1. Antibody Identification
Rh System
Antibody Identification

2. Rh System
Most important blood group system in blood transfusion medicine.
(after ABO)

3. 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

4. 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

5. 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

6. 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.

7. Rh Genetics
PS1
RHAG
PS CDE LW
RH genes LW genes

8. Rh Biochemistry Extends 12 spans of the RBC membrane
Integral part of the red cell membrane
Linked 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 antigen
Cells 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
IIIc
Iva
Ivb Va Vb Vc VI

11. 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.

12. 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+

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+ cells
Reacts weaker with Rh- cells
Anti-LW reacts strongly with Rh+ or Rh- Cord cells
Anti-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
DTT 1 + 2+ 2 3 4 5
w/0 6 7 8 9 PC
Rh= cord

16. 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.

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

18. 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.

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

20. 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

21. e Variants
hrs
hrb
e mosaic e
V/VS

22. 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

23. e Variant Antibodies hrs antibody is similar to anti-ce
hrb antibody is similar to anti-Ce
Anti-VS can be naturally occurring

24. Anti-hrs 1 + 2+ 2 3 w+ 4 5 6 7 8 9 PC Patient types e+ Cell D C E c eK 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+

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

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

27. 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

28. 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

29. 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

30. 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 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.