requires sufficient quantity of patient RBCs for removal of autoantibody |
| Adsorption—allogeneic | • Removes IgG autoantibody (when incubated at 37 °C) from patient plasma using phenotyped donor RBCs; then adsorbed plasma tested to identify underlying alloantibody. • Removes IgM autoantibody (when incubated at 4C) from patient plasma using donor RBCs; then adsorbed plasma tested to identify underlying alloantibody. | • Typically, 3 donor RBCs of known phenotype (eg, R1R1, R2R2, rr) are selected • May miss antibody to high prevalence antigen |
| Advanced: | ||
| Genotype | • Molecular methods used to determine patient's probable phenotype. | • More accurate than serologic phenotyping; generally, provides more comprehensive antigen profile • Recommended when patient has been transfused in last 3 months • Unusual, rare genetic variants or partial antigens may not be detected |
| Prewarm technique | • Wash patient RBCs with 37 °C saline prior to testing. • Warm patient plasma and reagent RBCs at 37 °C separately prior to mixing and performing testing to avoid CAA reactivity. | • Avoids CAA from binding to patient or donor RBCs • Clinically significant, weakly reactive alloantibodies may not be detected in a prewarm IAT |
| Thermal amplitude study | • Incubate patient serum with reagent RBCs for 2 hours at 30 °C and 37 °C. • Determines if CAA has ability to bind to reagent RBCs at warm body temperature. | • Should be performed with adult RBCs (positive for I) and cord blood RBCs (positive for i) • Typically, only need to perform once to aid in diagnosis |
| Method . | Description/purpose . | Comments/pitfalls . |
|---|---|---|
| Routine: | ||
| ABO group/RhD type | Determine patient's ABO group and RhD type | • Presence of high titer CAA may cause autoagglutination at room temperature. Steps to resolve this may include: o Warm/wash patient RBCs at 37 °C o Perform testing at 37 °C o Treat RBCs with thiol reagent (DTT/2-ME) to remove IgM coating patient RBCs |
| DAT • Polyspecific AHG | Detects IgG and/or C3 (indirectly detects IgM) on patient's RBCs | • Positive in >90% of cases of AIHA • Screening assay; when positive must repeat testing with anti-IgG and anti-C3d to define protein coating patient RBCs |
| DAT • Monospecific o Anti-IgG o Anti-C3d | Individual reagents to detect whether IgG, C3d, or both on patient's RBCs | • Both reagents plus control must be performed • False positives can occur due to autoagglutination (most common with IgM) |
| Antibody detection (screen) | Use of 2-3 reagent RBCs of known phenotype to screen for auto and/or alloantibody in patient's serum • Detects IgM CAA when performed at IS/RT (direct agglutination) using a test tube method • Detects IgG WAA at IAT with any method. | • Detection depends on test method used (test tubes, CAT, SP) • May be negative if all autoantibody bound to patient’s RBCs |
| Antibody identification | Use of panel reagent RBCs (10-16) of known phenotype to determine specificity of antibody. • Reactivity at IS/RT (direct agglutination) confirms CAA. Some show autoanti-I or -i specificity. • Confirms broad reactivity or specificity of WAA at IAT. | • Subsequent testing when antibody is detected to identify antibodies of clinical significance • Identifies specificity of any underlying alloantibody once autoantibody removed from patient serum |
| Crossmatch | Confirms ABO compatibility by either serologic (IS) or electronic/computer. | • If known clinically significant alloantibody, must perform serologic crossmatch at IAT (also known as AHG or full crossmatch) |
| Phenotype | Serologically type patient RBCs using known antisera. | • Performed when patient has not been transfused in last 3 months • Strong positive DAT may cause false positive; not all antisera able to give accurate results when DAT is positive • Partial antigens or variant antigens may not be detected |
| Advanced: | ||
| Elution | • Elution removes IgG bound to the patient's RBCs using a dilute acid solution. Resulting eluate is then tested against a panel of reagent RBCs to check for specificity of the RBC bound IgG. o Positive with all cells confirms WAA. o If negative, suspect drug-dependent antibody. | • Once diagnosis of AIHA, no need to perform with each subsequent pretransfusion test • Also used in suspected delayed transfusion reaction to detect binding of alloantibody to circulating transfused RBCs |
| Adsorption—autologous | • Removes IgG autoantibody (when incubated at 37 °C) from patient plasma using patient RBCs; then adsorbed plasma tested to identify underlying alloantibody. • Removes IgM autoantibody (when incubated at 4C) from patient plasma using patient RBCs; then adsorbed plasma tested to identify underlying alloantibody. | • Performed when patient has not been transfused in last 3 months • Patient RBCs are first treated to remove bound autoantibody to allow sites for autoantibody in plasma to bind • Requires sufficient quantity of patient RBCs for removal of autoantibody |
| Adsorption—allogeneic | • Removes IgG autoantibody (when incubated at 37 °C) from patient plasma using phenotyped donor RBCs; then adsorbed plasma tested to identify underlying alloantibody. • Removes IgM autoantibody (when incubated at 4C) from patient plasma using donor RBCs; then adsorbed plasma tested to identify underlying alloantibody. | • Typically, 3 donor RBCs of known phenotype (eg, R1R1, R2R2, rr) are selected • May miss antibody to high prevalence antigen |
| Advanced: | ||
| Genotype | • Molecular methods used to determine patient's probable phenotype. | • More accurate than serologic phenotyping; generally, provides more comprehensive antigen profile • Recommended when patient has been transfused in last 3 months • Unusual, rare genetic variants or partial antigens may not be detected |
| Prewarm technique | • Wash patient RBCs with 37 °C saline prior to testing. • Warm patient plasma and reagent RBCs at 37 °C separately prior to mixing and performing testing to avoid CAA reactivity. | • Avoids CAA from binding to patient or donor RBCs • Clinically significant, weakly reactive alloantibodies may not be detected in a prewarm IAT |
| Thermal amplitude study | • Incubate patient serum with reagent RBCs for 2 hours at 30 °C and 37 °C. • Determines if CAA has ability to bind to reagent RBCs at warm body temperature. | • Should be performed with adult RBCs (positive for I) and cord blood RBCs (positive for i) • Typically, only need to perform once to aid in diagnosis |
CAT, column agglutination technique; IS, immediate spin (direct agglutination at room temperature); 2-ME, 2-mercaptoethanol; RT, room temperature, direct testing; SP, solid phase technique.