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Dr. Mohammed H Saiemaldahr
Antibody Screening

   Antibody Detection and Identification

 The purpose of the antibody screen is to detect red blood cell
  antibodies other than anti-A or anti-B.
 These antibodies are called “unexpected” because only
  0.3 to 2 % of the general population have positive
  antibody screen.
 Once an unexpected antibody is detected, antibody
  identification studies are performed to determine the
  antibodies specificity and clinical significance.
Antibody Screening

  All red blood cell antibodies are significance if they cause
  shortened survival of antigen positive red blood cells. For
  example, anti-D is a clinically significant antibody,
  because it will bind to D-positive red blood cells,
  resulting in immune destruction or hemolysis.

  Proper detection and identification of red blood cell
  antibodies is important for the selection of appropriate
  blood for transfusion and in the investigation of
  hemolytic disease of the new born, and immune
  hemolytic anemia.
Antibody Screening

 Antibody screening test involve testing patient’s serum
  against two or three reagent red blood cell samples called
  screening cells

 Screening cells are commercially prepared group O cell
  suspensions obtained from individual donors that are
  phenotype for the most commonly encountered and
  clinically important red blood cell antigens.
Antibody Screening

 Group O cells are used so that naturally occurring anti-A
  or anti-B will not interfere with detection of unexpected

 The cells are selected so that the following antigens are
  present on at least one of the cell sample;
  D, C, E, c, e, M N, S, s, P, Lea, Leb, K, k, Fya, Fyb, and Jkb.
Antibody Screening

 An anti-gram listing the antigen makeup of each cell
  provided with each lot of screening cells issued from a
  manufacture. It is important that the lot number on the
  screening cells matches the lot number printed-On the
  anti-gram because antigen make up will vary with each lot.

 The “ideal” screening cells have red cells with homozygous
  expression of as many antigens as possible.
Antibody Screening

 There is no requirement that screening cells contain red
  cells with homozygous expression of antigens, however,
  the most workers prefer that such red cells are included
  in screening cells sets because many antibodies,
  especially JK and M antibodies, show dosage effect and
  give stronger reactions when tested against cells with
  homozygous expression of their corresponding antigen.
Antibody Screening

 As a result of dosage, weakly reacting antibody may not
  be detected if serum samples are not tested against red
  cells with homozygous expression of the their
  corresponding antigen.
Antibody Screening

  Screening Cells.
 The screening cells are available in three form
  1- a single vial of no more than two donors pooled
  together in one vial.
  2- two vials each with a different donor.
  3- 3 vials representing three different donors.

  Two or three cells screening sets are required for
  detection of antibodies in pre-transfusion testing.
Antibody Screening

  Detection of very low levels of antibody in a
  recipient’s serum is important because
  transfusion of antigen-positive red cells may
  result in a secondary immune response with
  rapid production of antibody and subsequent
  destruction of transfused red blood cells.
Antibody Screening

  Red blood cells should be re-suspended by gentle
  shaking or tilting the tube until the cells no longer adhere
  to the sides. Agglutination is graded once the red blood
  cells are re-suspended.

   Agglutination reactions are routinely graded as negative
  (no agglutination).

  Weakly positive, and 1+ through 4+. The degree of the
  positive reaction generally indicates the amount of Ab
  present not its significance.
Antibody Screening

  Auto-logous Control.
 Autologous control is considered as part of the Ab
  screening, it can be performed in parallel with the Ab
  screen and involves testing the patient’s serum against
  the patient’s red blood cells.

 A positive auto-logous control is an abnormal finding
  and usually means that patient has a positive direct
  antiglobulin test (DAT).
Antibody Screening

  Grading Reactions.
 Aggregation or hemolysis of test red blood cells is the
  visible end point of an Ab-Ag interaction.

 Test results should be read immediately after
  centrifugation as delays in reading may cause elution of
  antibody and false- negative test results

 The first step in reading hem-agglutination reactions is
  inspection of the supernatant for signs of hemolysis (red
  or pink coloration).
Antibody Screening

  Evaluation of the antibody screening and auto-
  logous control results can provide clues and give
  direction for the identification and resolution of
  the Ab or Abs. The investigator should consider
  the following questions;

 1- In what phase (s) did the reaction (s) occur?

 Low temperature: Abs of the IgM class will react best at
  low temperatures and are capable of causing
  agglutination of saline-suspended red blood cells
  (immediate spin reading (IS). Of the commonly
  encountered Abs are, anti-N, -I. and -P (IgM)

 Abs of the IgG class will react best at the AHG Phase
  (37°C). Of the commonly encountered Abs are; anti-Rh,
  kell, Kidd, and Duffy (IgG).

 Where as Lewis, M, Abs may be IgG, IgM, or a mixture of

 2- Is the auto-logous control negative or positive?

 A positive Ab screen and a negative auto-logous control indicate an
   allo-antibodv has been detected.

 A positive auto-logus control may indicate the presence of auto-
   antibodies, antibody to medication or it may idiopathic

 If the patient has been recently transfused, the positive
   auto-logous control may be due to alloantibody coating
   circulating donor red blood cells.

 3- Did more than one screening cell react and if so, did
  they react at the same strength and phases?

 If the patient has multiple Abs, when the Abs
  corresponding Ag is found on more than one screening
  cell, or when the patient’s serum contains an
  autoantibody, more than one screening cell will be
 A single Ab specificity should be suspected when all cells
  react at the same phase and strength.
 Multiple Abs are most likely when cells react at different
  phases and strengths and auto-antibodies are suspected
  when the auto-logous control is positive.

 4-Are the cells truly agglutinated or rouleaux present?

 Serum from patients with multiple myeloma or who have
  received high molecular weight plasma expanders
  (dextran) may cause non-specific aggregation of red
  blood cells known as rouleaux.

 Rouleaux is not a significant finding in Ab screening
  tests but it is easily confused with Ab mediated
  agglutination. To differentiate between rouleaux and

 1- Rouleaux cells have “a stacked coin” appearance when
  viewed microscopically.

 2- It will not interfere with the AHG phase of testing
  because the patient’s serum is washed away prior to the
  addition of the AHG reagent.

 3- It is observed in Ab tests containing the patient’s
  serum including the auto-logous control and the reverse
  ABO typing.
 4- Rouleaux is dispersed by the addition of 1 to 3 drops of
  saline to the test tube.
           Reagent Red Blood Cell Screening Cell
           Sectional Listing of Antigens Present

                                   F   F   J   J        L
                                                   L                      P   I   3
       D     C   E c   e   K   k   y   y   k   k        e   S   s   M N               AHG   CC
No                                 a   b   a   b   ea   b                 1   S   7
I      +    + 0 ++         + 0 0 + + + 0 + + + + 0 + 0 0 2+

II     +    0 + +0         0 + + 0 + 0 0 + 0 + + 0 + 0 0 0                                  2+

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