Practical Blood Bank

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Practical Blood Bank
Rh Grouping
Lab 3
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Practical Aspects of Rh Grouping

• Rh grouping in routine use for donors and
  patients involves testing for Rh (D) antigen only
• However tests for other important Rh antigens
  e.g. C,c,E and e may be done for Rh genotyping.
• The method of Rh grouping mainly depends on the
  type of reagents available, for which the
  manufacturers instructions have to be strictly
  followed.

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Reagents for Rh (D) Grouping

• Polyclonal human anti-D serum (IgG)
• Potentiating or enhancing substances such as
  albumin, enzymes and AHG reagent are used to
  bring about agglutination with human IgG anti-D.
• Anti-D serum (IgG) for saline or rapid tube test
  (high protein medium) This contains
  macromolecular additives and give reliable
  results.
• Anti-D for saline tube test 2 types
• Anti-D IgM Anti-D IgG, Chemically modified

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Monoclonal antibodies

• IgM anti-D monoclonal reagent
• IgM and IgG anti-D monoclonal reagent
• Blend of IgM monoclonal IgG polyclonal reagent
• These antibodies are highly specific, react
  equally well at 20C as well as 37C and are
  reliable for slide and rapid test tube technique.
• IgM anti-D monoclonal reagent cannot be used for
  Du testing by indirect antiglobulin test (IAT)
  while IgM IgG monoclonal reagent and blend of
  IgM monolconal and IgG polyclonal can be used for
  Du testing.

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Controls for Rh (D) grouping

• Known 0 Rh (D) positive and 0 Rh (D) negative
  cells may be used as controls with monoclonal
  anti-D reagent.
• Alternatively, AB serum or diluents control
  provided with the anti-D reagent or 22 bovine
  serum albumin may be used as negative control
  with the test cells

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Rh (D) Grouping

• In most of the blood transfusion laboratories, Rh
  (D) grouping is performed along with the ABO
  grouping and same techniques as used for ABO
  grouping may also be employed for Rh typing
•  

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Slide Technique

• This technique may be used in emergency Rh (D)
  typing if a centrifuge is not available.
• The slide test is not recommended for routine
  test as it may not pick up weak reactions, thus
  giving negative results.

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Tube Technique

• Saline Agglutination test for Rh (D) Typing
• Procedure
• Prepare 2-5 washed red cell suspension of test
  sample.
• Place 1 drop of anti-D in cleaned tube labelled
  D.
• Place 1 drop of 22 bovine albumin /control
  reagent in another tube labelled C.
• Add 1 drop of 2-5 test cell suspension to each
  tube.
• Mix well and centrifuge at 1000 rpm for 1 minute
  (in case of using IgG anti-D, incubate at 37C
  for 10mm. and centrifuge (spin tube method) or
  incubate at 37C for 60 minutes (sedimentation
  method).
• Resuspend the cell button and look for
  agglutination. All negative results must be
  confirmed under microscope

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Interpretation

• Positive test Agglutination in anti-D and
  smooth suspension in control tube.
• Negative test Smooth suspension in all the
  tubes (test and control)
• Test is considerable invalid if both test and
  control tubes show a positive reaction.In such
  case, the test should be repeated using saline
  IgM anti-D.
• For all microscopically negative reactions in
  donor grouping, Du testing should be performed,
  hereas some workers suggest that if the two anti
  D reagents used are potent and specific, it is
  not necessary to perform Du testing.

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Albumin technique for Rh (D) typing

• Principle Albumin increases the dielectric
  constant of the medium and thus reduces the zeta
  potential. Due to this effect, the electrical
  repulsion between the red blood cells is less and
  the cells agglutinate. Mostly 22 bovine albumin
  is used, as higher concentrations can cause
  rouleaux formation.

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Enzyme agglutination technique for Rh (D) typing

• Proteolytic enzymes such as papain, trypsin,
  bromelain and ficin digest the cell membrane
  partially and expose Rh antigens to react with
  IgG antibodies.
• When the membrane is partially removed, it brings
  about a loss of negative electric charge on the
  red cell which is responsible for keeping the
  cells a set distance apart, hence small IgG
  molecules are able to span the gap between cells
  and bring about agglutination.

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Rh(D) Grouping In Haemolytlc Disease of the
Newborn

• In haemolytic disease of the newborn, the babys
  red cells may be coated with immunoglobulin and a
  saline reactive Rh antiserum is usually necessary
  for testing. When the cells are heavily coated
  with antibody, no free antigenic sites remain for
  reaction, resulting in a negative test. This is
  suspected when the infants cells show a positive
  direct antiglobulin test (DAT) and a negative
  test with anti-D reagent. In such instances it
  is recommended that the antibody should be eluted
  by gentle elution (heating at 45C for 30
  minutes) to expose the antigenic sites before
  testing.

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Rh Discrepancies

• Rh ve persons mistyped, transfused with Rh ve
  blood have 70 chance of becoming immunized
• False ve reactions can be identified by testing
  an Rh control with the patients red cells each
  time an Rh typing is performed

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Causes of false positive reactions

1. Positive direct antiglobulin test
2. Polagglutinable red cells
3. Cold agglutinins or Rouleaux formation

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1- Positive direct antiglobulin test

• The presence of Ab on patients red cells can
  cause a false ve reaction with slide and tube
  anti-D
• High protein medium reduces zeta potential
  allowing red cells to move closer
• The cell bound Ab can cross link cells and cause
  agglutination

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2- Polagglutinable red cells

• Rhve red cells that are polyagglutinable due to
  T or Tn activation
• Agglutination occurs if anti-T or anti-Tn present
  in the anti-D reagent
• Most anti-D reagents do not contain these
  antibodies

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3- Cold agglutinins or Rouleaux formation

• Rh typing is performed using serum suspended red
  cells
• If individuals serum contains cold agglutinin or
  abnormal protein, false positive reactions can
  occur

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False Negatives

• False negatives are not readily identifiable, but
  can occur in the following instances
• The most common is the result of too heavy cell
  suspension due to too many cells for the amount
  of antibody in the antisera.
• They may also rarely be caused by extremely
  strong positive DAT. In this case a the patient's
  D antigen sites are coated in vivo and there are
  no sites left for commercial anti-D to attach to.
  This can be fixed by heating cells gently to
  elute off antibody without damaging cells, then
  re-test.

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Resolving Rh Problems

• Erroneous Results in Rh Grouping
• Perform clerical checks for validity of labels
  and requisition forms.
• Obtain a fresh blood sample of patient.
• Check patient records for history, diagnosis,
  pregnancy, medication and previous transfusion.
• Check equipment and reagents for proper quality
  control.
• Check the antisera and controls.
• Perform alternative procedures such as washing of
  red cells with warm saline, enzyme treatment of
  red cells, absorption / elution studies.
• Carry out family studies.

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Thank You
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Stages of Antigen-Antibody Interaction

• The first stage is sensitization. 
• Sensitization occurs when antibodies react with
  antigens on the cells and coat the cells.
• The second stage of the reaction is
  agglutination. 
• Agglutination occurs when antibodies on coated
  cells form cross-linkages between cells resulting
  in visible clumping.

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FACTORS AFFECTING SENSITIZATION

• Specificity depends on the spatial and chemical
  "fit" between antigen and antibody
• Since the immunoglobulins and the red cell
  membranes both have an electrical charge, there
  is an optimum pH.  pH differences cause
  differences in chemical structures of
  antigens/antibodies, affecting the "fit".
• The optimum temperature depends on the type of
  antibody involved.  IgG antibodies react best at
  37oC IgM react best at 4oC.
• Optimum incubation time  you need to incubate
  long enough to reach equilibrium, but not too
  long
• The antigen's accessibility is also important
  since the antibodies must be able to reach
  antigens.  Those antigens, like the ABO antigens,
  are on the surface of the red cell while others
  may be hidden in the crypts of the cell membrane.
  

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FACTORS AFFECTING AGGLUTINATION IN VITRO

• Number of Antigen Sites
• Size and Structure of the Antibody
• Distance between Cells
• Antigen-Antibody Ratio
• Prozone - antibody excess Antibodies saturating
  all antigen sites no antibodies forming
  cross-linkages between cells no agglutination
• Zone of equivalence antibodies and antigens
  present in optimum ratio, agglutination formed
• Zone of antigen excess (Post-zone) too many
  antigens - any agglutination is hidden by masses
  of unagglutinated antigens