Immunohematology - 11 Antibody Identification

IMMUNOHEMATOLOGY ANTIBODY PANEL INTERPRETATION BLOOD BANKING PROCEDURES: ANTIBODY IDENTIFICATION Thermal Characteristic Stronger at cold and weaker at warm temp OUTLINE • Antibody Identification o Antibody Identification Reactions o Important Blood Group Antigen Reactions o General Process o Antibody Panel Interpretation o Additional Procedures in Antibody Identification o Neutralization and Inhibition o Antibody Titer Determination o Elution o ANTIBODY IDENTIFICATION ANTIBODY IDENTIFICATION REACTIONS Reactions Possible interpretation Same strength in 1 phase only Single antibody Varying strength Multiple antibodies, antibodies exhibiting dosage, ags of different strengths In different phases Combination of warm & cold antibodies (IgM & IgG), ab with wide thermal range All cells in AHG, AC negative Warm-reacting alloantibody, high frequency antigen All cells in AHG, AC positive Warm autoantibodies All cells at 37C, Rouleaux formation neg AHG, AC positive Patter on Reactivity One of few cells positive Stronger at cold & All cells weaker at warm temp positive Stronger at cold & All cells weaker at warm temp positive Negative in cold & One or few positive at warmer cells temp positive Negative in cold & positive at warmer temp All cells positive Negative in cold & positive at warmer temp All cells positive • 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 • • • • • • • • • • 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 Check autocontrol. If negative, proceed. Positive, autoabs Look at general pattern. (IS/37/AHG) Look at what’s not there (cross-outs) Look at what is there. Use special techniques as necessary Ensure statistical significance Negative Consider cold antibody such as MN, P, Le, etc. Negative Consider Vel, Tj, etc. Positive Cold autoantibody such as anti-I Negative Consider clinically significant alloantibody such as Rh, Kell, Duffy, Kidd, Ss, etc. Negative Consider alloantibody to high frequency antigen KP, k, Lu, Js, Lan, Ge, At, U, etc. Positive Consider autoantibody with or without alloantibody Serum Procedures o Hemagglutination inhibition o Adsorption o Titration o Prewarming o Saline replacement o Use of sulfhydryl reagents Red Cell Procedures o Enzyme treatment o Use of AET/DTT o Chloroquine diphosphate o Elution o ZZAP o Lectin o Cell separation techniques NEUTRALIZATION AND INHIBITION GENERAL PROCESS (c/o Blood Bank Guy) • Interpretation ADDITIONAL PROCEDURES IN ANTIBODY IDENTIFICATION IMPORTANT BLOOD GROUP ANTIGEN REACTIONS • • AC • • Other substances in the body have antigenic structures similar to RBC antigens (these can neutralize abs in serum allowing separation) Procedure: o Serum + Neutralizing agent → treated serum for ab identification Sources of fluids o Hydatid cyst fluid → anti-P1 o Plasma or serum w/ Le subs. → anti-Lea & anti-Leb o Pooled serum or plasma → anti-Chido, anti-Rogers o Urine → anti-Sda o Saliva of “secretors” → anti-ABH o Human milk → anti-I Adsorption o Used to remove unwanted antibodies from serum o If an autoantibody such as I, H, IH are defined, it can be adsorbed onto the patient’s enzyme pretreated cells at 4ºC. o Rabbit cells may also be used as adsorbents for anti-I since they are rich in I antigen. • • • • • • • • Sulfhydryl Agents o 2-Mercaptoethanol (2-ME) or Dithiothreitol (DTT) o Destroys pentameric structure of agglutinating IgM antibodies, effectively bypassing cold reactive abs to allow for detection of underlying IgG Prewarming o Keep all components at 37degC prior to testing and use monospecific IgG AHG rgt o Bypasses cold autoagglutinins or alloaagglutinins to prevent direct agglutination and/or in vitro complement activation in ab adetection Saline Replacement o For test in which patient red cells are suspended in serum suspected with rouleaux o Serum is removed and replaced with equal volume of saline Enzyme Treatment o Ficin, trypsin, papain, bromelin o Neuraminidase (or removal of sialic acid) AET and DTT o AET (2-aminoethylisothiouronium bromide) and DTT (dithiothreitol) to create RBCs negative for all antigens of the Kell blood group systems except Kx ZZAP (Cysteine-Activated Papain + DTT) o Removal of autoantibodies from patient cells provides free antigen sites for adsorption of autoantibodies from serum Chloroquine Diphosphate o Dissociates IgG from the patient RBCs with a positive DAT so that they may be typed with blood grouping reagents that require IAT Lectins and Prolectins o Sourced from animal and plant substances ANTIBODY TITER DETERMINATION • • • For semiquantitative determination of ab amount in a given serum/plasma. For human T leukocyte antigen (HTLA) abs with titer 1:64 or greater Used to measure increase in production of IgG abs during pregnancy ELUTION • • • • • Used to dissociate IgG Abs from sensitized red cells The recovered antibody, eluate, can be tested like serum to determine the antibody’s specificity Techniques include: o Heat o Freeze-thaw process o Use of organic solvent o Acid eluates o By using ZZAP or chloroquine diphosphate 3 Types of Elution Techniques o 1st Generation o 2nd Generation o 3rd Generation 1st Generation Elution Technique o Landsteiner Miller Heat ▪ RBC sensitized w/ IgG is washed using NSS or protein heated at 56C ▪ After spinning, the dissociated IgG is harvested in the eluate (liquid product of eluate technique) o • • Lui-Freeze Thaw ▪ RBC sensitized with IgG is rapidly frozen then thawed which disrupts ag-ab binding ▪ IgG will dissociate with RBC 2nd Generation Elution Technique o Use of organic solvents which decreased the surface tension of the suspending medium thus lowering the bond between antigens and antibodies o Organic solvents: ▪ Ether ▪ Xylene 3rd Generation Elution Technique o Use of non-hazardous chemical agents ▪ Citric acid ▪ Glycine ▪ CCAP