Life's Blood


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CLASS  NOTES

ANTIBODY SCREENING

Review of Types of Antibodies

Immunoglobulins are the antibodies formed as a result of immune stimulus (exposure to foreign antigen).  In Blood Bank we are referring to those antibodies that will attach to blood cells.  We are most concerned about those antibodies that are known to cause transfusion reactions and hemolytic disease of the newborn. Except for anti-A, anti-B, and anti-A,B these are usually IgG immunoglobulins.  An old term that referred to IgG antibodies that can not cause agglutination of red cell antigens in saline is "incomplete antibodies."

The A and B antibodies are naturally occurring since they are formed without previous exposure to foreign blood cells.  These antibodies are expected and can be used to confirmed the antigen typing for ABO grouping.

The antibody screening cells are used to detect unexpected antibodies.  In most cases these are alloantibodies, which formed to foreign antigens on cells from other individuals within the same species.  Therefore for an individual to make an alloantibody they would lack the antigen for which they made a specific antibody due to its foreignness to the individual. 

Autoantibodies can also be detected with the antibody screening procedure.  These antibodies are ones a person makes toward their own antigens.  These are not a normal occurrence and may indicate autoimmune hemolytic anemia.  The autocontrol tube within the antibody screening will detect this type of antibody and other causes of a positive direct antiglobulin test.

Clinically significant antibodies are those antibodies known to cause transfusion reactions and hemolytic disease of the newborn.  Other the AB antibodies, which are saline agglutinins and IgM, the rest of clinically significant antibodies are IgG antibodies that are warm-acting and may only be demonstrated at the antiglobulin stage of testing. Antibodies that show up at the immediate spin phase are most likely nuisance antibodies that won't cause transfusion reactions.  These would also be referred to  as saline agglutinins since it is an antibody capable of causing direct agglutination of antigens suspended in a saline medium without requiring any enhancement techniques.  Most clinically significant antibodies are are warm antibodies whose optimal temperature of reactivity is greater than 35oC.

 

Antibody Screening Procedure

Uses

Antibody screening is used to test:

  1. Donor plasma to make sure no unexpected antibodies will be transfused.  Since an unit of blood  is usually divided into various components (red cells, fresh frozen plasma, and platelets), blood centers need to make sure none of these unexpected antibodies are present in the fresh frozen plasma.  If these antibodies are present, they could attach to the recipient's cells cause a positive direct antiglobulin test and potentially a transfusion reaction.
     
  2. Patient serum before transfusion to make sure patient has no unexpected antibodies to react with donor cells.  In this case we do not want the recipient's antibodies to attach to the donor cells causing a transfusion reaction.
     
  3. Maternal serum to make sure pregnant mother has no antibodies to react with fetal cells.  Hemolytic disease of the newborn is caused by the mother's IgG antibodies crossing the placenta and attaching to the baby's red blood cells.  The physician wants to know as early as possible in the pregnancy whether HDN can be a possibility.  This is also necessary if the mother is Rh negative and would be a candidate for RhoImmune Globulin.  Since  RhoImmune Globulin is anti-D given to the Rh negative mother who do not already have anti-D so we need to rule out the presence of anti-D already.

Follow up for a positive antibody screening

If antibody screening is  positive, we must do additional tests to specifically identify antibody using the antibody identification panel and red cell antigen typing. 

Screening Cells Characteristics

Screening cells are cells from two or three individual donors.  These cells are Group O and in order to licensed by the FDA must contain the following antigens: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, Jkb.  The most common clinically significant antigens must be present in order to detect the clinically significant antibodies.  It is preferred that as many antigens as possible be homozygous on the red cells because a double dose of the antigen results in stronger reactions and therefore can detect weaker antibodies.  Screening cells outdate every 4 weeks. Each new lot number of screening cells will vary related to antigen typings.  Always consult package insert  to determine which cells have the specific antigens.  Below is an example of a package insert.

 

Rh

MNSs

P Lewis

Kell

Duffy

Kidd

Sex-linked

37oC AGT CCC
  D C E c e M N S s P1 Lea Leb K k Kpa Jsa Fya Fyb Jka Jkb Xga Sex
1 + + 0 0 + + 0 + + + + 0 0 + 0 0 + 0 + + + /      
2 + 0 + + 0 + 0 + + + 0 + 0 + 0 0 + + + 0 + /      
3 0 0 0 + + + + 0 + + 0 + + + 0 0 0 + 0 + + /      

 

Procedure for Antibody Screening

Antibody Screening will involve various phases to allow for antibody-antigen agglutination.

Phase 1:   Immediate Spin:

 Immediate spin phase may be omitted but it may also give additional information as to whether the reaction is due to an IgM antibody instead of IgG or complement activation.

  • 3 tubes - Recipient serum plus saline suspension Screening Cell I, Screening Cell II, and the recipient's own cells for the auto control.
  • Centrifuge these three tubes  and read for agglutination.
  • Detects IgM antibodies (usually considered "nuisance" antibodies)

Phase 2:  37oC Incubation:

37oC phase is required since IgG clinically-significant antibodies are warm-acting antibodies. 

Can add enhancement media if desired (LISS or albumin). 

  • LISS is Low Ionic Strength Solution composed of NaCl, glycine and phosphate buffer along sodium preservative.  This solution speeds up antigen-antibody reaction but unfortunately enhances "nuisance" antibodies, so add after immediate spin step.
  • Albumin  is added to lower zeta potential so cells can agglutinate without Coombs step and may detect Rh antibodies. 

Whether adding an enhancement media or not we must do 37oC incubation, but we do not need to read at this step.  We- can proceed directly to Coombs (AHG or AGT) phase.

Phase 3:  Coombs phase (AHG or AGT)

The Coombs phase (AHG or AGT) is required since a number of these clinically significant antibodies may only show up at this phase.  The following steps are part of this third phase.

  1. After 37oC incubation, wash the cells 3-4 times
  2. Remove the saline and add AHG.
  3. Mix and centrifuge
  4. Read for agglutination.
  5. Add Coombs Control Cells to all negative results to confirm negative reactions.

This phase detects IgG antibodies, most of which are considered clinically significant and capable of causing Hemolytic Disease of the Newborn or Hemolytic Transfusion Reactions.

Goals of antibody screen

  1. Detect as many clinically significant antibodies as possible
     
  2. Minimize detection of nuisance antibodies
     
  3. Prompt delivery of blood to patient

Now try answering the questions on this Antibody Screening Worksheet.

OBJECTIVES - ANTIBODY SCREENING

  1. Discuss why antibody identification is necessary for transfusion recipients, blood donors, and obstetrical patients.
     
  2. Distinguish between alloantibodies and autoantibodies.
     
  3. Define a clinically significant alloantibody.
     
  4. Explain when an antibody screen should be followed by an antibody identification.

Performance objectives:

  1. Correctly perform and interpret an antibody screen.
     
  2. Use antigen phenotyping results of  the patient, including correct use of positive and negative controls,  to identify possible antibodies that may be giving a positive antibody screening.

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Clinical Microbiology Syllabus