Antibody Identification 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. 1939 – Levine & Stetson, ab in woman who delived stillborn infant 1940 – 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 factor 1960 – proven that Landsteiner ab was not anti-D but anti-LW. Rhesus factor shortened to Rh – Rh Blood Group System

Rh Nomenclature Fisher Race – D, C, E, d, c, e Weiner – R1, R2, r 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 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 RHAG acts on PS1 to form PS2. Without PS2, will not produce Rh or LW antigens. RH null phenotypes result from mutations in the RHAG.

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 Category II IIIa IIIb IIIc Iva Ivb Va Vb Vc VI

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 Changes in monoclonal abs used can help explain discrepancy in reactions from history Increased sensitivity of these reagents helps explain previous D- now typing D+

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

Anti-LW Cell + 2+ w/0 D C E c e K k Fya Fyb Jka Jkb M N S s AHG Gel DTT 1 + 2+ 2 3 4 5 w/0 6 7 8 9 PC Rh= cord

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 Can only tell if on same haplotype by genetic or family studies. We have anti-f unlicensed antisera to type for the f antigen.

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 Jka Jkb M N S s AHG Gel 1 + 2 3 4 2+ 5 6 7 8 9 PC

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. 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.

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 Jka Jkb M N S s AHG Gel 1 + 3+ 2 3 4 5 6 7 8 9 PC

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 Same scenario with dCe cells. Will adsorb out anti-C and leave anti-D if truly separate antibodies

e Variants hrs hrb e mosaic e V/VS

e Variants e, hrs ,hrb, V, and VS 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

Anti-hrs 1 + 2+ 2 3 w+ 4 5 6 7 8 9 PC Patient types e+ Cell D C E c e K k Fy a b Jk M N S s AHG Gel 1 + 2+ 2 3 w+ 4 5 6 7 8 9 PC Patient types e+

Rh Antibodies - Detection Saline Albumin Enzyme 37°C Incubation AHG Albumin reduces zeta potential around cells

Rh Antibodies AHG Sample Saline Albumin LISS PeG Gel Anti-D w+ 1+ 2+ Anti-E 3+ 4+ Anti-C

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 Bioarray Rh Variant BeadChip CICBC should have the new Bioarray kit available by end of next year.