IHLAB

• Saliva + antisera + RCS-agglutination or no agglutination


  Secretor or nonsecretor
• Antiglobulin Test


  Detects bound red cell antibodies that do not produce direct agglutination
• Described in 1945 by Coombs, Mourant, and Race
• Antiglobulin Test


  • Based on the principle that anti-human globulin obtained from immunized non-human species bind to human globulins such as IgG or complement in free state or attached RBCs
  • Primarily detects IgG and/or Complement-sensitized RBCs
• Primary Reagent


  Antihuman Globulin Reagent
• Antiglobulin Test


  Uses a secondary antibody, made in another species and directed against human globulins, that attaches and agglutinates sensitized red cells
• Preparation of Antiglobulin Reagent

  The antibody is produced by injecting animals with human globulins to stimulate antibody production against the foreign human protein (AHG serum)
• Types of AHG Reagents

  • Polyspecific AHG
  • Monospecific AHG
  • Polyclonal AHG
  • Monoclonal AHG
• Direct Agglutination Test


  Detects in vivo sensitization of RBCs with IgG and/or complement components
• Direct Agglutination Test Procedure

  1. 2-5% RCS (Sample) (2 DROPS)

  2. Add AHG reagent (2 DROPS)

  3. Centrifuge for 15 second @3400RPM

  4. Agglutination
• Coombs control calls should be added to confirm the Validity of NEGATIVE RESULTS
• Direct Coombs Test / Direct Antiglobulin Test

  Blood sample from a patient with Immune mediated haemolytic anaemia: antibodies are shown attached to antigens on the RBC surface. The patient's washed RBCs are incubated with antihuman antibodies (Coombs reagent). RBCs agglutinate, antihuman antibodies form links between RBCs by binding to the human antibodies on the RBCS.
• Applications of Direct Antiglobulin Test

  • Hemolytic Disease of the Newborn
  • Hemolytic Transfusion Reaction
  • Autoimmune Hemolytic Anemia
  • Drug-induced Hemolytic Anemia
• Indirect Agglutination Test


  Detects in vitro sensitization of RBCs with IgG and/or complement components
• Applications of Indirect Agglutination Test

  • Antibody Detection (Compatibility tests and Antibody screening)
  • Antibody Identification (Antibody Panel Tests)
  • Antibody Titration
  • Red cell phenotyping
  • Weak D testing
• Indirect Agglutination Test Procedure

  1. 2 drops serum + 2 drops RCS

  2. Centrifuge: 15 seconds @3400 RPM

  3. Place in water bath (37 C for 15 minutes)

  4. Centrifuge: 15 seconds @ 3400RPM

  5. Washing phase

  6. Add 2 drops of AHG

  7. Centrifuge: 15 seconds @3400 RPM

  8. OBSERVE
• Coombs control should be added to confirm the validity of NEGATIVE RESULTS
• Weak D Antigen (Du Variant)

  Weak D red cells have historically been defined as having a reduced amount of D antigen. In Rh phenotyping, the absence of agglutination reaction in slide and tube methods is not immediately reported as Rh negative. Some red blood cells express the D antigen so weakly that most anti-D reagents do not directly agglutinate them. Thus, test for weak expression of the D antigen must be performed before reporting Rh negative results.
• Types of Weak D

  • Partial D (D Mosaic)
  • C trans
  • Genetic weak D
• Principle of Testing for Weak D


  Some weak D antigens are recognized only by an IAT procedure
• Procedure for Testing Weak D

  1. Unknown tube with NO Agglutination

  2. Washing phase (3 times)

  3. Add 2 drops of AHG reagent

  4. Centrifuge for 15 seconds @3,400 RPM

  5. Examine for agglutination
• Absence of agglutination confirms the blood group to be Rh negative. Presence of agglutination means presence of weakly reacting D.
• For each negative tube, add 1 drop of check cells. A positive result is expected after the addition of check cells, indicating the test has been properly performed.
• If the reaction is positive for the test of Du, the person must be given proper designation because the person might have weakened D reaction due to missing part in the D antigen (D mosaic).
• Du positive individuals are classified as Rh positive when they donate blood and Rh negative when they receive blood.
• ISBT Numeric Terminology


  Universal language (both eye and machine readable). Adopted a 6-digit number for each authenticated blood group specificity. First three number = represents "system", Second three number = represents "antigen specificity".
• Rh Antibodies


  IgG in nature; react at 37°C or in AHG. Exposure to less than 1mL of Rh (+) red cells can stimulate antibody production in an Rh-negative person. Rh antibodies DO NOT BIND COMPLEMENT.
• Mechanism of red cell destruction by Rh antibodies

  EXTRAVASCULAR HEMOLYSIS
• Rh Typing: Rationale


  The terms "Rh positive" and "Rh negative" refer to the presence or absence of the red cell antigen D. After the A and B antigens, D is the most important red cell antigen in transfusion practice for it has greater immunogenicity than other red cell antigens. In contrast to A and B, however, persons whose red cells lack the D antigen do not regularly have anti-D. Formation of anti-D results from exposure, through transfusion or pregnancy, to red cells possessing the D antigen.
• Rh Typing Reagents


  • anti-D
  • anti-C
  • anti-c
  • anti-E
  • anti-e
• Rh Typing: Procedure


  1. Slide method

  2. Tube method
• Rh Typing: Interpretation


  Agglutination in the anti-D tube, combined with a smooth suspension in the control tube, indicates that the red cells under investigation are D (+)→ Rh positive. A smooth suspension of red cells in both the anti-D and the control tubes is a negative test result.
• Donor blood must be further tested for the presence of weak D antigen → perform antihuman globulin test (indirect) to differentiate weak D expression from Rh negative phenotype.
• Rh Genetics


  The genes are autosomal codominant located on the short arm of chromosome 1. The Rh antigens are inherited as codominant alleles.
• Rh Biochemical Structure


  Non-glycosylated CHON (No carbohydrate attached to the protein). Rh antigens are transmembrane polypeptides and are integral part of RBC membrane.
• Gene frequency of Rh antigen: D 85%, d 15%, C 70%, c 80%, E 30%, e 98%.
• Rules for conversion of Wiener to Fisher-Race terminology


  R= D;r-d, 1 or' = C+e, 2 or = c+E, O or (no prime) =c/e, Z or y= C+E, Rho = D
• Screening Test for Low Titer Group "O" Blood: Rationale is that type O individuals are the universal red blood cell donors due to the absence of A and B antigens on their red blood cells, but their serum/plasma contains naturally occurring anti-A and anti-B that can react to patient's red cells having the corresponding antigens.
• Screening Test for Low Titer Group "O" Blood: Procedure

  1. Prepare 1:50 dilution of group "O" serum/plasma

  2. Label 4 test tubes: UA, UB, DA, DB

  3. Mix the contents of the tubes and cover all tubes with Parafilm

  4. Centrifuge for 15 seconds at 3,400 rpm

  5. Gently dislodge the cell button and examine for agglutination or hemolysis

  6. Interpret and grade the agglutination reaction
• Interpretation of Screening Test for Low Titer Group "O" Blood

  • Low Titer O: Antibody titer is less than 1:50. Whole blood can be safely transfused irrespective of the ABO blood type of the recipient.
  • High Titer O: Antibody titer is more than 1:50. Packed RBC can be safely transfused irrespective of the ABO blood type of the recipient.
  • Inconclusive: Absence of agglutination reaction suggests doubtful results. Further investigation is suggested.