Ch 10 Detection and Identification of Antibodies PDF

Detection and Identification of Antibodies Chapter 10 Preamble  PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY.  PowerPoints DO NOT cover the details needed for the Unit exam  Each student is responsible for READING the TEXTBOO...

Detection and Identification of Antibodies Chapter 10 Preamble  PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY.  PowerPoints DO NOT cover the details needed for the Unit exam  Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES  Unit Objectives are your study guide (not this PowerPoint)  Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  Introduction  Pretransfusion Testing 1. ABO Grouping  Forward and Reverse 2. Rh Testing 3. Antibody Screening  Screening Cells I and II (III) 4. Compatibility Testing  Donor Cells with Patient’s Serum Screening Cells  WHY DO SCREENING CELLS?  DETECTION OF UNEXPECTED ANTIBODIES  Only 0.3 to 2% of the population have unexpected antibodies  Patient lacks antigen, been exposed to antigen, makes antibody  Unexpected antibodies are usually alloantibodies 2 Antibodies Sub classified by temperature of reactivity Usually an immune response, either by transfusion, pregnancy, or sometimes with certain medications Once detected – antibody identification must be performed Not always easy to identify Reagent Red Blood Cells  Test the patients’ serum or plasma against two or three reagent RBC’s – Screening Cells (SC I, II or III)  Commercially prepared Group O cell suspensions obtained  Donors phenotyped for most commonly encountered and clinically significant RBC antigens 3  Reagent Red Blood Cells  Group O is used so “Expected” anti-A and anti-B will not interfere  Common antigens present  D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, and Jkb  If the antigen is not on the RBC, the antibody would not be detected Reagent Red Blood Cells  Antigens are different on every lot  Ideal SC’s have homozygous expressions of as many antigens as possible. “Double Dose”  Which antibodies show dosage?  What is dosage? 4 Enhancement Reagents  Added to  promote antigen-antibody binding thus agglutination  Decrease incubation time  Increase sensitivity  Detect lower levels of antibody Enhancement Reagents LISS (low ionic strength saline)  Enhance antibody detection at AHG phase by increasing rate antibodies bind to RBC antigens PEG (polyethylene glycol)  Same as LISS Albumin  Reduces Zeta potential, bringing cells closer together 5 Antihuman Globulin Reagents  Promotes agglutination of RBC’s sensitized with IgG or complement  Polyspecific or monospecific?  Most labs use monospecific due to:  Interference from naturally occurring cold agglutinins  Controversy about complement – most believe these antibodies are rare and benefits of monospecific outweigh risks Coombs Control RBC’s  Check cells  Coated with human IgG antibody  Used to insure all AHG tests with negative results are not false negatives.  Free AHG in test should agglutinate the RBC’s  IF CHECK CELLS DO NOT AGGLUTINATE – TEST INVALID - REPEAT 6 Screening Cell and Antibody Panel: Methodology Label tube appropriately Add 2 drops of patient serum to each tube Add 1 drop of appropriate screening cells to each tube Centrifuge and gently resuspend and read for agglutination of hemolysis Methodology  Add 2 drops of enhancement reagent  Incubate for 15 to 30 minutes depending on manufacturer’s directions.  Centrifuge and resuspend and read for agglutination or hemolysis 7 Methodology  Wash cells by filling tubes with saline, centrifuge and remove supernatant, resuspend button and repeat three times.  This removes the unbound antibody  Remove the last drop of saline completely  Add 2 drops of AHG Methodology  Centrifuge, gently resuspend cell button and read for agglutination or hemolysis  Negative tests are read microscopically  Add 1 drop of Coombs control cells (check cells)  Centrifuge and read for agglutination 8  Panel Acronym I Immediate Spin Eat Enhancement Ice Cream Incubate Covered Centrifuge With WASH Almonds AHG Carmel Centrifuge Chocolate Chips Coombs Cells Autologous Control  Patient’s serum and Patient’s washed RBC’s  Positive auto control means  Patient has a positive DAT or  Patient has free auto antibody  Provides useful information in interpretation of positive antibody screen  Is antibody auto or alloantibody  Not required 9  Interpretation  In what phase did the reaction occur?  COLD or WARM  Is the autologous control negative or positive?  Neg = Allo; Pos = Auto  Did more than one screening cell sample react, and, if so, did they react at the same strength and phase?  Is hemolysis or mixed-field agglutination present?  Are the cells truly agglutinated, or is rouleaux present? Fig 10-4 10 Limitations  Antibody screening tests are designed to detect significant RBC antibodies  Cannot detect all antibodies Resolving Antibodies  Patient History  Reagent Red Blood Cells – Antibody Panel  Same manner as antibody screen  Expanded Screening Cells  8 to 16 Group O cell suspension  Antigen profile that lists the antigenic makeup of each RBC sample is provided and serve as worksheets 11 Antibody Panel  Profile states whether each donor cell tests positive or negative for each antigen  Specificity of antibodies in a serum sample is determined by comparing the pattern of positive and negative reactions with the antigen profile Antibody Panel Evaluation  What type of antibody do I have?  Cold Autoantibody  Cold Alloantibody  Warm Autoantibody  Warm Alloantibody 12 Evaluation of Panel Results  Evaluation should be carried out in a logical, step-by-step method  Is the Auto control negative or positive?  Neg – Alloantibody  Pos - Autoantibody (could mask an alloantibody)  In what phase(s) did positive reactions occur?  IS; 37 C; &/or AHG (cold or warm)  At what strength(s) did positive reaction occur?  multiple antibodies OR dosage effect Review Antibody Tutorial 13  Rule out  What antibodies can be ruled out or eliminated as possibilities  Antibodies are ruled out when patient’s serum fails to react with an RBC sample known to carry the corresponding antigen.  Circle the symbols at the top of the antigram if the antigens were not ruled out  Look for homozygous (double dose) or antigen – may weakly react and not react with heterozygous Review circled antigens Is there a pattern? If reactions do not fit exactly, review dosage. When a pattern that fits from those that are circled is found, check to see if there are a minimum of three antigen-positive cells that react and a minimum of three antigen negative that do not react. (3 + 3 rule) 14 Patterns  Does the serum reactivity match any of the remaining specificities  Pattern usually matches a pattern of antigens exactly when only one alloantibody is present  Cannot rule out Cw or Lua based on ruling out all where there no reaction Are all commonly encountered RBC antibodies ruled out?  Patient’s serum may contain more than one antibody  One specificity may mask ID of another  Low-frequency antigens (