CLS 3311 Advanced Clinical Immunohematology

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CLS 3311Advanced Clinical Immunohematology

• Antihuman Globulin Testing
• (AHG)

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Antihuman Globulin

• Definition
• Antihuman antibodies against human antigens
• Globulin all antibody molecules are globulins
• Therefore Antihuman Globulin is antibody
  directed against the Fc portion of human
  antibodies and/or complement components.

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Antihuman Globulin

• In the past AHG was made by injecting rabbits (or
  sheep, etc.) with human globulin and complement
  (C). The rabbit would then make antibody to the
  human globulin and C components.
• The antihuman antibody (polyclonal) would then be
  harvested, fractionated and purified for use in
  the blood bank as anti-A, anti-B, etc. Each lot
  was quite unique.
• Today we use, almost exclusively, Monoclonal AHG
  produced using mouse hybridoma cells to make very
  specific AHG. Figure 4.1, page 75 Harmening

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Antihuman Globulin Reagents

• Polyspecific
• Monospecific
• Table 4.1, page73 Harmening

• Contains anti-IgG and anti-C3d (Complement)
• Contains only one specificity either anti-IgG
  or anti-C3d. NOT both, only one.

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• AHG Reagents Include
• Anti-IgG (Anti-IgG)
• Anti-C (Anti-C3/C4)
• Anti-IgG C (Polyspecific AHG)
• These Anti-Human Globulins must be diluted to
  achieve optimum reactivity.
• In doing this anti-IgM is diluted BELOW
  detectable level.
• The presence of IgM on the RBC is NOT detected
  using AHG reagents.

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Table 12.1, page 260, AABB Technical Manual, 13th
Edition
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AHG Techniques What is the relevance?

• Some very clinically significant unexpected
  antibodies (Kidd, Duffy, etc.) attach to red cell
  antigen or activate complement to do so, but do
  NOT cause agglutination at immediate spin or 37o
  phase.
• Yet, these antibodies are capable of causing
  severe hemolytic transfusion reactions or
  hemolytic disease of the newborn.

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Relevance of AHG

• AHG techniques enable the detection of these
  antibodies or C components that otherwise went
  undetected. AHG enables detection of both in
  vitro (Indirect Antiglobulin Test) and in vivo
  (Direct Antiglobulin Test) antibody attachment.
• AHG testing is the primary method of detecting
  all Antibodies except those of the ABO System.

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Expected Vs. Unexpected Antibodies

• Expected Antibodies
• If an ABO antigen is missing from an individuals
  red blood cell membrane then it is EXPECTED that
  the individual will produce an antibody to that
  antigen. These have also been called naturally
  occurring.
• Example Group A individual does NOT have B
  antigens on their red blood cell membrane. They
  will normally produce anti-B antibodies.

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Unexpected Antibodies

• In ALL other blood group systems if the antigen
  is missing from the red cell, the individual is
  NOT expected to produce an antibody against it,
  normally.
• When these antibodies are produced they are
  termed UNEXPECTED antibodies.
• EXAMPLE An individual lacking the D antigen
  on their RBC membrane is NOT expected to have
  anti-D antibodies in their plasma, normally. It
  requires exposure to the D antigen from a foreign
  RBC either by transfusion or pregnancy.

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• CONCLUSION Only ABO antibodies are expected to
  be produced in the absence of foreign RBC Ag.
• All other blood group systems require, with a few
  exceptions, exposure to a foreign red cell
  antigen (transfusion or pregnancy) to stimulate
  production of an unexpected antibody.

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Indirect Antiglobulin Test (IAT)

• Another name is Antibody Screen
• Patient serum or plasma containing unexpected
  Antibodies is incubated with RBCs possessing the
  corresponding antigen (Screen Cells).
• The unexpected IgG class Antibody will bind to
  the corresponding RBC Antigen during incubation.

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• OR
• The Patients unexpected IgM or IgG class antibody
  will bind C to the RBC membrane with the
  corresponding antigen during incubation.
• The RBCs are then washed with saline to remove
  all UNBOUND serum proteins including IgG C.
• An Anti-Human Globulin reagent is added to the
  washed, dry RBC button.

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• If unexpected antibodies (or C) are present on
  the red cell membrane AHG will bind and cross
  link the red blood cells causing agglutination.

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Set of Problems

• Even 10 mLs of AHG can be neutralized by a tiny
  amount of free serum protein.
• A technologist can forget to add AHG to a test
  tube.
• A couple of drops of residual saline can dilute
  the AHG reagent below detectable levels.
• Normally people do NOT produce unexpected
  Antibodies. Therefore the test should normally
  be negative.
• HOW DO YOU KNOW A NEGATIVE AHG TEST IS A TRUE
  NEGATIVE?

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Coombs Control or Check Cells

• Addition of IgG coated red cells to all negative
  AHG tests is required by AABB Standards for Blood
  Banks and Transfusion Services for antibody
  screen and crossmatch tests.
• Negative AHG test should contain FREE AHG
  reagent still capable of binding to IgG coated
  RBCs
• If IgG coated RBCs are added to negative test
  and centrifuged, then the free AHG should bind
  to them and cause agglutination.

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If the Check Cells agglutinate, it indicates
three (3) things

• The test was adequately washed prior to addition
  of the AHG reagent.
• AHG reagent was added to the test tube.
• The AHG reagent that was added was in an ACTIVE
  form.

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What agglutinated Check Cells DOES
NOT mean!

• That the test was performed correctly!!
• Why? Because
• Patient Serum could have been left out of tube
• The WRONG Patient Serum could have been added
• The WRONG test cells could be used
• Incubation time or temperature may be incorrect
• Enhancement media (LISS) may not have been added
• The tube could be mis-labeled
• The results could be mis-read
• The results could be recorded incorrectly

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Purpose of the IAT

• The purpose of the Indirect Antiglobulin test is
  to detect unexpected antibodies in patient or
  donor serum.
• Screening test only, not for antibody
  identification.
• RBCS used are called SCREEN CELLS
• Reason theyre used to screen for unexpected
  antibodies present in the donor or recipient
  serum.
• Use a set of cells - Usually 2 or 3 cells, each
  cell from a different source/donor so each has a
  unique phenotype

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Purpose of the IAT

• What type of cells are used as the Screen
  Cells?
• Group O cells Why?
• The cells MUST have antigens that most commonly
  stimulate production of unexpected antibodies
  (Kell, Kidd, Duffy and Rh).
• SOURCE OF CELLS Individual donors with specific
  phenotypes

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DIRECT ANTIGLOBULIN TEST (DAT)

• DAT tests for in vivo sensitized red blood cells
• Attachment of antibody or complement in vivo
• Problem
• Clotted samples still contain serum with Ca
  present so that Ccan be activated
  non-specifically after collection causing false
  positive results.
• Solution Perform test on EDTA sample. EDTA
  chelates calcium stopping C activation. The
  blood in the EDTA sample represents in vivo C
  activity.

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DIRECT ANTIGLOBULIN TEST

• Collect sample in EDTA tube
• Make 3-4 RBC suspension and wash 3-4 times
• Cord blood needs additional washing.
• Take 1 drop of 3-4 RBC suspension and centrifuge
  to get dry button
• Add AHG to the dry button
• If IgG or C attached to the RBCs in vivo, the
  AHG will cause agglutination (Autoimmune process,
  HDN)
• NORMAL RESULT Negative
• Again, how do we know that the Negative result is
  a TRUE Negative?

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Application of AHG testing include detection of

• Hemolytic Anemia's
• Transfusion Reactions
• Hemolytic Disease of the Newborn
• Antibody Screening of donors and recipients
• Final step in Antibody Identification

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This represents a general layout for an Antibody
Screen report and suggested interpretation of an
IAT without reactivity. Can I report it out as
negative? Are we able to detect all unexpected
antibodies with this system?
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Why do we suspect that this unexpected antibody
is IgG class? Why not IgM? Is it clinically
significant?
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