DAT (Direct Antiglobulin Test) PDF

18 Multiple Questions CollceraiDg Alltibody war blood X...

18 Multiple Questions CollceraiDg Alltibody war blood X r ~ l-r':.--1 Characteristics Alltibody Identification ~ AddAHG/ /□"- Including Select Cell PaJJel Sample Used ~ &z] for DAT II II no agglutination agglutination DAT (DIRECT ANTIGLOBUL/N TEST) Neg DAT Pos DAT 1. Antiglobulin added to 3-4 times washed cells 2. If cells coated in vivo, antiglobulin will AHG REAGENTS r eact with the IgG antibody and/or 1. Polycolonal- derived from innoculated complement (depending on ty pe of animals targeting different epitopes AHG used) 2. Monoclonal - derived from hybridoma 3. EDTA sampJe is optimum; EDTA targeting a single epitope chelates Ca++ preventing complement activation by plama antibody (causes a 3. Polyspecific- mixture of antibody to false positive DAT) l gG and complement components ( usually C3b and C3d) 4. Add check cells to all negative antiglobulin tests- confirms negatives 4. Monospecific- antibody to either lgG or with lgG coated or complement coated a specific complement component check cells (proves AHG added and not ( usually C3h or C3d) neutralized due to insuffici ent washing) 5. The polysp ecific and monspecific can be blended to en.sure detection of all epitopes Positive DAT TESTS WITH AHG Immune Hemolytic Anemias 1. P erform DAT with polyspecific to Protein Coating scr een and monospecific to characterize Condition Red Cell the globulin as lgG or complement. AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA) Warm Autoantibodies (WAIHA) lgG and/or Complement 2. Perform IAT with monosp ecific a nti-. Cold Hemagglutinin Disease (CHO) Complement I gG to avoid cold, complement-binding, clinically insignificant antibodies. Mixed Type AIHA lgG and Complement. Paroxysmal Cold Hemoglobinuria (PCH) Complement 3. Use IgG coated or complement coated check cells to confirm negative DRUG INDUCED HEMOLYTIC ANEMIA {DIHA) l2G and/or Comolement antiglobulin tests; this confirms AHG added and not neutralized (insufficient. ALLOIMMUNE HEMOLYTIC ANEMIA Hemolytic Disease of the Newborn lgG washing to r emoval serum globulins. Transfusion Reaction lgG prior to addition of AHG)  19 ELUTIONS NEUTRALIZATION (INHIBITION) TESTS 1. Break antigen-antil)ody bond to release 1. Soluble antigen can bind with antibody the antibody into solution (eluate) to inhibit a r eaction with RBCs; allows detection of alloantibodies " masked" b y 2. Test eluate to determine antibody the following antibodies: specificity in cases of positive DAT due a. Lewis substances - in saliva to IgG antibody(ies), e.g., hemolytic b. Pl substan ce- in h ydatid cyst fluid transfusion r eactions, HDFN, and pigeon egg whites a utoimmune and drug-induced c. Sda substan ce - most abundant in hemolytic anemia urine 3. Cannot elute off complement d. Chido and Rodger s substan ces - epitopes of C4 (complem e11t) 4. T ypes a. o single method b est for all INACTIVATION antibodies 1. Sulfhydryl r eagents b. Lui freeze-thaw and heat - ABO a. AET and DTT - destroys or antibodies weakens Kell system antigens c. Acid or organic solvent methods b. ZZAP : enzyme+ DTT - destroys work b est for auto- and allo- warm Kell antigen s and those antigens r eacting antibodies destroyed by enzymes (M,N, S, s, 5. Last wash control Fya , and Fyh); can r emove immunoglobulins and complement a. Prior to elution, r ed cells coated from RBCs to enhance adsorption with antibody should be thorou ghly c. DTT and 2-ME - destroys or washed to remove an y r esidual diminishes activity of IgM serum (antibody) antibodies- cleaves disulfide b onds b. T est " last wash" (s upernatant) before performing elution or in 2. Chloroquine diphosphate parallel with eluate a. Removes IgG from RBCs ( does n ot c. "Last wash" should show NO r em ove compl em ent) r ea ctivity with r eagent cells b. With IgG r emoved, cells can b e d. Positive test results using " last phenotyped wash" indicate serum antibody c. May cause some denaturation of Rh contamination of supernatant; if antigen s performed before elution, wash again; if performed in pa rallel, test 3. Acid glycine/EDTA invalid: r epeat a. Dissocia tes antibodies from RB Cs b. Destroys Kell system antigens ADSORPTION 1. Used to: a. Separate multiple antibodies b. Remove autoantibody - r eveal alloantibody " m asked " b y autoantibody c. Confirm antigen existence on RBC d. Confirm antibody specificity Interpret Eluate Results 2. Autoadsorption (patients own serum and cells) can be used for patients not recently transfused Recognize Problems when 3. Allogeneic adsorptions (patients serum Testing Eluate Last Wuh and other cells) can be u sed on patients r ecently transfused 20 Additional Technologies to Detect 2. Positive reactions have cells adhering Antigen-Antibody Reactions to sides of microwell plate COLUMN AGGLUTINATION ( GEL TESTING) 3. Negative reactions have RBC pellet at bottom of plate since no attachment 1. Perform serological wor-k in special chamber with controlled centrifugation a. Gel acts as a filter - unagglutinated cells pass through gel; agglutinated cells cannot. ❖ Negative reactions, the cells settle at the bottom; ❖ Positive reactions, the cells remain on the top or only partially travel through the gel depending on agglutinate size NEGATIVE STRONG POSmVE WEAKPOSmVE b. Cells are phenotyped when reagent Copyrighted material used with the pem,lsSJOn of lmmucor, Inc. antisera (e.g. , anti-A, -B, -D, etc.) Copying or repnnting without permlssit>n is express/)' forbidden, is in the gel; cells agglutinate on exposure to antibody during Pretransfusion Testing centrifugation 1. Sample accompanying r equest c. Antiserum/serum/plasma and cells a. Serwn or plasma from recipient are added in the upper chamber; labeled with 2 unique identifiers sensitized cells agglutinated by anti- b. Must have system to identify IgG in the gel and cannot pass -IAT phlebotomist and the date of d. DAT by gel- no washing needed; collection only RBCs go through gel and c. Retain sample and an RBC segment sensitized cells agglutinate when for 7 days after transfusion exposed to anti-IgG in the gel d. Patients transfused with allogeneic RBCs or pregnant within the last 3 months or transfusion history unknown, a n ew sample must be drawn within 3 days of transfusion. Day of draw is considered day 0. 2. Tests a. ABO group and Rh type b. Rh typing; weak D testing confirmation not required for patients c. Antibody screens d. Crossmatch e. Autocontrol not r equired 4+ s+ 2+ 1+ 0 Reprinted wdh Permission, copyright Ortl,o.Clinlcal Diagooslks. Inc. 2014 3. Compare current r esults with prior testing SOLID PHASE a. ABO group and Rh type b. Any prior difficulties in blood 1. Antibody or antigen - fixed to a typing microwell plate a. Antibody fixed to plate ❖ RBCs with the antigen are added ❖ Antigens adhere to antibody on sides of micrnplate b. Antigen s adhere ❖ Plasma with the antibody is added Evaluate Results of ❖ Antibody will adhere to antigen on Incompatible Crossmatcb sides ❖ Wash; add check cells to attach to antihody  21 r eacting cells with commercial c. A list of any clinically significant preparations of the antibody antibodies d. QC rarely used antisera on day of use d. Any history of transfusion reactions ❖ Positive contJ"ol: heterozygous e. Any special transfusion cell (e.g., anti-K tested with a Kk requirements cell rather than a KK cell) ❖ Negative control: cell without 4. Crossmatch antigen (e.g. , anti-K tested with a a. Patient serum mixed with donor kk cell) RBCs b. Observe for agglutination or hemolysis c. Demonstrate ABO compatibility d. Carry through to 37C incubation with AHG if current antibody screen positive or prior history of ' 1, clinically significant antibodies Select Appropriate Controls : for Anti&ea TypiDg 5. Immediate spin or electronic ( computer) crossmatch if current antibody screen negative and no prior When ABO Identical is NOT available. antibody history 1. For RBCs a. Decide what antihody(ies) is (are) in a. Only a test for ABO compatibility is the patient's plasma required b. Transfused cells must lack b. Computer only ABO incompatibility corresponding antigens check can he used if: c. 0 rbcs can be given to any ❖ Computer system is validated alternative blood group- A, B or AB ❖ Second ABO check on another d. AB can receive A, B and O RBCs current sample; checking previous e. D positive can receive D positive or records; or retesting same sample D negative RBCs ❖ System can verify correct data f. D negative should only receive D entry a11d contains logi.c to alert if negative RB Cs. In an emergency, D discrepancies occur between positive can be administered if no D donor label and confirmatory tests negative is available. Rhig should and between donor unit label and follow especially if the recipient is a recipient ABO type woman of child bearing age. 6. Antigen typing 2. For Plasma a. Patients with significant antibodies a. Decide what antigen(s) is (are) on receive antigen negative units that the patient's RBCs crossmatch compatible b. Transfused plasma should lack the b. Probability of finding antigen corresponding antibody(ies) negative units c. AB plasma can be given to any ❖ Multiply antigen n egative alternative blood group - A, B, or 0 ( compatible) % converted to d. (;roup O can 1·eceive A,.H, or AH decimal plasma C = (70% pos; 30% neg); Jka = (75% pos; 25%neg).30 x.25 =.075 or 7.5%* * probably fmd 8 units/100 neg for C & REMEMBER! For Decisions Involving Jka Compatibility Testing Results ❖ Only need 3 units Selection of Units for 8/100 = 3/X X = 300/8 = 37.5* Transfusion when ABO Identical is NOT Available: * need to screen -38 units to find 3 units C negative and Jka negative Decide what ABO antibody (ies) is (are) in the patient's plasma. c. Confirm antigen negative status by Any red cells LACKING that {those) antigen(s} will be compatible. REMEMBER! NO WHOLE BLOOD!