Rh System Antibody Identification
Rh System • Most important blood group system in blood transfusion medicine. • (after ABO)
Rh Discovery • 1939 – Levine and Stetson 1st discovered antibody • 1940 – Landsteiner and Weiner discovered antibody developed by using Rhesus monkey cells. • 1960’s- Discovered the antibody produced by the pregnant woman and the Rhesus monkeys were actually two different, distinct antibodies.
Rh Nomenclature • Fisher Race – D, C, E, d, c, e • 3 sets of genes produce the antigens • Examples: • DCe/dce • DcE/DCE • Weiner – R1, R2, r • Inheritance of all Rh antigens lies under control of one gene • Examples: • R1R2 • R1r’ • R0r
Rh ISBT Nomenclature • International Society of Blood Transfusion • Uniform nomenclature both eye and machine readable • D = RH1 • C = RH2 • E = RH3 • c = RH4 • e = RH5
Rh Genetics • RHD and RHCE genes located on Chromosome 1 • Over 100 RHD and 50 RHCE alleles have been identified • RHAG (RHAG) Rh associated glycoprotein located on Chromosome 6 • LW gene located on Chromosome 4
Rh Genetics • PS1 RHAG PS2 CDE LW RH genes LW genes
Rh Biochemistry • Extends 12 spans of the RBC membrane • Integral part of the red cell membrane • Linked to membrane skeleton
Rh Antigens • D antigen is comprised of multiple epitopes • Persons with one or more epitopes missing from the red cells can produce an immune response when exposed to the common form of the D antigen • Cells generally type normally as D+ since typing reagents are designed to detect multiple epitopes
Rh Antigens • Classification of Partial D Epitopes
Rh Typing Reagents • Early reagents relied on antibodies produced by women sensitized by pregnancy or in hyperimmunized volunteers. • Monoclonal antibody technology was introduced in the 1980’s. • But monoclonal antibodies are specific for a single D epitope • Does not detect all D-positive red cells.
Rh Typing Reagents • Current reagents are blends containing monoclonal IgM antibody plus monoclonal or polyclonal IgG antibody • IgM allows for RT reactivity • IgG allows for AHG testing and detection of Weak D • Must read package insert to see which variants it detects • Gammaclone – reacts at AHG with DVI, DBT, DHar, Crawford • Immucor Series 4 and 5 reagents do not react with Crawford • OrthoBioclone does not react with Dhar or Crawford
Rh Antibodies • Enhanced by enzymes • Not affected by DTT, Chloroquine, EGA treatment
Rh Antibodies • Anti-D vs Anti-LW • Anti-LW reacts with all adult cells • Reacts stronger with Rh+ cells • Reacts weaker with Rh- cells • Anti-LW reacts strongly with Rh+ or Rh- Cord cells • Anti-LW destroyed by DTT
Rh Antibodies • Anti-f • f antigen is expressed on RBCs having c and e on the same haplotype (cis). • R1r DCe/dce • f antigen is not expressed when c and e occur on separate haplotypes (trans). • R1R2 DCe/DcE • 65% Caucasian population, 92% African Americans, 12% Asians
Rh Antibodies • Anti-G • Inseparable anti-CD • G antigen is present on ANY cell with the C or D antigen, or both • But there have been cases of D-C-G+ and D+G- • Must perform adsorb/elution studies to confirm presence • Patient can have both anti-G plus anti-D or anti-C.
Anti-G • Ro (Dce) • Will adsorb out true anti-D while leaving separate anti-C • Antibody will be coating cells after adsorption • Perform elution to harvest coating antibody • Perform antibody identification on eluate • If shows anti-D plus anti-C pattern = ANTI-G • If shows only anti-D, then have separate anti-D and anti-C antibodies
e Variants hrs hrb e mosaic e V/VS
e Variants • The e antigen is considered to be a mosaic • Correct terminology is Partial • The antigens in the mosaic are • e, hrs ,hrb, V, and VS
e Variant Antibodies • hrs antibody is similar to anti-ce • hrb antibody is similar to anti-Ce • Anti-VS can be naturally occurring
Rh Antibodies - Detection • Saline • Albumin • Enzyme • 37°C Incubation • AHG
Enzymes • Enzymes: Ficin, Papain, Bromelin • Rh antibodies show enhanced reactivity with enzyme-treated cells • Enzyme treatment removes structures from the red cell membrane that otherwise interfere with the antigen-antibody complex
Rh Antibodies • Other sources of Rh antibodies • RhIg • WinRho • IVIg • Rutuximab/Rutixan • Other sources of red cell stimulation • Renal transplantation • Red cells still in organ • B cells in graft producing anti-D • Bone grafts • Needle sharing
Rh Antibodies • RhIg/WinRho • Used for the treatment of ITP • Used as a prophylaxis in Rh negative mothers • May contain anti-A, anti-B, anti-C, anti-E, Duffy and Kidd antibodies
Rh Antibodies • IVIg • Manufactured from a pool of 1000 to 100,000 donors • May contain anti-A, anti-B, anti-D, and other antibodies • Used for treatment of WAIHA • Cost - $10,000 a dose (220 lb person @ 2g/Kg)
Rh Antibodies • Rutuximab (Rituxan) • Antibodies directed against CD20 (B cell marker) • Used for treatment of TTP, lymphoma, leukemia, transplant rejection, autoimmune diseases
On the Horizon • Partial RhD typing kit • BioarrayRh Variant BeadChip • CICBC should have the new Bioarray kit available by end of next year.