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    10th and 11th Direct Coomb s Test Finals

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    Kaila Cabran

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    © All Rights Reserved
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    10th and 11th Direct Coomb s Test Finals

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    Kaila Cabran
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    10th and 11th Direct Coomb s Test Finals

    Uploaded by

    Kaila Cabran

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 1

    IMMUNOHEMATOLOGY LECTURER: Mr. Earl Joseph D.

    Catampatan, RMT, MPH


    TRANSCRIBED BY: Renzy Estrada – MLS 3B-7
    IMHM311 – LABORATORY
    UNIT X & XI: DIRECT COOMB'S TEST
    ANTI-HUMAN GLOBULIN TEST / ANTIGLOBULIN  The DAT does not require the incubation phase
    TEST because of the antigen-antibody complexes formed in
     Commonly known as Coomb’s Test vivo.
     identify unknown, unidentified, weakly reacting
    antibodies AHG Principle:
     It is based on the principle that antihuman globulins  Detects in-vivo sensitization of RBCs. (Sensitized
    (AHGs) obtained from immunized nonhuman species with IgG and/or C3b/C3d)
    bind to human globulins such as IgG or complement,  AHG reagent is added on patients sample (Washed
    either free in serum or attached to antigens on red RBCs).
    blood cells (RBCs). o unwashed RBC – yields false negative
     There are two major types of blood group antibodies, o If Patients RBC is sensitized with IgG and/or
    IgM and IgG. C3b/C3d, AHG will bind to these components and
    o Because of their large pentamer structure, IgM will agglutinate the red blood cells.
    antibodies bind to corresponding antigen and
    directly agglutinate RBCs suspended in saline.
    o IgG antibodies are termed nonagglutinating
    because their monomer structure is too small to
    agglutinate sensitized RBCs directly. Procedure:
    1. Prepare a test tube and label it with patient’s name
    Detection of In Vivo or In Vitro Sensitization of RBCs 2. Deliver 2 drops of well-mixed anticoagulated blood
     The use of AHG to detect in-vitro sensitization of into the tube.
    RBCs is a two-stage technique referred to as the 3. Wash the anticoagulated blood three times with
    indirect antiglobulin test (IAT) NSS.
     In-vivo sensitization is detected by a one-stage 4. Decant completely the supernatant after the last
    procedure, the direct antiglobulin test (DAT). washing
    5. Add 2 drops of AHG reagent
    AHG REAGENTS 6. Centrifuge for 15 seconds at 3400 rpm
    Polyspecific AHG 7. Gently dislodge the cells and examine for hemolysis
     Anti-IgG and Anti-C3b or Anti-C3d or agglutination.
     Other anticomplement antibodies, such as anti-C3b,  Note: Coomb’s check cells are also added to
    anti-C4b, and anti-C4d, may also be present. validate a negative reaction and confirm AHG
     Anti-IgG – Reacts with Fc region of the gamma heavy was previously added. If AHG was not added, no
    chain of the IgG molecule agglutination will be observed after the addition of
     If positive with polyspecific AHG ➔ use check cells. It is suggested that the procedure
    monospecific AHG to determine which component should be repeated
    reacts with the red cell 8. Interpretation:
    Monospecific AHG  With agglutination: DAT POSITIVE
     Anti-IgG or Anti-C3b or Anti-C3d o Proceed to antibody panel if DAT positive
    Licensed monospecific AHG  No Agglutination: DAT NEGATIVE
     reagents in common use are anti-IgG and anti-C3b- o DAT negative – validate using coombs check
    C3d. cells (composed of O cells sensitized with
    IgG)
    DIRECT AHG TEST
     The DAT detects in-vivo sensitization of RBCs with For test to be valid for negative AHG test
    IgG and/or complement components.  Group O RBCs sensitized with IgG are added
     Clinical conditions that can result in in-vivo coating of  Valid if: There is agglutination
    RBCs with antibody and/or complement are:  Invalid if: There’s no agglutination
    1. Hemolytic disease of the newborn (HDN) or the
    erythroblastosis fetalis
    o Maternal antibody coating fetal RBCs
    2. Hemolytic transfusion reaction (HTR)
    o Recipient antibody coating donor RBCs
    3. Autoimmune and drug-induced AIHA
    o autoantibody coating individual’s RBCs

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