Antibody Identification

Most important blood group system in
blood transfusion medicine.
 (after ABO)



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.

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

International Society of Blood Transfusion
◦ Uniform nomenclature both eye and machine
readable
D = RH1
 C = RH2
 E = RH3
 c = RH4
 e = RH5


RHD and RHCE genes located on
Chromosome 1
 Over 100 RHD and 50 RHCE alleles have been
identified

RHAG (RHAG) Rh associated glycoprotein

LW gene located on Chromosome 4
located on Chromosome 6

PS1
RHAG
PS2
CDE
RH genes
LW
LW genes

Extends 12 spans of the RBC membrane

Integral part of the red cell membrane

Linked to membrane skeleton



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

Classification of Partial D Epitopes
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.

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

Enhanced by enzymes

Not affected by DTT, Chloroquine, EGA
treatment

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
Cell
D
1
+ + 0 0 + 0 + + 0 + 0 + +
+ + 0 0 + + + 0 + 0 + 0 +
+ 0 + + 0 0 + 0 + + + + +
+ 0 0 + + + + 0 0 + 0 0 +
+ 0 2+
0 + 2+
0
0 + 2+
0 + 2+
0
0 + 0 + + 0 + + 0 + 0 + +
0 0 + + + 0 + + 0 + + + 0
0 0 0 + + + + 0 + + + + 0
0 0 0 + + 0 + 0 + 0 0 + +
0 + w/0
+ 0 w/0
0
+ + w/0
0 + w/0
0
2
3
4
5
6
7
8
9
PC
Rh=
cord
C
E
c
e
K
k
Fy
a
Fy
b
Jk
a
Jk
b
M
N
S
s
AHG
Gel
DTT
0
0
0
0
+ + 0 0 + 0 + + + + 0 0 + + 0 2+
0
0
2+
0
0


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
Cell
D
C
E
c
e
K
k
Fy
a
Fy
b
Jka
Jk
b
M
N
S
s
AHG
Gel
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-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+GMust perform adsorb/elution studies to
confirm presence
Patient can have both anti-G plus anti-D or
anti-C.
Cell
D
C
E
c
e
K
k
Fy
a
Fy
b
Jka
Jk
b
M
N
S
s
AHG
Gel
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





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
hrs
hrb
e mosaic
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

hrs antibody is similar to anti-ce

hrb antibody is similar to anti-Ce

Anti-VS can be naturally occurring
Cell
D
C
E
c
e
K
k
Fy
a
Fy
b
Jk
a
Jk
b
M
N
S
s
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
AHG
Gel
0





Saline
Albumin
Enzyme
37°C Incubation
AHG
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+



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

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

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





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)



Rutuximab (Rituxan)
Antibodies directed against CD20 (B cell
marker)
Used for treatment of TTP, lymphoma,
leukemia, transplant rejection, autoimmune
diseases

Partial RhD typing kit

Bioarray Rh Variant BeadChip
 CICBC should have the new Bioarray kit available by
end of next year.