2 Rh SystemMost important blood group system in blood transfusion medicine.(after ABO)
3 Rh Discovery 1939 – Levine and Stetson 1st discovered antibody 1940 – Landsteiner and Weiner discoveredantibody developed by using Rhesusmonkey cells.1960’s- Discovered the antibody produced bythe pregnant woman and the Rhesusmonkeys were actually two different,distinct antibodies.1939 – Levine & Stetson, ab in woman who delived stillborn infant1940 – Landsteiner, believed same ab as one discovered by Levine. Landsteiner, et al used Rhesus monkey cells to stimulate Ab prod in guinea pigs and rabbits. Originally called the Rh factor1960 – proven that Landsteiner ab was not anti-D but anti-LW. Rhesus factor shortened to Rh – Rh Blood Group System
4 Rh Nomenclature Fisher Race – D, C, E, d, c, e Weiner – R1, R2, r 3 sets of genes produce the antigensExamples:DCe/dceDcE/DCEWeiner – R1, R2, rInheritance of all Rh antigens lies under control of one geneR1R2R1r’R0r
5 Rh ISBT Nomenclature International Society of Blood Transfusion Uniform nomenclature both eye and machine readableD = RH1C = RH2E = RH3c = RH4e = RH5
6 Rh Genetics RHD and RHCE genes located on Chromosome 1 Over 100 RHD and 50 RHCE alleles have beenidentifiedRHAG (RHAG) Rh associated glycoprotein located on Chromosome 6LW gene located on Chromosome 4RHAG acts on PS1 to form PS2. Without PS2, will not produce Rh or LW antigens. RH null phenotypes result from mutations in the RHAG.
8 Rh Biochemistry Extends 12 spans of the RBC membrane Integral part of the red cell membraneLinked to membrane skeleton
9 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 antigenCells generally type normally as D+ since typing reagents are designed to detect multiple epitopes
10 Rh Antigens Classification of Partial D Epitopes Category II IIIa IIIb IIIcIvaIvbVaVbVcVI
11 Rh Typing ReagentsEarly 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 epitopeDoes not detect all D-positive red cells.
12 Rh Typing ReagentsCurrent reagents are blends containing monoclonal IgM antibody plus monoclonal or polyclonal IgG antibodyIgM allows for RT reactivityIgG allows for AHG testing and detection of Weak DMust read package insert to see which variants it detectsGammaclone – reacts at AHG with DVI, DBT, DHar, CrawfordImmucor Series 4 and 5 reagents do not react with CrawfordOrthoBioclone does not react with Dhar or CrawfordChanges in monoclonal abs used can help explain discrepancy in reactions from historyIncreased sensitivity of these reagents helps explain previous D- now typing D+
13 Rh Antibodies Enhanced by enzymes Not affected by DTT, Chloroquine, EGA treatment
14 Rh Antibodies Anti-D vs Anti-LW Anti-LW reacts with all adult cells Reacts stronger with Rh+ cellsReacts weaker with Rh- cellsAnti-LW reacts strongly with Rh+ or Rh- Cord cellsAnti-LW destroyed by DTT
15 Anti-LW Cell + 2+ w/0 D C E c e K k Fya Fyb Jka Jkb M N S s AHG Gel DTT1+2+2345w/06789PCRh= cord
16 Rh AntibodiesAnti-ff antigen is expressed on RBCs having c and e on the same haplotype (cis).R1r DCe/dcef antigen is not expressed when c and e occur on separate haplotypes (trans).R1R2 DCe/DcE65% Caucasian population, 92% African Americans, 12% AsiansCan only tell if on same haplotype by genetic or family studies.We have anti-f unlicensed antisera to type for the f antigen.
17 Anti-f Cell AHG Gel 1 + 2 3 4 2+ 5 6 7 8 9 PC D C E c e K k Fya Fyb JkaJkbMNSsAHGGel1+2342+56789PC
18 Rh Antibodies Anti-G Inseparable anti-CD G antigen is present on ANY cell with the C or D antigen, or bothBut there have been cases of D-C-G+ and D+G-Must perform adsorb/elution studies to confirm presencePatient can have both anti-G plus anti-D or anti-C.Use r’ (dCe) cell or an Ro (Dce) cell to adsorb plasma. r’ cell will adsorb the C but not D. Ro cell will adsorb D but not C. Perform elution on the adsorbing cells. If can adsorb off both D and C, have anti-G. Will show anti-D and anti-C in the eluate.Important to distinguish in prenatal tests. If true anti-D and anti-C, won’t need RhIg. But if Anti-G, patient can still produce anti-D and will need RhIg.
19 Anti-G Cell AHG Gel 1 + 3+ 2 3 4 5 6 7 8 9 PC D C E c e K k Fya Fyb JkaJkbMNSsAHGGel1+3+23456789PC
20 Anti-GRo (Dce)Will adsorb out true anti-D while leaving separate anti-CAntibody will be coating cells after adsorptionPerform elution to harvest coating antibodyPerform antibody identification on eluateIf shows anti-D plus anti-C pattern = ANTI-GIf shows only anti-D, then have separate anti-D and anti-C antibodiesSame scenario with dCe cells. Will adsorb out anti-C and leave anti-D if truly separate antibodies
26 Rh Antibodies AHG Sample Saline Albumin LISS PeG Gel Anti-D w+ 1+ 2+ Anti-E3+4+Anti-C
27 Enzymes Enzymes: Ficin, Papain, Bromelin Rh antibodies show enhanced reactivity with enzyme-treated cellsEnzyme treatment removes structures from the red cell membrane that otherwise interfere with the antigen-antibody complex
28 Rh Antibodies Other sources of Rh antibodies RhIgWinRhoIVIgRutuximab/RutixanOther sources of red cell stimulationRenal transplantationRed cells still in organB cells in graft producing anti-DBone graftsNeedle sharing
29 Rh Antibodies RhIg/WinRho Used for the treatment of ITP Used as a prophylaxis in Rh negative mothersMay contain anti-A, anti-B, anti-C, anti-E, Duffy and Kidd antibodies
30 Rh Antibodies IVIg Manufactured from a pool of 1000 to 100,000 donors May contain anti-A, anti-B, anti-D, and other antibodiesUsed for treatment of WAIHACost - $10,000 a dose (220 lb 2g/Kg)
31 Rh Antibodies Rutuximab (Rituxan) Antibodies directed against CD20 (B cell marker)Used for treatment of TTP, lymphoma, leukemia, transplant rejection, autoimmune diseases
32 On the Horizon Partial RhD typing kit Bioarray Rh Variant BeadChip CICBC should have the new Bioarray kit available by end of next year.