Practical Blood Bank

1. Practical Blood Bank Weak D testing (Du) Lab 4

2. D antigen: • The D antigen has greater immunogenicity than other red cell antigens. • Rh antigens are present on red cells only and are not detectable on platelets, lymphocytes, monocytes, neutrophils, or other tissues • The predicted products of both RHDand RHCE are proteins of 417 amino acids that traverse the red cell membrane 12 times and display only short exterior loops of amino acids. Within the red cell membrane. • The Rh polypeptides form a complex with the Rh associated glycoprotein (RhAG),

4. D phenotypes: • D+ persons have full RHD gene expression. • D– persons either lack RHD, which encodes for the D antigen, or have a nonfunctional RHDgene. • Weak D red cells have the D antigen, but have fewer D antigens per cell than normal Rh positive cells • Red cells lacking components of the D antigen have been referred to in the past as “D mosaic” or “D variant.” Current terminology more appropriately describes these red cells as “partial D.” • There is 5 phenotype D (D+, D-, Weak D, partial D, Del)

5. Inheritance • The weak D phenotype was believed to arise from three mechanisms, • Gene Interaction (Position Type) • A C gene in the trans position to D can suppress the D gene. For example, in the genotypes CDe/Cde or cDe/Cde the C gene that is trans to the D gene can suppress it and the person will have a weak form of the D antigen. • Mosaic Type (partial D) • The D antigen is a mosaic consisting of several parts, e.g., DABCD. Most people who are D-positive inherit a gene which produces all parts of the D mosaic. Weak D people inherit a gene which produces only part of the mosaic, e.g., DAB , or DBC • Hereditary • This type of weak D is not well delineated, but it is different from the other two types. It is controlled by D genes that produce a smaller number of D antigens per cell than the normal D gene. However, it is not influenced by a position effect (C in trans to D), and it produces all parts of the D antigen mosaic.

6. The term week D (DU) is widely used to describe cells which have • A quantitative reduction in the expression of their RhD antigen. • Or qualitative variation in RhD antigen expression, these are referred to as partial D.

7. Diagram of amino acid changes in RhD proteins shown as circles.

8. When Du Test should be done • When weak or 1+ reactions are found. Microscopic readings should only be done if mixed field agglutination* is suspected. • When Rh typing discrepancies are found between current and previous results. • When Rh negative neonates born to Rh negative mothers. If the weak D testing is positive, the neonatal Rh type would be reported as "D positive" and the mother would be a candidate for Rh Immune Globulin (RhIG). • By definition, weak D phenotype is characterized by • Negative reaction with anti-D reagent at immediate spin (IS), • Negative reaction after 37C incubation, and • Positive reaction at anti-human globulin (AHG) phase.

9. PRINCIPLE • To test for a weak expression of the D antigen. • Red cells that react weakly or not at all in direct agglutination tests with anti-D may react with anti-D by the indirect Antiglobulin test (IAT). • Red cells that fail to react 2+ in direct agglutination tests with anti-D are incubated with anti-D at 37° C and examined for agglutination. The red cells are washed to remove unbound antibody (IgG anti-D), then tested with anti-IgG.

10. Specimens • Clotted or anticoagulated whole blood Reagents & Equipments • 37oC incubator • Wash bottle with normal saline • Coombs serum - either polyspecific or anti-IgG • Coombs control cells (IgG coated control cells ). • All reagents, equipment, and supplies used in the Rh testing procedure

11. Recommended techniques SLIDE TECHNIQUE • Add to a clean, labeled slide: • One drop of anti-D IgM and IgG blend. • One drop of the test red cells suspension. • Mix well by gently and continuously rocking the slide for approx. 30 seconds and incubate the slide for 5 minutes at room temperature, with occasional mixing, we can put the slide on source of light as heat source (Box Lamp) • Examine macroscopically for agglutination. A diffuse light source may aid reading.

12. TUBE TECHNIQUE – IMMEDIATE SPIN • Prepare a suspension of test washed red cells 2-3% or 1.5 - 2% in LISS. • Place in a small, labeled test tube: • 1 volume of anti-D IgM & IgG blend. • 1 volume of suspended red cells. • Mix well, then Centrifuge immediately for 10 seconds at 1000g or for a suitable alternative force and time. • Agitate the tube gently to dislodge the cell button and examine macroscopically for agglutination. • Apparently negative tests which are to be tested for week D should be further tested by the DU test method.

13. TUBE TECHNIQUE – LISS • Place in a small, labeled test tube: • 1 volume of Anti-D blood grouping reagent • 1 volume of red cells suspended 1.5-2% in LISS. • Mix well and incubate for 15-20 minutes at 37°C. • Centrifuge immediately for 10 seconds at 1000g or for a suitable alternative force and time. • Agitate the tube gently to dislodge the cell button and examine macroscopically for agglutination.

14. DU TEST METHOD (indirect antiglobulin test (IAT). ) • After reading the immediate spin results, re-incubate the test for a further 20 minutes at 37°C before completing the DU test method described below. OR • After reading the LISS tube test, complete the DU test, without further incubation, following the procedure given next.

15. DU TEST METHOD • Wash the test 4 times with a large excess of PBS (e.g. 4ml of PBS per 12 (or 10) x 75mm glass tubes). NOTE: Allow adequate spin time to sediment the red cells make sure that most of the residual saline is removed at the end if each wash to leave a ‘dry’ cell button. • Add two drops of anti-human globulin reagent to each tube. • Mix thoroughly. • Centrifuge at 1000g for 10 seconds or for a suitable alternative force and time. • Agitate the tube gently to dislodge the cell button and examine macroscopically and microscopically for agglutination. • Add 1 drop of IgG-coated control cells to the tube(s) with negative results. Centrifuge, resuspend cells, read macroscopically and record results. Agglutination (2+) shall be present or the test shall be repeated.

16. Procedural Notes • Tests should be read immediately after centrifugation. Delay may cause bound IgG to dissociate from red cells and either leave too little IgG to detect or neutralize AHG reagent causing false negative results. Interpretation • A negative result in the immediate spin phase but agglutination in the D tube following incubation (with no agglutination in the DC tube) indicates a positive test for weak D. • Lack of agglutination is a negative test and the patient is considered truly D negative. Agglutination in the DC tube invalidates the test.

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