Immunohematology - 10 Antibody Screening

IMMUNOHEMATOLOGY 2. BLOOD BANKING PROCEDURES: ANTIBODY SCREENING 3. OUTLINE • Antibody Screening o Antibody Screening o Traditional Tube Method o RBC Reagents o Phases on Antibody Screen o Enhancement Reagents o Gel Test Technology o Solid-Phase Red Cell Adherence ANTIBODY SCREENING • • • • For the detection of antibodies directed against red blood cell antigens Investigating potential hemolytic transfusion reactions and immune hemolytic anemias. Aids in detecting and monitoring patients “irregular” or “unexpected” antibodies RBC REAGENTS • • • • • Antibody Screening Procedure o Test to detect clinically significant IgG antibody outside the ABO system Performed in the following: o Pretransfusion test o Antenatal screening o For HDFN diagnosis Detects 2 types of Ab o Alloantibodies → PS or DS is tested with screening O cells = (+) o Autoantibodies → PS or DS + RBC of P or D = (+); patient’s serum is tested against patient’s O cell Group O cells are used so that anti-A and anti-B will not interfere The RBCs are suspended at a concentration between 2% and 5% in a preservative diluent Screen Cells – packaged in sets of two or three cell suspensions PHASES ON ANTIBODY SCREEN • • ANTIBODY SCREENING • Incubate at 37 deg C ▪ Coating the RBCs with enhancement media may be added ▪ Centrifugation is done Observe for hemolysis and agglutination after incubation ▪ (+) = ag-ab interaction • Immediate Spin o May/may not be required o Detects clinically significant cold antibodies (IgM) o Immediate centrifugation of mixture at RT → 20s 37 incubation phase with enhancement medium o Aka thermophase o Detects warm reacting IgG abs o EM + cell-serum mixture → 37C incubation in waterbath (to increase test sensitivity) Antiglobulin Phase o Detects non-agglutinating, warm-reactive IgG antibodies that possibly not detected in 37C incubation period o Detects abs that have coated/sensitized RBCs but were not visible as agglutinates ENHANCEMENT REAGENTS • • • 22% Albumin – reducing the zeta potential and dispersing the charges Low Ionic Solution (LISS) – increase the uptake of antibody onto the RBC during the sensitization phase Polyethylene Glycol (PEG) – PEG in a LISS solution removes water from the test system, concentrating any antibodies present; increases RBC sensitization GEL TEST TECHNOLOGY • • • • Screening O Cells o Contains all possible antigen that could react with unknown abs from the patient sample TRADITIONAL TUBE METHOD • • Materials o RBC reagents o Enhancement reagents o AHG reagents Procedure: 1. Patient’s serum or plasma mixed with RBCs with known antigen ▪ May include an immediate spin phase • • • • • • Innovative approach to red cell serology Invented by Dr. Yves Lapierre in 1985 It is developed to minimize conventional techniques of blood grouping. Addresses issues of standardization and documentation with unmatched sensitivity, specificity and efficiency. Part of compatibility testing Can be used for antibody screening Main advantage - Standardization o Standardization removes the use of tube, shaking, resuspension and it is stable up to 3 days Disadvantage o Requires purchase of special equipment Principle of Gel Test Technology o To trap agglutinates formed when antigen react to antibody o To detect agglutination reaction o • The agglutination occurs when the red blood cell antigens react with the corresponding antibodies, present in the gel solution or in the serum or plasma sample (in the case of reverse grouping test) o Gel Test Card o 5 x 7 cm card with 6 microtubules o Microtubules contain dextran acrylamide gel o Incubated for sensitization o Centrifuged after incubation o Dextran acrylamide gel ▪ Provides prolonged contact with the RBC during centrifugation ▪ Reaction filter will sieve and not allow large RBC clump to travel through the gel ▪ Large agglutinates will be trapped at the top ▪ Unagglutinated form will form a pellet at the bottom after centrifugation • • Advantages o Standardization ▪ Well-defined and point of result ▪ Stable for 3 days o Decreased volume needed (microsampling) o Cell washing is no longer performed o Enhanced specificity and sensitivity o Minimizes contamination and sources of error Disadvantages o Sample restrictions ▪ Hemolyzes sample, icteric sample, suspect of agglutination/rouleaux formation, lipemic sample are not allowed (false positive/negative) o Need for a special equipment SOLID-PHASE RED CELL ADHERENCE • Gel Matrix o Highly sensitive o Component o Component: acts as sieve ▪ Sephadex ▪ Dextranacrylamide gel o Principle: based on size exclusion chromatography ▪ Bigger clump = higher location in gel matrix ▪ • • • • • • Procedure of Gel Test 1. Addition of cells 2. Addition of plasma/serum 3. Incubation (15/30-60 mins) 4. Centrifugation 5. Results Developed commercially for the detection of RBC and platelet related antibodies. The ability of plastics, such as polystyrene to absorb proteins from solutions and bind them irreversibly made SPRCA possible. o Polystyrene - component of solid phase If test plasma contains antibodies to antigen = attach to the fixed antigen. If the test plasma contains no antibodies to the antigen = no attachment to the fixed antigen. Solid phase reactions are stable for observation or review for 2 days LUMINEX BASED ASSAY • • • • • • SOLID-PHASE IMMUNOSORBENT ASSAY (ELISA) • Principle o The solid-phase ELISA test (MACE®) is used primarily for compatibility testing. o To perform MACE® tests, patient serum or plasma is incubated with intact platelets to allow antibody, if present, to bind to the platelet glycoproteins. o To perform PAK® tests, patient serum or plasma is added to microwells that are coated with platelet glycoproteins, allowing antibody, if present, to bind. Unbound antibodies are then washed away. o After a 30-minute incubation period, the reaction is stopped by a sodium hydroxide solution. o Finally, the optical density of the color that develops is measured in a spectrophotometer o Color is directly proportional to color intensity/ absorbance RESULTS • • SOLID-PHASE PROTEIN A TECHNOLOGY • • • IgG antibodies are captured in microwells that are coated with Protein A Solidscreen II – an assay that uses traditional antiglobulin (AHG) technique Solid-phase protein A testing is only available in the United States as an automated technology on the TANGO optimo instrument Gen-Probe GTi Diagnostics has developed ELISA assays to detect and identify HLA (human leukocyte antigen) antibodies (both class I and class II MHC). They have also developed a new Luminex-based assay for platelet and HLA antibodies. Luminex is a bead-based assay that uses fluorescence and flow cytometry to achieve a high level of sensitivity The Luminex assay uses a mixture of up to 100 different colored beads; each bead is coated with a different protein. The flow cytometer distinguishes between each of the 100 beads by the amount and color of the internal dyes Points to Remember o Luminex-based assay is a bead-based assay that uses florescence and flow cytometry to test for platelet/HLA antibodies. o Luminex assay uses a mixture of 100 different colored beads, each bead coated with a different protein o The flow cytometer distinguishes between each of the 100 beads by the amount and color of the internal dyes. Interpretation of Results will be based on: o Phase of reaction ▪ Phases: IS, 37C, AHG (IAT) • IS - IgM • 37C - IgG • AHG - IgG (not agglutinated by 37C) o Clinically significant (HDN, HTR, AHA) o Autocontrol ▪ Identify presence of alloantibody/autoantibody ▪ (+) = autoantibody ▪ (-) = alloantibody o Strength of reaction ▪ Guide on deciding on result of panel testing ▪ 3+/4+ - true agglutination ▪ 3+ on AHG = IgG ▪ 3+ on IS = IgM Ab screening result o Positive → proceed to ab identification o Negative → proceed to crossmatching