Detection and identification of alloantibodies to red cell antigens
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Detection and Identification of alloantibodies to Red Cell Antigens. Unexpected Alloantibodies : Any Red Cell Alloantibodies other than naturally occurring anti-A or anti-B Detected by : Performing an antibody screen test

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Unexpected Alloantibodies Antigens:

Any Red Cell

Alloantibodies other than naturally occurring anti-A or anti-B

Detected by: Performing an antibody screen test

Found in: 0.3-38% of the population

- Depending on group of patient or Donor Studied

- Sensitivity of the test methods used

Reaction: Only with allogenic red cells VS. Auto- antibodies that reacts with red cells from other individuals

Immunization to red cell Ag Antigens:

Results from pregnancy-Transfusion- Transplantation- Injection with immunogenic material-No specific- Immunizing event

Initial detection of alloantibodies

In tests that use serum of Plasma including:

  • ABO Test

  • Antibody Screen Test

  • Cross-match Test

  • Eluate

Once an antibody is detected Antigens:

  • Determine Specificity

  • Asses Clinical significance

    Clinically Significant antibodies defined as:

  • One that shortens the survival of transfused red cells

  • Cause Hemolytic disease of the newborn (HDN)

  • Hemolytic Transfusion reaction

    * Reported experience with other examples of antibody with the same specificity can be used in assessing clinical significance

NOTE: AntigensIf no data exists on certain antibody ,decision must be based on the temp. that one antibody is active

  • 37C

    And Or

  • Reactive by the Indirect Antiglobulin Test (IAT)

    Preparing Compatible blood for a recipient :


  • Detect as many clinically significant Antibodies

  • Detect as few clinically insignificant Antibodies

  • Complete the procedure in a timely manner

  • Patients with clinically significant antibodies should, receive red cells that are negative for the corresponding antigen.

  • In prenatal testing, the specificity and immunoglobulin class of an antibody influence the likelihood of HDN

  • Techniques: Antigens

    Techniques for Antibody detection Antibody identification are similar, but methods for:

    • Antibody detection is broad to detect antibodies with differing patterns of reactivity

    • Antibody Identification is more focused based on reactivity patterns identified in the antibody detection

    • Use of flowcharts as a guide to

      - expediting process

      - minimizing unnecessary tests.

    Specimen Requirements: Antigens

    Either 1- Serum


    2- Plasma

    10-ml aliquot is enough for identifying simple antibody specificities

    Medical History:

    When performing an antibody identification it is useful to know:

    1- Patient’s clinical diagnosis

    2- History of Transfusion

    3- History of pregnancies

    4- AntigensRecent drug therapy

    5- Ethnic background

    6- Sex M.F.

    7- Age


    Screening Cells:

    - Group O red cells

    commercially available

    - Available as sets of two or three

    vials of single-donor red cells

    - Pooled antibody screening cells are used for donor serum and not for recipients’ specimens

    Reagents Antigens:

    - Reagent cells licensed by the Food and Drug administration (FDA) must Express the following antigens D,C,E, c, e, M,N,S, s, P1,Lea ,Leb,K,K, Fya, Fyb, Jka, Jkb

    -weakly reactive antibodies react only with screening red cells from donors who are homozygous , a serologic phenomenon called dosage effect .

    - When not in use reagent should be refrigerated at 2-8ºС

    Red cell Panels Antigens:

    - Use panel of selected red cell with known antigen composition

    - Obtained commercially or institutionally

    (Horne made) from local

    - Group O cells

    except in special circumstances

    - Each cell from different individual

    - The pattern of reactivity should not overlap with any other,

    Example 0 Not all K+ should also be E+

    - Expiration for commercially prepared cells is every 2-4 weeks

    - Use only reagent phenotype listing sheet for the specific kit

    - Suspension of 2-5% Red Cells in a Preservative medium

    Saline – Suspended Red cells Technique:

    • Simplest serologic

    • Incubation of saline suspended cell with serum at


      • Immediate spin

      • Room Temperature

      • 37oC

    • Antibodies reacting below 37 specific kitoC are Anti-M, N, P1, Lea, Leb

    • This phase reading can be omitted to avoid finding little clinically significant antibodies.

    • 37 oC antibody detection

      anti – D, K, E also some

    • Some antibodies like anti- Lea, Jka can be detected in this phase by Lysing antigen- incompatible red cells

    Antiglobulin Reagents specific kit

    • Antiglobulin phase to detect clinically significant antibodies

    • Use antiglobulin reagent as

      • Polyspecific to detect antibodies that bind complement kidd antibodies

      • IgG- specific

    Enhancement media enhancement techniques
    Enhancement Media & Enhancement Techniques specific kit

    • Techniques

      • Temperature reduction

      • Increased serum to cell ratio

      • Increased incubation time

      • Alteration of PH

      • Inhibition tests

        • Lewis substances (saliva)

        • P1 substance (pigeon egg whites)

        • Sola substance (body fluids-urine)

        • Pooled plasma (Chido & Rodgers)

    Enhancement media enhancement techniques1
    Enhancement Media & Enhancement Techniques specific kit

    • Include incubating Serum/Plasma and reagent red cells in such media as:

      • Albumin 22% or 30%

      • LISS (low-ionic-strength saline)

      • PEG (polyethylene glycol)

      • Enzyme techniques

      • Polybrene

    Autologous control
    Autologous Control specific kit

    • To determine alloantibody from Autoantibody

    • Not performed in antibody screening

    • Used with panel cells

    Basic antibody identification techniques
    Basic Antibody Identification Techniques specific kit

    • Traditional serologic method in the united states are based agglutination performed in tubes or micro-plates

    • Other methods are not dependent on agglutination

      • Solid- Phase

      • Flocytometry

      • Agglutination test (gel tests) column techniques

      • Automated systems

    • Initial observation

    Basic antibody identification techniques1
    Basic Antibody Identification Techniques specific kit

    • Interpreting results

      • Positive and Negative

      • Exclusion or “crossing out”

    • Antibodies to High- Incidence Antigens anti-H, I, P, LW , Sda, Vel , Ch, Rg, U, Jsb

    • Antibodies to low-Incidence Antigens anti-Wra

    Selecting blood for transfusion
    Selecting Blood for Transfusion specific kit

    For a patient with clinically significant Antibodies (37oC and IAT)

    • Red cell should be tested and be negative for the appropriate antigen

    • Even if Ab. Is no longer detectable to prevent a secondary immune response

    • An antiglobulin cross-match is required

    • The absence of Ag should be confirmed with a potent commercial antisera

    • FDA requires use of licensed (commercial) reagents

    Selecting blood for transfusion1
    Selecting Blood for Transfusion specific kit

    • When rare type is needed

      • High- Incidence

      • Multiple antibodies frequency of random donors negative for each antigen should be used, Example: serum contains anti-C, Fya and s among random donors

        18% C Neg

        34% Fya Neg

        45% S Neg

    Selecting blood for transfusion2
    Selecting Blood for Transfusion specific kit

    The Frequency of compatible units would be 0.18×0.34 ×0.45= 0.028

    If patient is group O then 45% of random donors are group O then 0.028 ×0.45=1.3% of random donors would be compatible with the patient serum.