Rh System Antibody Identification

Most important blood group system in blood transfusion medicine.

(after ABO)

Rh System

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 Discovery

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 Nomenclature

International Society of Blood Transfusion◦ Uniform nomenclature both eye and machine

readable D = RH1 C = RH2 E = RH3 c = RH4 e = RH5

Rh ISBT Nomenclature

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 Genetics

Extends 12 spans of the RBC membrane

Integral part of the red cell membrane

Linked to membrane skeleton

Rh Biochemistry

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 Antigens

Category

II

IIIa

IIIb

IIIc

Iva

Ivb

Va

Vb

Vc

VI

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 Typing Reagents

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

Cell D C E c e K k Fya

Fyb

Jka

Jkb

M N S s AHGGel

DTT

1 + + 0 0 + 0 + + 0 + 0 + + + 0 2+ 02 + + 0 0 + + + 0 + 0 + 0 + 0 + 2+ 03 + 0 + + 0 0 + 0 + + + + + 0 + 2+ 04 + 0 0 + + + + 0 0 + 0 0 + 0 + 2+ 05 0 + 0 + + 0 + + 0 + 0 + + 0 + w/0 06 0 0 + + + 0 + + 0 + + + 0 + 0 w/0 07 0 0 0 + + + + 0 + + + + 0 + + w/0 08 0 0 0 + + 0 + 0 + 0 0 + + 0 + w/0 09 + + 0 0 + 0 + + + + 0 0 + + 0 2+ 0PC 0 0Rh= cord

2+ 0

Anti-LW

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

Cell D C E c e K k Fya

Fyb

Jka

Jkb

M N S s AHGGel

1 + + 0 0 + 0 + + 0 + 0 + + + 0 0

2 + + 0 0 + + + 0 + 0 + 0 + 0 + 0

3 + 0 + + 0 0 + 0 + + + + + 0 + 0

4 + 0 0 + + + + 0 0 + 0 0 + 0 + 2+

5 0 + 0 + + 0 + + 0 + 0 + + 0 + 2+

6 0 0 + + + 0 + + 0 + + + 0 + 0 2+

7 0 0 0 + + + + 0 + + + + 0 + + 2+

8 0 0 0 + + 0 + 0 + 0 0 + + 0 + 2+

9 0 0 0 + + 0 + + + + 0 0 + + 0 2+

PC 0

Anti-f

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.

Rh Antibodies

Cell D C E c e K k Fya

Fyb

Jka

Jkb

M N S s AHGGel

1 + + 0 0 + 0 + + 0 + 0 + + + 0 3+

2 + + 0 0 + + + 0 + 0 + 0 + 0 + 3+

3 + 0 + + 0 0 + 0 + + + + + 0 + 3+

4 + 0 0 + + + + 0 0 + 0 0 + 0 + 3+

5 0 + 0 + + 0 + + 0 + 0 + + 0 + 3+

6 0 0 + + + 0 + + 0 + + + 0 + 0 0

7 0 0 0 + + + + 0 + + + + 0 + + 0

8 0 0 0 + + 0 + 0 + 0 0 + + 0 + 0

9 + + 0 + + 0 + + + + 0 0 + + 0 3+

PC 0

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

Anti-G

e Variants

e mosaic

hrshrb

V/VS e

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 Variants

hrs antibody is similar to anti-ce

hrb antibody is similar to anti-Ce

Anti-VS can be naturally occurring

e Variant Antibodies

Cell D C E c e K k Fya

Fyb

Jka

Jkb

M N S s AHGGel

1 + + 0 0 + 0 + + 0 + 0 + + + 0 2+

2 + + 0 0 + + + 0 + 0 + 0 + 0 + 2+

3 + 0 + + 0 0 + 0 + + + + + 0 + w+

4 + 0 0 + + + + 0 0 + 0 0 + 0 + 2+

5 0 + 0 + + 0 + + 0 + 0 + + 0 + 2+

6 0 0 + + + 0 + + 0 + + + 0 + 0 2+

7 0 0 0 + + + + 0 + + + + 0 + + 2+

8 0 0 0 + 0 0 + 0 + 0 0 + + 0 + w+

9 + + 0 + + 0 + + + + 0 0 + + 0 2+

PC 0

Anti-hrs

Saline Albumin Enzyme 37°C Incubation AHG

Rh Antibodies - Detection

AHG

Sample Saline Albumin LISS PeG Gel

Anti-D w+ w+ 1+ 2+ 2+

Anti-E 2+ 2+ 2+ 3+ 4+

Anti-C 1+ w+ 1+ 2+ 2+

Rh Antibodies

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

Enzymes

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

Rh Antibodies

Partial RhD typing kit

Bioarray Rh Variant BeadChip CICBC should have the new Bioarray kit available by

end of next year.

On the Horizon