1. Rh BLOOD GROUP SYSTEM AHLS 311

2. HISTORY u Ab in serum of mother of stillborn child; responsible for the death of fetus? (1939, Levine and Stetson) u Rb-derived Ab to Rhesus monkey RBCs reacts with 85% of human subjects; same Ab as reported by Levine? (1940, Landsteiner and Weiner) u Erythroblastosis fetalis (HDN) linked with Anti-Rh (1941, Levine et al )

3. NOMENCLATURE: 4 VERSIONS u Fisher Race u Suggested 3 sets of closely linked alleles (D and d, C and c, E and e) u Each gene (except d, which is an amorph) causes production of an Ag u Inherited from parents in linked fashion as haplotypes u See Tables 6-1 and 6-2

4. NOMENCLATURE u Weiner u Multiple alleles at 1 complex locus u 1 locus encodes for production of an agglutinogen which has 3 factors (antigens or epitopes) u Abs can recognize single or multiple factors u See Table 6-3

5. WEINER’S THEORY

6. WEINER & FISHER-RACE TERMINOLOGY

7. 1 ( C) D C 2 ( E ) D c E 0 (neither C or E ) D c e Z (both C & E ) D C E ‘( C) d C e ‘’ ( E ) d cE (neither C or E ) d c e y (both C & E ) d C E D = R d = r

8. NOMENCLATURE u Rosenfield u No genetic assumptions made u Numerical system u If listed alone, the Ag is present (Rh:1 = D Ag) u If listed with a “-”, the Ag is not present (Rh:1, - 2, 3 = DcE) u If not listed, the Ag status was not determined u Adapts well to computer entry

9. COMMON Rh TYPES BY 3 NOMENCLATURES

10. NOMENCLATURE u Internatl. Soc. of Blood Transfusion u 6 digit number for each Ag specificity u First 3 indicate the blood group, eg., 004 = Rh u Last 3 indicates the Ag specificity, eg., 004001 = D Ag of Rh system u For recording of phenotypes, the system adopts the Rosenfield approach

11. Rh PHENOTYPING u Uses u Parentage testing u Predicting hemolytic disease of the newborn (HDN) u Confirmation of Rh Ab specificity u Locating compatible blood for recipients with Rh Abs u Protocol u Mix unknown RBCs with Rh antisera u Take tubes through phases (IS, heat/potentiator, AHG, CCC); record data u Use published frequencies and subject information to determine genotype

13. GENOTYPE FREQUENCIES u Dce (R1)0.42 u dce (r)0.37 u DcE (R2)0.14 u Dce (R0)0.04 u dCe (r’)0.02 u dce (r”)0.01 u DCE (Rz)<0.01 u dCE(r y )<0.01 The probability of 2 frequencies appearing together = the product of those 2 frequencies. For example, DCe/dce occurs with a frequency of 0.42 X 0.37 or 0.155.

14. Rh ANTIGENS u Nonglycosylated proteins (A,B,H are CHOs) u Transmembrane molecules u D and CE are epitopes of proteins with 417 Aas that traverse the membrane 12 X u DNA sequences of D and CE differ by only 44 base pairs; CE, Ce, cd and cE are even more similar to D u Integral part of RBC membrane (Rh null people have mild hemolytic anemia) u Density of Rh Ags on RBCs varies by phenotype (see Table 6-7)

15. MODEL OF Rh PROTEIN

16. D ANTIGEN VARIATIONS u Weak D u Some cells require addition of AHG (IDAT) to demonstrate agglutination with Anti-D u 3 mechanisms causing weak D expression u Genetic - inheritance of D genes which result in lowered densities of D Ags on RBC membranes u C trans - position effect; the D gene is in trans to the C gene, eg., Dce/dCe (C and D Ag arrangement causes steric hindrance weakening D expression) u D mosaic - 1 or more parts of the D Ag is missing; may result in production of Anti-D u People with weak D are considered Rh+ and receive Rh+ blood (except mosaics)

17. D ANTIGEN VARIATIONS u Enhanced D u When c and D are in double doses, eg., cDe/cDe, (C has limiting effect on expression of D) u D-- or D.. represent partial locus deletions; usually seen in consanguinous situations

18. D TESTING u Anti-D reagents u Saline-based - Low protein (fewer false positives); long incubation times; cannot convert to weak D testing u Protein-based - Faster, increased frequency of false positives; requires use of Rh control tube, converts to weak D testing u Chemically modified - “Relaxed” form of Anti-D in low protein medium; few false positives; saline control performed; converts to weak D testing u Blends of mAbs

19. D TESTING u Protocol u Add Anti-D to “D” tube; Rh control to “C” tube u Spin, read and record u If “D” is positive, cells are Rh positive u If “D” is negative, continue testing u Add 22% albumin and incubate for 20” at 37 o C u Spin, read, and record u Wash 3 X in saline u Add AHG, spin, read, and record u If “D” is positive after heat/albumin or AHG  cells are weak D positive; if negative, cells are Rh negative; “C” should always be negative u Add check cells to neg. tubes; spin, read & record

20. WEAK D Ag IN THE LAB u Differences from normal D expression u Quantitative (inherited weak D or position effects) u Qualitative (mosaic D; could produce Anti-D) u If cells are weak D, consider the person to be Rh + u D w not given to D negative recipients u D positives usually OK for D w recipients u D w mothers do not receive RhoGAM u Donors and expectant mothers should be tested for weak D; transfusion recipiencts +/- for weak D testing (D w people may receive D negative blood)

21. OTHER ALLELES AND ANTIGENS u Weak C (C w ) u Not allelic to C and c (C and C w usually seen together) u 2% of whites; very rare in blacks u Anti-C w may be naturally occurring and shows dosage u f (ce) u When c & e are in cis, eg., dce/DCe u Combination Ag u Anti-f may be helpful in phenotyping

22. OTHER ALLELES AND ANTIGENS u Ce u When C and e in cis u Compound Ag u Ab helpful in phenotyping u G u Always found with C-positive RBCs; usually with D-positive cells u Anti G appears to bind to D, C, and G u Many others

23. ALLELIC DELETIONS u No Cc and/or Ee epitopes u DC-, Dc-, D-E, D-- u Enhanced or exalted D Ag expression u Rh null (no Rh Ag expression at all) u ---/--- (double bar rr) u Or, because of independently inherited suppressor genes u If exposed to any Rh Ags, make Abs to those and to Rh 29 (“pan” or “total” Rh) u Causes a mild hemolytic anemia u Rh mod - weakened expression of all Rh Ags

24. Rh ANTIBODIES u Immune IgG Abs (IgG 1 and IgG 3 most important) u React optimally at 37 o C or with AHG u Order of immunogenicity: D > c > E > C > e u Do not bind complement (RBC destruction by Rh Abs is extravascular)

25. Rh Abs: CLINICAL SIGNIFICANCE u Severe HDN u Severe transfusion reactions