MLS Review - Blood Banking - Tests for Blood Component Compatibility

MEDICAL LABORATORY SCIENCE REVIEW • BLOOD BANKING TECHNIQUES: TESTS FOR BLOOD COMPONENT COMPATIBILITY OUTLINE • • Antibody Screening Procedure Compatibility Testing BLOOD BANKING TECHNIQUES ANTIBODY SCREENING PROCEDURE • • • • Performed to detect as many significant clinically significant antibodies outside the ABO system o IgG warm-reacting antibodies - target When the procedure was introduced, it replaced minor crossmatching in pre-transfusion testing o Current routine pre-transfusion procedures: ▪ ABO & Rh typing ▪ AB Screening ▪ Major X-match o Minor crossmatch = px red cells + donor serum o (-) Ab screening = (-) compatible o Minor crossmatch is only performed if requested Components o AHG - to target IgG o Enzyme - differentiates various types of antibodies ▪ Enzymes used: ficin, bromelain, papain, trypsin ▪ Enzymes may enhance or destroy antibodies ▪ Two types of antibodies • Antibodies enhanced by enzyme treatment (reaction grade is enhanced) • Antibodies destroyed by enzyme treatment (reaction grade is weakened/absent) Types o One-cell pool (donors) o Two cells o Three cells (recommended) o Six cells • • Procedure o Patient serum ▪ Patient serum and donor serum is tested in search of clinically significant antibodies outside of ABO system o Screening cell ▪ Group O cells with known antigens (pooled) • Type O is used to prevent anti-A, anti-B, antiAB in px sample o ABO = High titer antibodies ▪ Known red cell antigens: D, C, e, k, Fya, Jkb, Lea, P, M, N, S • Specifically react with antibody if the antibody is present in the sample ▪ Screening cells is used to detect IgG alloantibodies • Alloantibodies - antibodies of other individuals of the same species ▪ In suspect of autoantibody, patient serum is tested against patient RBC o Agglutination of patient antibody with corresponding red cell antigens ▪ (+) agglutination = positive If the antibody screening is positive, sample to proceed in antibody specificity/identification Example o Px 2 has alloantibody which must be identified ▪ To proceed with antibody identification ANTIBODY IDENTIFICATION • • • • If the Antibody Screen is reactive/positive, the antibody specificity must be determined. So safe blood can be administered to the Recipient. 11 reagent panel cell are to be used for identification Example 7 ADSORPTION & ELUTION TECHNIQUES • • • • • • Principle: Rule in rule out procedure o Eliminate antigens that not in the target Rules o (+) = presence of antigen o (0) = absence of antigen Parts o Left side - for alloantibodies ▪ 1-10 alloantibodies ▪ 11th cell - autocontrol (AC) o Upper part - red cell antigens ▪ Immediate spin at room temp (IS) - IgM detection • Expected to be negative always • Always after IgG • (+) IS = insignificant ▪ AHG - IgG Detection ▪ CC - control cell/check cell/Coomb’s cell • Tested when AHG is negative • To confirm/validate negative AHG result Other techniques may be used to eliminate clinically insignificant reactions and make identification of significant antibodies easier. o Use of AET, DTT, and ZZAP which inactivates some antigens especially Kell. ▪ Anti-K - commonly encountered • To eliminate when not target, Kell antigen must be inactivated to prevent reaction by AET and ZZAP reagents o Prewarm procedure. Clinically insignificant cold antibodies may be removed by this technique. Patient serum, reagent red cells and enhancement medium can be warmed separately at 37°C for 5-10 minutes prior to mixing ▪ IgM are inactivated with prewarming ▪ Prewarming allows reaction of IgG w/ RBCs o Use of sulfhydryl or thiol reagents (DTT and 2-ME) which denature IgM antibodies by breaking/denature disulfide bonds. ▪ Denature IgM can no longer react with RBCs o Use of adsorption and elution techniques to remove unwanted antibodies such as cold or warm autoantibodies, or to help resolve multiple antibodies o Another technique for facilitating antibody identification is Neutralization • Adsorption o Used to remove unwanted antibodies from serum o Antibodies are removed from the sample o If an autoantibody such as I, H, or IH are defined, it can be adsorbed onto the patient’s enzyme pretreated cells at 4ºC. Rabbit cells may also be used as adsorbents for anti-I since they are rich in I antigen. Elution o Antibodies are detached from the antigen o Used to dissociate IgG Abs form sensitized red cells o The recovered antibody, eluate, can be tested like serum to determine the antibody’s specificity o Techniques include heat, freeze-thaw process, use of organic solvent, acid eluates, or by using ZZAP or o chloroquine diphosphate o ZZAP- mixture of DTT and papain that is used to remove Ab from sensitized red cells and to enzyme treat them at the same time o Chloroquine diphosphate- reagent used to remove IgG Abs from the surface os sensitized cells; inactivates Bg antigens Types Of Elution Techniques: o First Generation ▪ Landsteiner-Miller Heat ▪ Lui-Freeze-Thaw o Second Generation ▪ Use of Organic solvents (chloroform, ether) o Third Generation ▪ Commonly used ▪ Uses Non Hazardous Chemical Agents: ▪ “Acid Elution” o o Eluate - contains the dissociated antibodies Note: the recovered antibody, the eluate can be tested like serum to determine the antibody specificity o RBC destruction is the common end result NEUTRALIZATION • • Commercial substances are available to neutralize or to inhibit reactivity of some antibodies. Targets clinically insignificant antibodies or unwanted antibodies present in the serum so that the serum is concentrated on target antibodies 8 • Sources of Substances for Neutralization of Antibodies: o Hydatid cyst fluid – anti-P1 ▪ From E. granulosus o Plasma or serum w/ Le subs. – anti-Lea & anti-Leb o Pooled serum or plasma – anti-Chido, anti-Rogers o Urine – anti-Sda (Sid) ▪ Tamm-Horsfall protein - part of urine that neutralize Sda o Saliva of “secretors” – anti-ABH o Colostrum/Human milk – anti-I • • CROSSMATCHING • • COMPATIBILITY TESTING • • • Aka Crossmatching Pre-transfusion procedure composed of a series of tests to ensure the safety of the recipient during blood transfusion Performed to select the appropriate donor blood unit for patient transfusion • COLLECTION AND PREPARATION OF SAMPLES • • • • Patient Identification. o Patient ID is very critical o Identify everything that needs to be identified before phlebotomy and performing any procedure o Identify correctly when release blood donor unit ▪ After compatible crossmatching, the donor blood will be named to the receiving patient o Crossmatch is a very important and critical Collection. Serum is the preferred specimen for compatibility testing. Hemolysis should be avoided. o Why SERUM and NOT PLASMA? o Plasma may cause small fibrin clots to form which may be difficult to distinguish from true agglutination. ▪ May cause spontaneous agglutination o Plasma may inactivate complement so that antibodies may not be detected. ▪ Anticoagulant may be anti-complementary Age of Specimen. The freshest sample possible should be used for compatibility testing. Specimens must be less than 3 days old if the patient has been transfused or pregnant within the past 3 months. o Age of specimen can correctly represent the current immunologic status of the patient Sample Storage. The AABB requires that patient samples must be stored between ref temp. (1-6ºC) for at least 7 days after transfusion. o Storage of sample after transfusion allows testing in the event of sudden or delayed transfusion reactions o Crossmatch samples are used in the investigation of transfusion reactions ▪ Some TRs may be very delayed Rh Typing o If Rh type of the recipient cannot be determined and transfusion is essential, Rh(-) blood should be given. Safest blood to provide is O Rh (-) packed RBC • • Major X-Match: Donor’s cells + Recipient’s serum o PS+DR = P ab + D ag o Part of pre-transfusion testing Minor X-Match: Donor’s serum + Recipient’s cells o PR+DC = P ag + D ab o No longer part of routine pre-transfusion testing o Replaced by antibody screening Purpose: o Final check of ABO compatibility between patient and donor to prevent transfusion reaction. o Detects presence of antibody in patient’s serum that will react to donor’s RBC that is not detected n antibody screen. 4 Crossmatching Techniques: o Saline Technique: px serum and donor red cells are tested in saline medium ▪ Detects IgM antibodies o High-Protein/Albumin Technique: px serum and donor red cells are tested in high protein media ▪ Detects IgG antibodies (eg. Rh abs) o AHG Technique: px serum and donor red cells are tested in AHG medium ▪ Detects non-agglutinating IgG antibodies o Broadspectrum Technique: composed of 3 steps ▪ Identifies IgG and IgM ▪ Usually performed in BB section to cover all abs ▪ 3 steps: IS (IgM) → Thermophase (IgG) → AHG 3 Phases of Crossmatching o Immediate Spin in saline at RT - detects IgM o Thermophase/37ºC incubation for 30 minutes with enhancement medium (e.g. albumin, LISS, PEG) – Detects IgG ▪ Enhancement medium - shortens incubation period and to enhance sensitivity through potentiators o AHG Phase after washing incubated cells with saline. o Check cells/Coombs control cells (IgG sensitized cells) should be added to tubes that demonstrate no agglutination. ▪ Performed when AHG tested negative ▪ To validate result ▪ (+) check cell for agglutination = (-) negative ▪ For results to be considered valid, agglutination must occur. ▪ AHG Phase COMPATIBILITY TESTING PROTOCOLS • ABO Grouping. Most critical pretransfusion serologic test. o ABO - most important o Included in crossmatching by default o If the patient’s ABO group cannot be satisfactorily determined and immediate transfusion is essential, group O packed red cells should be utilized. ▪ Applied for emergency situation requiring immediate transfusion 9 ▪ ▪ ▪ • • • Invalid: neg AHG, neg check cell Possible cause of invalid check cell • Failure of medtech to wash cells o May lead to unbound abs actively neutralizing reagent AHG • Reagent is already deteriorating or nonreacting ABO Crossmatching • Reporting of Results. o 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 RBC o Case 1 ▪ Major - Neg (compatible) ▪ Minor - Neg (compatible) ▪ Release donor blood unit o Case 2 ▪ Major - Neg (compatible) ▪ Minor - Pos (incompatible) ▪ Release donor unit as packed RBCs (remove the plasma) o Case 3 ▪ Major - Pos (incompatible) ▪ Minor - Neg (compatible) ▪ Do not release the blood to the ward due to risk to transfusion reaction o Case 4 ▪ Major - Pos (incompatible) ▪ Minor - Pos (incompatible) ▪ Never release the blood • Troubleshooting Incompatible Crossmatches o O - universal donor to RBCs ▪ Universal recipient of blood o AB - universal recipient of RBCs ▪ Universal donor of plasma Grading of Results o Presence of positive reaction indicates incompatibility o Sometimes the notations “S” for strong or “W” for weak are used to denot slight variations between reaction grades. For example, a reaction between 1+ and 2+ might be noted as 1+S or 2+w Grade % Ag Score Description of Appearance 4+ 100% 12 One solid clump, no free cells, clear supernatant 3+ 75% 10 Several large clumps, clear supernatant 2+ 50% 8 Several/Many medium-sized clumps, cloudy red supernatant 1+ 25% 5 Numerous small-sized clumps, cloudy red supernatant +/<25% 3 Numerous very small clumps easily (weak) dispersed, cloudy red supernatant + <25% 1 Appears negative macroscopically, but agglutination visible microscopically 0 0 0 Negative, no agglutination reaction seen macroscopically H 0 Complete hemolysis, no intact RBCs remaining PH 0 Partial hemolysis, some RBCs still intact but hemolysis visible in supernatant mf 0 Mixed field-mixtures of agglutinated and agglutinated cells • Ab Screen AC (-) (-) Major X-match (+) (+) (+) (+) (+) (-) (+) Possible Problem • ABO/Rh typing error • Donor unit w/ (+) DAT • Patient w/ low incidence Ab • Patient alloantibody • Patient autoantibody • Rouleaux Which of the following would likely be responsible for an incompatible major crossmatch? a. Recipient’s red cells possess a low-frequency antigen (minor x-match) b. Anti-K (Kell) antibody in donor serum (minor x-match) c. Recipients red cells are polyagglutinable (minor xmatch) d. Donor red cells have a positive direct antiglobulin test (major x-match) 10 THE FUTURE OF COMPATIBILITY TESTING • • • • • Red cell/Blood substitutes Biochemical modification of non-Oblood Galvanic biosensor – energy measured Dipstick method oftyping o (ex. Eldoncard Blood typing kit) Dry plate method BLOOD SUBSTITUTES • • • Substances that are able to carry oxygen in the absence of intact red cell Stroma-free Hb solutions/Hemoglobin-Based Oxygen Carriers (HBOCs) o Stroma - cytoskeleton of cell membrane ▪ Absence of this means there is no intact cell o No intact cells available to carry oxygen o These substnaces are capable of carrying oxygen without the actual RBCs o e.g. PHP, PEG-Hb, Hemolink, Polyheme, HemAssist, Hemopure, Optro o Advantages of SFHS ▪ Long shelf life ▪ Very stable ▪ Not immunogenic ▪ No reqt for bld typng procedures o Disadvantages of SFHS ▪ Short intravascular halflife ▪ Possible toxicity ▪ High O2 affinity ▪ High oncotic effect Perfluorochemicals (PFCs) o examples: Fluosol-DA-20, Oxygent o Excellent gas (O2 and CO2) solvents o Can dissolve gas better than donor whole blood unit o Advantages of PFCs ▪ Biologic inertness ▪ Lack of immunogenicity ▪ Easily synthesized o Disadvantages of PFCs ▪ Adverse clinical effects ▪ High O2 affinity ▪ Retention in tissues 11