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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Presentation Transcript
Unexpected Alloantibodies:

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:

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:
  • 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:If 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 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:

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- Recent 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


- 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:

- 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 oC 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
  • 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
  • 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
  • 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
  • To determine alloantibody from Autoantibody
  • Not performed in antibody screening
  • Used with panel cells
basic antibody identification techniques
Basic Antibody Identification Techniques
  • 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
  • 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

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
  • 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

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.