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The Rh System

The Rh System. Immunohematology. Objectives. Compare the three theories of inheritance of the Rh antigens. List the antigens and antibodies of the system using both Wiener and Fisher-Race nomenclature. Convert haplotype from Fisher-Race nomenclature into Wiener, and vice versa. Objectives.

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The Rh System

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  1. The Rh System Immunohematology

  2. Objectives • Compare the three theories of inheritance of the Rh antigens. • List the antigens and antibodies of the system using both Wiener and Fisher-Race nomenclature. • Convert haplotype from Fisher-Race nomenclature into Wiener, and vice versa.

  3. Objectives • Discuss key characteristics of antigens and antibodies in the Rh system. • Compare major characteristics of the Rh system to the ABO system. • List the three theories of the weak D antigen.

  4. Objectives • Evaluate reactions of Rh typing, using conventional reagents. • Explain the principle of the weak D test. • Discuss situations when weak D testing would be appropriate.

  5. DISCOVERY • 1939 – Levine and Stetson working with a woman with a fetus was suffering from Hemolytic Disease of the Fetus and Newborn (HDFN). • 1940 – Landsteiner and Wiener working with guinea pigs and rabbits that had been injected with red cell from Rhesus monkeys. • Source of term “Rh factor”. • 85% of human red cells were agglutinated by this antibody.

  6. Presence of D = Rh positive Absence of D = Rh negative May be missing the D gene (whites), or have an amorph at that location (blacks and other ethnicities).

  7. Inheritance • Genes located on chromosome 1. • Alleles are co dominant.

  8. Rh Inheritance: Wiener • Wiener proposed one gene that produced an “agglutinogen” with 3 distinct specificities. Rh0 rh Rh1 rh’ Rh2 rh” Rhz rhy

  9. Rh Inheritance: Fisher-Race • 3 closely linked genes. • Each responsible for expression of one antigen. • The major antigens of the Fisher Race system are: • D • C and its allele c • E and its allele e. D C or c E or e

  10. NOTE:There is no d antigen! d is used to denote the absence of D antigen.

  11. Rh Inheritance: Tippett • Using molecular techniques, Tippett showed in 1993 that Rh inheritance comes from 2 genes. • One gene controls production of the D antigen (RHD). • The second gene controls production of C/E antigen combinations (RHCE). RHD RHCE

  12. Haplotypes • Both Wiener and Fisher-Race nomenclature systems propose haplotypes- genes that are inherited as a unit. • A person inherits one haplotype from each parent. D Ce ce

  13. Antigens

  14. Ro R1 R2 Rz r r’ r” ry Dce DCe DcE DCE dce dCe dcE dCE Haplotypes Wiener Fisher Race

  15. Antigens • Integral transmembrane proteins. • Found only on red cells. • Expression is enhanced with enzyme treatment. • May show variability of expression. • Well developed at birth. • D is highly immunogenic.

  16. Weakened expression of the D antigen. Detected only when using an indirect antiglobulin test. May stimulate production of anti-D. 3 main causes of weak D: Inheritance Gene interaction Partial D (aka mosaic) Weak D

  17. Weak D - Inheritance • Associated with inheritance of Ro. • More commonly seen in Blacks. • The D antigen is normal, but decreased amounts of D antigen is found on the RBCs.

  18. Weak D – Gene Interaction • C inherited on chromosome opposite D. • C in trans position. • D antigen is normal. • Fewer antigens per RBC. D C

  19. Weak D – Gene Interaction Position of C and D antigens when C is inherited in cis position Position of C and D antigens when C is inherited in trans position • When C is inherited in trans position, expression of the D antigen is reduced due to steric hindrance from the C antigen.

  20. Weak D – Partial D • Formerly known as the mosaic model. • Portion of D antigen is missing. • Patient can make anti-D directed at portion of antigen that is missing.

  21. Other Rh Antigens • Cw – Low frequency antigen related to C/c. • G – Found on cells that are positive for either C or D. • Anti-G reacts as if it were anti-D plus anti-C. • ce – Compound antigen. • Formed when c and e are inherited on the same chromosome. • Reacts with anti-f.

  22. Deletions • Deletions – missing all or part of the RHCE gene • E/e “disappears” more frequently (DC -) • C/c “disappears” next (D- -)

  23. Rh Null • No Rh antigens on the RBC • Amorphic: • Both parents have one haplotype that is a total Rh deletion, for example Dce/--- • Each parent passes the deletion on to the offspring. (---/---) • Regulator: • Rh-associated glycoprotein gene (RHAG) missing • RHD and RHCE are normal • Creates a deformity in the RBC membrane leading to Rh Null Disease

  24. Rh Null Disease • Compensated Hemolytic Anemia • Stomatocytes • Increased reticulocytes • Increased HGB F • Can only receive red blood cells products from other Rh Null individuals.

  25. Antibodies • Immune • May react at 37oC • React best in antiglobulin phase • Clinically significant

  26. Antibodies • Do not activate complement • May show dosage • Enhanced by enzymes • Often appear in combinations

  27. ABO vs. Rh

  28. ABO vs. Rh

  29. Rh Typing

  30. TYPING SERA • The Rh typing sera in routine use is anti-D. • Anti-D anti-sera contains antibody to multiple D epitopes.

  31. TYPING SERA • Originally, anti-D was in a high protein medium that would cause spontaneous agglutination in patients whose cells were coated with antibody (Positive DAT). • A protein control (Rh-hr control) was run in parallel on the patient’s cells.

  32. TYPING SERA • More commonly used today is an anti-D in a low protein medium, which does not cause spontaneous agglutination, and therefore does not routinely require a protein control. • Saline based anti-D has also been used to avoid problems with spontaneous agglutination.

  33. Routine TestingTube Method 2 - 5% cells in saline ID D ID Centrifuge at 3500 rpm. Read, grade, record.

  34. Weak D D determination may include a test for weak D. • Incubate at 37oC for 15 to 30 minutes. • Wash with saline (x3) to remove unbound antibody. • Add 2 drops of AHG reagent. • Centrifuge, then read for agglutination. ID D

  35. Populations Requiring the Weak D Test • Donors. • Rh negative infants born to Rh negative mothers. • Any one who historically was typed as Rh positive, but currently is typing as Rh negative.

  36. TYPING SERA • Other Rh typing serum includes anti-C, anti-E, anti-c and anti-e. • These may be high protein reagents (requiring a protein control) or monoclonal reagents.

  37. Phenotype/Genotype • Phenotype: Type for presence of D, C, c, E and e antigens. • Determine most probable genotype based on phenotype results. • Example: A patient’s phenotype is D+, C+, c 0, E 0, e+ Determine the possible genotypes.

  38. Phenotype/Genotype • R1, r and R2 are the most common haplotypes.* • Ro r’ and r” are “mid-range” in frequency. • Rz and ry are rare. * In Caucasians

  39. ABO & Rh TestingGel Method • For the forward grouping, a 3-5% suspension of red cells is made in a diluent. • 10-12.5 uL of the cell suspension is added to the microtubes containing >A, >B, >D, and a control. • For the reverse grouping, 50 uL of a 0.8% suspension of A1 and B cells is added to the buffered gel microtubes, along with 50 uL of patient’s serum or plasma. • The reaction card with the microtubes is centrifuged for 10 minutes. • Read the card for agglutination. Courtesy Ortho-Clinical Diagnostics Raritan, NJ

  40. You are ready to perform ABO and Rh determinations in the lab!

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