IMMUNOHEMATOLOGY LABORATORY BLOOD BANKING PROCEDURES OUTLINE • Antihuman Globulin Test o Introduction o Stages of Antigen-Antibody Reaction o AHG Reagent o Antihuman Globulin Test Procedures o Direct AHG Test o Indirect AHG Test o Automated AHG Technique o Factors that May Affect AHG Testing o Sources of Error in Antihuman Globulin Testing • Antibody Screening o Antibody Screening Procedure o Traditional Tube Method o Gel Test Technology o Solid-Phase Red Cell Adherence o Solid-Phase Immunosorbent Assay o Solid Phase Protein A Technology o Luminex Based Assay • Compatibility Testing o Collection and Preparation of Samples o Compatibility Testing Protocols o Steps in Pre-Transfusion Testing o Pre-transfusion Testing in Special Cases o Crossmatching o Techniques in Crossmatch o Incompatible Crossmatches • Antibody Identification o Antibody Identification o Important Blood Group Antigen Reactions o General Process o Antibody Panel Interpretation • Antiglobulin test (Coomb’s Test) • Antibody Testing • Crossmatching ANTIHUMAN GLOBULIN TEST INTRODUCTION • Principle : o A technique for detecting cell-bound immunoglobulin. It is used to detect incomplete antibodies or non-agglutinating antibody (IgG) • Role of AHG o It links cell-bound IgG to promote agglutination reaction by lattice formation o Non-agglutinating antibodies can now agglutinate • IgM o Naturally occurring antibodies (eg. ABO, MN) o Complete antibodies, readily bound o Agglutinating antibodies o Cold reacting (room temp/4C cold) o Saline-reactive o ABO blood group system o Most potent in complement binding • IgG o Immune (eg. Rh) o Incomplete antibodies, requires AHG to interact with other IgG in promoting lattice formation o Requires coating and sensitization before agglutination as well as recognition o Warm-reacting (37C) o Requires albumin & AHG o Rh blood group system o Complement binding (IgG3 > IgG1 > IgG2) ▪ IgG3 & IgG1 - cross through placenta STAGES OF ANTIGEN-ANTIBODY REACTION • Hypervariable portion of Fab region attaches to the epitope of antigen • Strength of Interaction: Affinity • First Stage: Sensitization o Antibody reacts with antigens on cell and coats cell o Specific recognition between antibody and antigen • Second Stage : Lattice Formation o Antibodies on coated cells cross-linkages with other coated cells which leads to cell clumping o Results in agglutination reaction o IgM - immediately forms lattice formation o IgG - requires AHG to form lattice formation AHG REAGENTS (COMMERCIALLY PREPARED) • Polyspecific AHG Reagents o Consists of a pool of rabbit anti ‐ human IgG (anti-IgG) and mouse monoclonal (anti-C3b/C3d) o Also referred to as broad specific Coomb’s reagent • Monospecific AHG Reagents o Contains only one antibody specificity o Either: anti-IgG or anti-C3b/C3d ANTIHUMAN GLOBULIN TEST PROCEDURES • Also known as the Coomb’s Test • Principle : Antihuman Globulin (AHG) will bind immunoglobulins (or complement) in the serum or red cells • Target : IgG (or complement) • Direct AHG Test - Immunoglobulin-sensitized RBCs • Indirect AHG Test - Free floating immunoglobulins in the plasma MATERIALS • Test tubes 10 or 12 x 75 mm • Serofuge • Anti-human serum • Water bath • Magnifying mirror or microscope • Cord red cells/red cells • Patient’s serum • AHG Reagent 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 AHG TEST (DAT) • Principle: This technique is used to detect presence of antibody coated onto red cells in vivo, as it occurs in hemolytic disease of the newborn (HDN), hemolytic transfusion reactions (HTR), and acquired hemolytic anemia (AHA). • Procedure 1. Wash the cord blood/RBCs suspected of being coated with autoantibody 3-4 times in large volumes of saline 2. Decant completely at the end of the last washing 3. Add 2 drops of AHG to the sedimented cells remaining 4. Mix well and centrifuge at 1000-2000 rpm for 1 minute 5. Examine for agglutination. If desired, readings may be confirmed with the help of a small hand lens or magnifying mirror or the LPO of a microscope • Clinical Significance of Reactive DAT o Transfusion-Associated (HTR) ▪ Alloantibody in the recipient, donor antibody attacks patient RBC o Drug Induced (Hemolytic Anemia) ▪ Type I (hapten dependent) ▪ Type II (autoantibody) ▪ Type III (drug-dependent Ab) o Autoimmune Hemolytic Anemia ▪ WAIHA - Warm Autoimmune Hemolytic Anemia ▪ CAS - Cold agglutinin syndrome ▪ PCH - Paroxysmal Cold Hemoglobinuria o HDFN o Others ▪ Antilymphocyte globulin, high dose IV gammaglobulin, Hypergammaglobulinemia • Samples o Anti-complement o Patient RBC (Citrate, EDTA) ▪ Requires washing ▪ EDTA - chelates calcium • Things to Remember o The direct antihuman globulin test (DAT) is needed to demonstrate antibodies in the event of in vivo erythrocyte sensitization. o Thus, antibodies or complement components already fixed to the patient's erythrocytes are detected. o Following a triple washing process with the sensitised cells, the AHG serum is added. o If not washed (False Negative) INDIRECT AHG TEST (IAT) • A two-step procedure (sensitization by incubation and agglutination) that determines in vitro sensitization of red cells • Useful in the ff. situations: o Identification of ag specificity using a panel of RBC o Determination of red cell phenotype o Detection of incomplete ab in compatibility testing o Titration of incomplete antibodies • 2 steps o Incubation (thermophase in crossmatching) o Cell washing & addition of AHG ▪ Check for agglutination reaction • Delay in AHG Reagent addition can lead to spontaneous elution process which can cause the dissociation of the antibody on the epitope of antigen (false negative) • Sample: Patient serum • Procedure 1. Prepare 2% saline suspension of test cells containing the appropriate antigen 2. Add 2 drops of the suspension of cells in the small test tube 3. Add 2 drops of serum to be tested, centrifuge. 4. Incubate in water bath (37°C) for 15 minutes 5. Remove tube and wash 3 times with large volumes of saline. Decant completely after last washing 6. Add 2 drops of AHG and mix. Centrifuge for 1 minute 7. Examine for agglutination. Add check cells to confirm negative result. 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 that promotes active lattice formation and agglutination of sensitized RBCs • Enzyme Linked Anti ‐ globulin Test (ELISA) o RBC suspension with sensitized IgG, reacts with AHG label with enzyme and substrate
FACTORS THAT MAY AFFECT AHG TESTING • Temperature - 37C o Fluctuations may cause false reactions • Ratio of serum to cells o 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 o Reaction medium - 22% Bovine Albumin, LISS, PEG • Washing of cells - 3x • Saline for washing - fresh or buffered for pH 7.2 to 7.4 • Addition of AHG reagents - immediately after washing • Centrifugation - 1000-2000 rcf for 15-20 sec o Overcentrifugation - false positve SOURCES OF ERROR IN ANTIHUMAN GLOBULIN TESTING • 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 the test tube may mimic agglutination. o Cells with a positive DAT will yield a positive IAT. o Polyagglutinable 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 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 ANTIBODY SCREENING PROCEDURE • Test to detect clinically significant IgG antibody outside the ABO system • Performed in the following: o Pre-transfusion testing o Antenatal screening o HDFN diagnosis • Detects 2 types of Ab o Alloantibodies ▪ Patient or donor serum is tested with increasing O cell (+ for agglutination) o Autoantibodies ▪ Patient or donor serum + donor/patient RBC = (+) TRADITIONAL TUBE METHOD • Materials : o RBC reagents o Enhancement reagents o AHG reagents • Procedure o Patient serum or plasma is mixed with RBC reagent or screening O cells. o Incubate at 37C. (thermophase to detect IgG) ▪ Enhancement reagent can be used. o Add AHG reagent. o Observe for agglutination/
• RBC reagents o Group O cells are used because anti-B & anti-A will not interference with the results o The RBCs are suspended at a concentration between 2% and 5% in a preservative diluent o Screen Cells – packaged in sets of two or three cell suspensions • Phases on Antibody Screening o Immediate spin (IS) ▪ IgM antibody detection (immediate agglutination) ▪ Immediate centrifugation at room temp for 20s ▪ When positive, report the reaction ▪ When negative, proceed to thermophase o 37C incubation phase with enhancement medium ▪ Aka thermophase ▪ To detect IgG antibodies (warm-reacting) ▪ Enhancement medium + cell-serum (IS) will be incubated at 37C. Then, check for agglutination. ▪ When positive, report the reaction. ▪ When negative, proceed to the next phase. o Antiglobulin phase ▪ Detects non-agglutinating warm-reacting IgG abs ▪ Detects antibodies that have coated/sensistized RBCS are were not visible as agglutinates ▪ Utilizes AHG reagent • Enhancement Reagents o 22% Albumin – reducing the zeta potential and dispersing the charges o Low Ionic Solution (LISS) – LISS increases the uptake of antibody onto the RBC during the sensitization phase. o Polyethylene Glycol (PEG) – PEG in a LISS solution removes water from the test system, concentrating any antibodies present. Increases RBC sensitization. • Dosage effect - increased reaction • If screening is positive, proceed to antibody identification • If screening is negative, proceed to crossmatching • AC positive = presence of autoantibodies • Case 1 Cell IS 37C AHG Possible Interpretation SC I Neg Neg Neg 1. Single alloantibody 2. 2 alloab, but w/ dosage 3. IgG abs SC II Neg Neg 2+ AC Neg Neg Neg o Proceed to ab panel • Case 2 Cell IS 37C AHG Possible Interpretation SC I Neg 1+ 3+ 1. Multiple abs 2. Single ab (dosage) 3. IgG abs SC II Neg Neg 1+ AC Neg Neg Neg o Proceed to panel • Case 3 Cell IS 37C AHG Possible Interpretation SC I 1+ 1+ 3+ 1. Multiple abs 2. IgM or IgG or both SC II 2+ Neg 1+ AC Neg Neg Neg • Case 4 Cell IS 37C AHG Possible Interpretation SC I 2+ Neg 3+ 1. Multiple abs 2. Potent cold IgM ab 3. IgG ab SC II 2+ 1+ 1+ AC Neg Neg Neg • Case 5 Cell IS 37C AHG Possible Interpretation SC I Neg Neg 3+ 1. Single ab 2. Warm autoantibody 3. IgG SC II Neg Neg 2+ AC Neg Neg 3+ GEL TEST TECHNOLOGY • 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. • 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 o To trap agglutinates formed when antigen reacts with an antibody o To detect agglutination reactions Patient + Donor cell + screening O cell • Procedure : o Incubate at 15-60 mins o Centrifuge for 10 mins o Results may have top or bottom settling. • Gel Matrix o Highly sensitive o Component : acts as sieve ▪ Sephadex ▪ Dextranacrylamide gel o Principle : based on size exclusion chromatography ▪ Bigger clump = higher location in gel matrix
• Gel test card o 5x7cm card w/ 6 microtubules o With dextran acrylamide gel ▪ Provides prolonged contact with RBC during centrifugation ▪ Reaction filter will sieve RBC, larger RBC will not pass through o Incubated to promote sensitization ▪ After incubation, centrifugation follows o Large agglutinates - trapped at the top o Unagglutinated form - forms pellet at the bottom after centrifugation • Procedure of Gel Test o Addition of cells (px/screening cell) o Addition of plasma/serum (from px/donor) o Incubation (15/30 - 60 minutes) o Centrifugation o Results • 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 • 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, one of the important innovations in BB SOLID-PHASE IMMUNOSORBENT ASSAY (ELISA) • ELISA is one of the common tests in the clinical lab • 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 the absorbance
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 technique • Solid-phase protein A testing is only available in the United States as an automated technology on the TANGO optimo instrument LUMINEX BASED ASSAY • Gen-Probe GTi Diagnostics has developed ELISA assays to detect and identify HLA (human leukocyte antigen) antibodies (both class I and class II). • 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 • The 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. COMPATIBILITY TESTING • A pretransfusion procedure performed to ensure the safety of the patient during and after blood transfusion • Called Crossmatching because the patient’s sample are tested against the donor’s sample COLLECTION AND PREPARATION OF SAMPLES • Patient Identification. o Identify based: ▪ Tubes received must be identical w/ the request ▪ Freshness of sample • Collection • Serum is the preferred specimen for compatibility testing because it lacks fibrinogen which fibrin clots may cause in vitro clots causing false positive • Hemolysis should be avoided. • Lipemic and icteric sample should also be avoided • Why serum and not plasma? o Plasma may cause small fibrin clots to form which may be difficult to distinguish from true agglutination o Plasma may inactivate complement so that antibodies may not be detected • Age of Specimen o The freshest sample possible should be used for compatibility testing o Specimens must be less than 3 days old if the patient has been transfused or pregnant within the past 3 months o Must be 3 days old because it represents the current or actual immunologic status of the patient o If autologous priocedure, it must always be retyped o In cases of emergency, safest blood to give is bloo type “O” - pRBC because it lacks highly immunogenic ABO Ag (AB plasma lacks high titer anti-A, anti-B, anti- AB that why it’s not safe for transfusion • Sample Storage o AABB requires that patient samples must be stored between 1-6C for at least 7 days after transfusion o The crossmatch sample store at ref. temp. is used in the investigation of transfusion reaction if it appears/occurs in the patient • Use of Check Cell/Control Cell o Used to validate all negative AHG test reaction o To be valid, check cell must give a positive agglutination reaction o To check for the reaction of reagent • Invalid Check Cell Result (negative; w/o agglutination) o Reagent is deteriorating o AHG is already neutralized o Not washed properly COMPATIBILITY TESTING PROTOCOLS • ABO Grouping o Most critical pretransfusion serologic test o If the patient’s AB group cannot be satisfactorily determined and immediate transfusion is essential, group O packed RBCs (pRBC) should be utilized • Rh Typing o If Rh type of the recipient cannot be determined and transfusion is essential, Rh-negative blood should be given STEPS IN PRE-TRANSFUSION TESTING • Request for transfusion • Identification of transfusion recipient and blood specimen collected o Identical with one another o To avoid clerical/procedural errors • Testing of transfusio n recipient’s blood specimen: o Blood specimen acceptability (check for hemolysis, lipemia, ictericia) o ABO group and Rh type o Antibody detection testing o Antibody identification o Comparison of current and previous test results • Donor RBC unit testing: o ABO group confirmation and Rh type confirmation for 5.12 and Rh-negative RBC units • Donor red cell unit selection: o Selection of components of ABO group and Rh type that are compatible with the transfusion recipient and with any unexpected allogeneic antibodies • Compatibility testing (crossmatch): o Serologic o Computer or electronic • Labeling of blood or blood components with the recipient’s identifying information and issue
PRETRANSFUSION TESTING IN SPECIAL CASES • Emergency (For immediate transfusion, O, Rh-) • Non-blood group specific blood • Plasma products • Intrauterine transfusions (HDFN) • Neonatal transfusions (0-4 months old) • Massive transfusions (8-10 units in less than 24 hrs or 4-5 units in 1hr) • Prolonged clotting time • Preoperative autologous blood NEONATAL TRANSFUSION • Compatibility Tests for Infants Once per Admission o Routine o ABO o Rh o Antibody screen ▪ Using maternal serum or ▪ Using infant’s serum, especially when: • No maternal specimen is available • Mother has clinically insignificant abs or ▪ Using infant’s eluate o Additional ▪ IAT using infant serum and A1 or B cells • Cells can be reagent or donor (i.e., major crossmatch) • Must be done if non-group O cells will be transfused • Antigen typing donor unit • While infant antibody screen is positive • Donor units must lack antigen corresponding to antibody o Every 3 Days ▪ Same tests as above when: • ABO- or Rh-incompatible units are transfused or • Unexpected antibodies are demonstrating via antibody screen CROSSMATCHING (X-MATCH) • Serves as final check for ABO compatibility between donor and patient; presence of agglutination indicates incompatibility of crossmatching • Major X-Match : donor’s cells (ag) + recipient’s serum (ab) (PS + DR) • Minor X-Match : donor’s serum (ab) + recipient’s cells (ag) (PR + DS) o Replaced w/ antibody screening procedure • After 14 days is longest transfusion reaction o Watch out for 14 days for transfusion rxns 3 PHASES OF CROSSMATCHING • Immediate Spin in saline at Rt → detects IgM • Thermophase/37ºC - incubation for 30 minutes with enhancement medium (e.g. albumin, LISS, PEG) → detects IgG • AHG Phase after washing incubated cells with saline. • *Check cells/Coombs control cells(IgG sensitized cells) should be added to tubes that demonstrate no agglutination. For results to be considered valid, agglutination must occur TECHNIQUES IN CROSSMATCH • Saline technique o PS + Dr in saline medium o IgM detection at RT o Detects ABO incompatibilities • High protein (albumin) o PS + DR in albumin medium o IgG detection at 37C o Detects Rh incompatibilities • AHG Technique o PS + DR in AHG medium o IgG detection at 37C o Detects Duffy, Kidd, Kell antibodies • Broad Spectrum Technique o IgM and IgG are detected REPORTING OF RESULTS • A compatible crossmatch is indicated by absence of agglutination and/or hemolysis at any stage of the crossmatch • The absence of agglutination indicates that the patient has no demonstrable antibodies with a specificity for any antigen on donor’s RBCs • Major: Px ab + Donor ag • Minor: Px ag + Donor ab • Sample 1: o Major: Neg o Minor: Neg o Release the blood to the ward (compatible) • Sample 2: o Major: Neg o Minor: Pos o Release the blood as packed RBCs (remove plasma) • Sample 3: o Major: Pos o Minor: Neg o Do not release the blood to the ward • Sample 4: o Major: Pos o Minor: Pos o Never release the blood • Sample 5: o Patient: Type O ▪ Antigen: H ▪ Antibody: anti-A, anti-B o Donor: Type B ▪ Antigen: B, H ▪ Antibody: anti-A o Major Crossmatch (PS + DR) ▪ PS = Anti-A, Anti-B ▪ DR = B, H ▪ PS Anti-B will react with DR B = (+) agglutination ▪ Incompatible for major crossmatch o Minor crossmatch (PR + DS) ▪ PR = H ▪ DS = Anti-A ▪ No ag will react w/ ab = (-) agglutination ▪ Compatible for minor crossmatch o Reporting: ▪ Major: Incompatible ▪ Minor: Compatible ▪ Do not release the unit
• Sample 6: o Patient: Type A ▪ Antigen: A, H ▪ Antibody: Anti-B o Donor: Type AB ▪ Antigen: A, B, H ▪ Antibody: No ab o Major Crossmatch (PS + DR) ▪ PS = Anti-B ▪ DR = A, B, H ▪ PS Anti B react with DR B = (+) agglutination ▪ Incompatible for major crossmatch o Minor crossmatch (PR + DS) ▪ PR = A, H ▪ DS = None” ▪ No ag react with ab = (-) agglutination ▪ Compatible for minor crossmatch o Reporting: ▪ Major: Incompatible ▪ Minor: Compatible ▪ Do not release the unit INCOMPATIBLE CROSSMATCHES • (from Quick Review Cards for MLS by V.D. Polansky) Reactions Possible Cause Resolution Neg ab screen, Incompatible IS Xmatch ABO incompatibility Retype donor and recipient. Xmatch with ABO compatible donor 1 ab screening cell and 1 donor pos in AHG Alloantibody Identify ab. Xmatch units neg for corresponding ag Ab screening cells and all donors except 1 neg at 37degC and in AHG. 1 donor pos in AHG only. Positive DAT on donor Perform DAT on unity (if positive, return to collecting facility) Ab screening cells, donors and AC positive in AHG Warm autoantibody Not to transfuse. If unavoidable, find “least incompatible unit” Ab Screening cells, donors and AC positive at 37degC, neg AHG Rouleaux Saline replacement technique • SUGGESTED ABO GROUP SELECTION ORDER FOR TRANSFUSION OF RBCS Recipient ABO Group 1st Choice 2nd Choice 3rd Choice 4th Choice AB AB A B O A A O B B O O O • Type specific - the priority • Universal donor - “type O” • Universal recipient - “type AB” ANTIBODY IDENTIFICATION • o Positive → ab identification pane o Negative → crossmatching • Interpretation of the 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 ▪ Positive - presence of autoantibody ▪ Negative - presence of alloantibody o Strength of reaction ▪ 3+/4+ - true agglutination ▪ Ex. 3+ IAT = IgG CS ab ANTIBODY IDENTIFICATION Reactions Possible interpretation Same strength in 1 phase only Single antibody Varying strength Multiple antibodies, antibodies exhibiting dosage In different phases Combination of warm & cold antibodies (IgM & IgG) All cells in AHG, AC negative Warm-reacting alloantibody, high frequency antigen All cells in AHG, AC positive Warm autoantibodies All cells at 37ddegC, neg AHG, AC positive Rouleaux formation IMPORTANT BLOOD GROUP ANTIGEN REACTIONS • Antibody panel - 10-11 cells • High frequency: almost in all cell = Jsb, Kpp (can be ruled out in most situations) • Usual reactions o 37degC or AHG - Rh o AHG only - Kell, Kidd, Duffy, S/s o Cold/IS or RT - Lewis, MN, P o Cold-reacting: Lea, Leb, MN, P GENERAL PROCESS (c/o Blood Bank Guy) 1. Check history - Anti-D in pregnancy (HDN), Bacterial infection (antibiotic induced warm autoab) viral illness - (anti I or i, cold reacting), recent transfusion o Note: Consider also race: Asians are D+ majority, African Americans lack Duffy ag, Whites lack high frequency antigens 2. Check autocontrol. If negative, proceed. Positive, autoabs 3. Look at general pattern. (IS/37/AHG) 4. Look at what’s not there (cross-outs) 5. Look at what is there.
6. Use special techniques as necessary 7. Ensure statistical significance ANTIBODY PANEL INTERPRETATION Thermal Characteristic Patter on Reactivity AC Interpretation Stronger at cold and weaker at warm temp One of few cells positive Negative Consider cold antibody such as MN, P, Le, etc. Stronger at cold & weaker at warm temp All cells positive Negative Consider Vel, Tj, etc. Stronger at cold & weaker at warm temp All cells positive Positive Cold autoantibody such as anti-I Negative in cold & positive at warmer temp One or few cells positive Negative Consider clinically significant alloantibody such as Rh, Kell, Duffy, Kidd, Ss, etc. Negative in cold & positive at warmer temp All cells positive Negative Consider alloantibody to high frequency antigen KP, k, Lu, Js, Lan, Ge, At, U, etc. Negative in cold & positive at warmer temp All cells positive Positive Consider autoantibody with or without alloantibody • Single alloantibody, IgG ab • Anti-D antibody • Alloantibody, IgG antibody • Anti-Kidd (anti-Jkb) = IgG • Alloantibody, IgG Antibody • Anti-Kell (anti-K) ANTIGLOBULIN TEST (COOMB’S TEST) INTRODUCTION • The antiglobulin test, which is also referred to as the anti-human globulin test (AHG) or the Coombs test, is the cornerstone of detecting clinically significant unexpected antibodies that have coated cells either in vivo or in vitro • The test aims to detect globulins (IgG) immunologically bound to red cells of a blood sample • The test involves the principle that if human antibody (a globulin) is coated onto red cells, its presence there can be detected by the reaction of the antiglobulin serum with the anti-body coated cells which are thus agglutinated. The antiglobulin reagent is usually prepared in rabbits by the injection of human globulin either as serum or as purified protein fractions. • Performance of the indirect test requires that the serum to be used is fresh to provide a source of complement; therefore, the serum must not be inactivated. In event that this is not possible, an equal volume of group compatible fresh serum must be added to the serum being tested. PRINCIPLE AND MATERIALS IN COOMB'S TEST • Principle of Antiglobulin Test o Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged. These cells are said to be sensitized with IgG or complement. • There are two types of antiglobulin tests: o Direct Antiglobulin Test (DAT) - Detects antibodies or complement coating patient's cells in vivo. o Indirect Antiglobulin Test (IAT) - Uses a 37oC incubation step so antibodies in serum can react with antigens on cells in vitro, After washing the cells antiglobulin reagent is used to detect antibody coating of cells. • Materials o Test tubes 10 or 12 x 75 mm o Serofuge o Anti-human serum o Water bath o Magnifying mirror or microscope o Cord red cells/red cells o Patient’s serum DIRECT ANTIGLOBULIN TEST • Principle : This technique is used to detect presence of antibody coated onto red cells in vivo, as it occurs in hemolytic disease of the newborn and in acquired hemolytic anemia.
• Procedure : 1. Wash the cord red cells or those red cells suspected of being coated with autoantibody three to four times in large volumes of saline. 2. Decant completely at the end of the last washing. 3. Add 2 drops of anti-human serum to the sedimented cells remaining. 4. Mix well and centrifuge at 1000-2000 r.p.m. for 1 minute. 5. Examine for agglutination. If desired, readings may be confirmed with the aid of a small hand lens or magnifying mirror or the LPO of a microscope. INDIRECT ANTIGLOBULIN TEST • Principle : This technique is used to detect the presence of antibodies in sera or, by using unknown antisera, to demonstrate the presence of antigens not detectable by other means, e.g. D ᵁ • Procedure : 1. Prepare a 2 % saline suspension of test cells containing the appropriate antigen. 2. Add 2 drops of suspension of cells to a small test tube. 3. Add 2 drops of the serum to be tested. 4. Incubate in the water bath at 37°C for 15-50 minutes. 5. Remove the tube from the water bath and wash three times with large volumes of saline; decant completely after the last washing. 6. Add 2 drops of anti-human serum and mix well 7. Centrifuge at 1000-2000 r.p.m. for 1 minute. 8. Examine for agglutination. If desired, readings may be confirmed with the aid of a small hand lens or magnifying mirror or the LPO of a microscope. ANTIBODY TESTING INTRODUCTION • The test aims to detect the presence of clinically significant antibodies other than anti-A and anti-B in the patient’s/donor’s serum. • The occurrence of isoagglutinins, anti-A and anti-B, in the serum of individual is a normal finding. However, the presence of antibodies to other blood group antigens is usually the result of immunization and the detection of these antibodies to other blood group antibodies is of obvious importance. This is concern to the obstetrical patient in whom, detection and identification of the antibody prior to delivery allows adequate time for preparations to be made for the possible transfusion of the newborn infant. The second instance in which sera are tested for atypical antibodies is in the case of crossmatch or the investigation of a transfusion reaction. In either case, the detection of the antibody is especially important so that only compatible blood will be selected for the patient. Also, the screening of sera from blood donors for the presence of antibodies is increasingly utilized instead of a minor crossmatch. This donor-screening can be done at the same time as the other routine tests are performed on the donor blood and, in this way, valuable time required for crossmatch is held at a minimum. • Bovine Albumin, 22% solution, is an ideal suspending medium for a variety of serological tests that are performed in the blood bank. The primary use of this medium is in the detection of blood group antibodies, as well as in the compatibility tests. Once antibodies have been detected, 22% Bovine Albumin may be used in the titration of those antibodies. In addition, this solution is used as a control of possible error in Rh typing. • Bovine Albumin, 22% solution, is prepared by a unique, patented manufacturing process. The final product is adjusted to a pH of 7.5; the salt and protein concentrations are also adjusted within a narrow range. In addition, a proper ration of alpha globulin to albumin is maintained to provide optimal avidity. • When Bovine Albumin is used in conjunction with the antiglobulin (Coombs) test it will markedly enhance reactions in which antibodies of other blood group systems are involved. • Rh antibodies are generally referred to either as (1) saline agglutinins, which agglutinate red cells in saline suspension; and (2) albumin agglutinins, which do not agglutinate saline suspended red cells, but will agglutinate the cells when they are suspended in albumin. Antibody detection test – either for Rh or other atypical blood group antibodies are conveniently performed by employing SELECTOGEN Reagent Red Blood Cells (Human). ANTIBODY TESTING PROCEDURES MATERIALS • Test tube • Serofuge • Waterbath • Selectogen Reagent Red Cell (Human) • Saline • Bovine Albumin, 22% • Anti-Human serum • Patient’s serum PROCEDURE • Part I. 1. Add two drops of the serum to be tested to each of 4 test tubes. 2. Add 1 drop of unwashed SELECTOGEN1 to 2 of the test tubes approximately labeled; add 1 drop of SELECTOGEN II to other 2 tubes. (For example, the test tubes might be labeled – using “S” for saline and “A” for albumin – as I-S, I-A, II-A,II-S). 3. Add 2 drops of Bovine Albumin, 22% solution to those labeled “A” 4. Centrifuge for 1 minute at 1000-2000 r.p.m. Examine all tubes macroscopically for agglutination or hemolysis.
• Part II. 1. Mix all tubes and incubate for 15 minutes at 37°C. 2. Remove all tubes from waterbath. 3. Centrifuge at 1000-2000 r.p.m. for 1 minute. Examine macroscopically for agglutination. If there is no agglutination or hemolysis in the “Saline” tubes, they may be discarded. • Part III. 1. The cell- serum mixtures in the “Albumin” tubes are washed thoroughly with large volumes of saline. Perform this washing for a minimum of 3 times. Decant the saline completely after the last washing. 2. Add 2 drops of anti-human serum to the sedimented cells and mix well. 3. Centrifuge immediately for 1 minute at 1000-2000 r.p.m. Examine for agglutination macroscopically INTERPRETATION OF RESULTS • Part I. o Many of the more strongly Rh and Hr antibodies including those that prozone – will be detected by immediate centrifugation. Certain other antibodies particularly in the Lewis System (anti-Le ͣ, anti-Leb) may be detected at lower temperatures, e.g., anti-M, anti-P, etc., are of little clinical significance but may also be detected in this place. • Part II. o The more weakly reactive anti-Rh and anti-Hr are detected best with albumin suspended red cells and will be revealed in Part II. These, as well as the stronger Rh and Hr antibodies detected in Part I, are the most frequent cause of hemolytic disease of the newborn as well as hemolytic transfusion reactions. • Part III. o The indirect antiglobulin (Coombs) test will detect anti-Kell, anti-Duffy, anti-Kidd and antibodies of other blood group systems that are seldom revealed by other methods. Such antibodies are frequently the cause of transfusion reactions and may be involved in hemolytic disease of the newborn. • Note: This procedure detects only the presence of a blood group antibody. For identification a similar method is followed using IDENTIGEN reagent. • How do you record your own reading in the laboratory? Screening Cells (Pooled O Cells) Immediate Spin 37°C 15’ Coombs Antibody Screening Vs Patient’s serum CROSS MATCHING CROSSMATCHING • Determine compatibility of blood unit from the donor with the recipient • Prevent transfusion reactions • 2 procedures: o Major Crossmatch (PSDR) o Minor Crossmatch (DSPR) STEPS IN PRE-TRANSFUSION TESTING • Request for transfusion • Identification of transfusion recipient and blood specimen collected • Testing of transfusion recipient’s blood specimen: o Blood specimen acceptability o ABO group and Rh type o Antibody detection testing o Antibody identification o Comparison of current and previous test results • Donor RBC unit testing: o ABO group confirmation and Rh type confirmation for 5.12 and Rh-negative RBC units • Donor red cell unit selection: o Selection of components of ABO group and Rh type that are compatible with the transfusion recipient and with any unexpected allogeneic antibodies • Compatibility testing (crossmatch): o Serologic o Computer or electronic • Labeling of blood or blood components with the recipient’s identifying information and issue CLINICAL SIGNIFICANCE OF 37C-REACTIVE ANTIBODIES USUALLY* VERY UNUSUAL (IF EVER)† SOMETIMES • ABO • Rh • Kidd • Duffy • S, s, U • P • Bg (HLA) • Ch/Rg (complement C4) • Le b • JMH • Xg a • Cartwright (e.g., Yt a )‡ • Lutheran (e.g., Lu b +)‡ • Gerbich‡ • Dombrock‡ • M, N‡ • Lea • Vel • LW • Ii • H • Ata • Inb • Mia • Csa SUGGESTED ABO GROUP SELECTION ORDER FOR TRANSFUSION OF RBCS Recipient Abo Group 1st Choice 2nd Choice 3rd Choice 4th Choice AB AB A B O A A O B B O O O
CROSSMATCH INCOMPATIBILITIES • Incorrect ABO grouping of patient and donor • Alloantibody in the patient’s serum reacting with donor’s RBC • Autoantibody on patient’s serum • Protein/antibody sensitization of donor’s RBC • Other abnormalities in patient’s serum • Contamination PRETRANSFUSION TESTING IN SPECIAL CASES • Emergency • Non-blood group specific blood • Plasma products • Intrauterine transfusions • Neonatal transfusions (0-4 months old) • Massive transfusions (8-10 units in less than 24 hrs or 4-5 units in 1hr) • Prolonged clotting time • Preoperative autologous blood NEONATAL TRANSFUSION • Compatibility Tests for Infants Once per Admission o Routine o ABO o Rh o Antibody screen ▪ Using maternal serum or ▪ Using infant’s serum, especially when: ▪ No maternal specimen is available ▪ Mother has clinically insignificant antibodies or ▪ Using infant’s eluate o Additional ▪ IAT using infant serum and A1 or B cells • Cells can be reagent or donor (i.e., major crossmatch) • Must be done if non-group O cells will be transfused • Antigen typing donor unit • While infant antibody screen is positive • Donor units must lack antigen corresponding to antibody o Every 3 Days ▪ Same tests as above when: • ABO- or Rh-incompatible units are transfused or • Unexpected antibodies are demonstrating via antibody screen PROCEDURE • Prepare a 2- 5% cell suspension of donor’s red cell • Place 2 drops of recipient’s serum in each of the two labeled tubes • Add 2 drops of 2- 5% of donor’s RCS in each tube • Place 2 drops of 22% BSA in one tube. Do not add on the other. • Centrifuge for 1 minute. Examine for agglutination/hemolysis • Incubate tubes at 37 degrees Celsius for 15 minutes • Remove the tube with albumin and centrifuge for 1 minute. • Examine for agglutination/hemolysis. • The contents of the second tube are washed 3-4 times in large volumes of saline. At the end of the last washing, decant saline completely. • Add 2 drops of AHG to the sedimented cells. Mix and centrifuge for 1 minute. • Examine for agglutination/hemolysis. Phase Interpretation Protein Phase +/- Thermo Phase +/- AHG Phase +/- ANTIBODY TESTING ANTIBODY SCREENING • Test to detect clinically significant IgG • antibody outside the ABO system • Detect the presence of “unexpected”/ “unusual” antibodies (aka. Indirect Coomb’s Test) • Unexpected antibodies o Result of immunization/prior exposure to RBC with such antigens o May also be passively acquired o May or may not be clinically significant o IgG or IgM in nature • Screen the donor’s plasma (similar function with MINOR crossmatch) • After screening, ANTIBODY PANEL is performed to identify the specific antibody present in the sample ANTIBODY SCREENING (INDIRECT COOMB’S TEST) RBC REAGENT CELLS • Type O blood that contains the most common and clinically significant RBC antigens • Set of 2-3 red cell suspensions • ENCHANMENT REAGENTS • 22% Bovine Serum Albumin (BSA) • Low Ionic Strength Solution (LISS) • Polyethylene Glycol (PEG)
BLOOD SCREENING PROCEDURE • Part 1 (Immediate Spin) 1. Add 2 drops of the serum to be tested to each of the 4 tubes 2. Add 1 drop of unwashed SELECTOGEN 1 to 2 of the test tubes approximately labeled and 1 drop of SELECTOGEN2 to other 2 tubes. 3. 1st tube will only have saline and second will have albumin for each SELECTOGEN tubes 4. Centrifuge for 1 minute at 1000-2000 rpm. Examine all tubes macroscopically for agglutination or hemolysis o Strongly reacting Rh and Hr antibodies will react in these stage o Prozone reactions o Anti-Le (a) and Anti-Le (b) o Anti-M and Anti-P • Part 2 (Thermo) 1. Mix all tubes and incubate for 15 minutes at 37 degrees Celsius 2. Remove all tubes after 15 minutes 3. Centrifuge for 1 minute and observe for agglutination or hemolysis o More weakly reactive Rh and Hr antibodies are detected especially in albumin tube • Part 3 (AHG) 1. The cell-serum mixtures in the albumin tubes are washed thoroughly with large volumes of saline. Perform washing 3 times. Decant the saline completely after last washing. 2. Add 2 drops of AHG to sedimented cells and mix well 3. Centrifuge immediately for 1 minute and observe for agglutination o Detects Kell, Duffy and Kidd antibodies. o Other antibodies may also be detected. o Some clinically significant antibodies (HTR and HDFN) will be detected in this stage • Gel Method • • Solid Phase Adherence Method • ANTIBODY PANEL TESTING/IDENTIFICATION • Done after the antibody screening test • Identify the specific clinically significant antibody that caused agglutination in the thermo and AHG phase of the screening • Reagent Red cells: 11 to 20 RCS • Worksheets should never be interchanged with other set/lot • Optimal Phase of Reactivity for Some Common Antibodies Phase Immediate Spin (Room Temp.) 37°C Incubation Antiglobulin Phase Antibodies Cold autoantibodies (I, H, IH) Potent cold (IgM) antibodies (especially those causing hemolysis) Rh antibodies Le a , Le b Some warm antibodies, if high in titer(e.g., D, E, and K) Kell M, N Duffy P 1 Kidd Lu a S,s Lu b Xg a Immunoglobulin Class IgM Usually IgG IgM that activate complement IgG Clinically Significant No Yes Yes •