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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.
REAGENTS       FOR   RH (D) GROUPING
I    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
    II 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 20°C as well as 37°C 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.
III 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
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
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.
   TUBE TECHNIQUE
a. Saline Agglutination test for Rh (13) Typing

  Procedure

  1. Prepare 2-5% washed red cell suspension of test sample.

  2. Place 1 drop of anti-D in cleaned tube labelled D.

  3. Place 1 drop of 22% bovine albumin/control reagent in another tube
  labelled C.

  4. Add 1 drop of 2-5% test cell suspension to each tube.

  5. Mix well and centrifuge at 1000 rpm for 1 minute (in case of using IgG
  anti-D, incubate at 37°C for 10mm. and centrifuge (spin tube method) or
  incubate at 37°C for 60 minutes (sedimentation method).

  6. Resuspend the cell button and look for agglutination. All negative
  results must be confirmed under microscope
     TUBE TECHNIQUE
   Interpreation

    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.
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.
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.
 RH(D) GROUPING IN HAEMOLYTLC DISEASE OF
 THE NEWBORN

In haemolytic disease of the newborn, the baby’s 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 infant’s 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 45°C for 30 minutes) to
expose the antigenic sites before testing.
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 patient’s red cells each time an Rh
  typing is performed
CAUSES OF FALSE POSITIVE REACTIONS




  1.   Positive direct antiglobulin test
  2.   Polagglutinable red cells
  3.   Cold agglutinins or Rouleaux formation
1- POSITIVE DIRECT ANTIGLOBULIN TEST


  The presence of Ab on patient’s 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
2- POLAGGLUTINABLE RED CELLS


  Rh –ve 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
3- COLD AGGLUTININS OR ROULEAUX FORMATION



    Rh typing is performed using serum suspended red
     cells
    If individual’s serum contains cold agglutinin or
     abnormal protein, false positive reactions can
     occur
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.
RESOLVING RH PROBLEMS
   Erroneous Results in Rh Grouping

    1. Perform clerical checks for validity of labels and requisition
    forms.

    2. Obtain a fresh blood sample of patient.

    3. Check patient rçcords for history, diagnosis, pregnancy,
    medication and previous transfusion.

    4. Check equipment and reagents for proper quality control.

    5. Check the antisera and controls.

    6. Perform alternative procedures such as washing of red cells with
    warm saline, enzyme treatment of red cells, absorption / elution
    studies.

				
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posted:2/10/2012
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