RH Blood Group System

Rh Blood Group System
Rules for converting

Wieners :shorthand into
Fisher-Race terminology
R = D, r = d
1 or prime = C
2 or double prime = E
0 or blank = ce
Any superscript letter =
CE
R1, R2, R0, and r. (~97% of blacks

and whites use only these four)
How to remember?
R0 is most common in blacks, least
common in whites.
r is always second in frequency.
R1 always comes before R2.
Weak D Phenotype
Most D+ rbcs react macroscopically w/ Rgt
anti-D at IS = Rh positive (1+ to 3+ or >)
HOWEVER, some DO NOT = require further
testing (37oC &/or AHG) to determine D status
Further test for Weak D Status

If neg at IS, PCs + anti-D rgt incubated at 37 o C
X 20 mins. (Do not add enhancement media)
After incubation, Centrifuge, observe for
agglutination
If positive, report as Rh+
If negative wash 3X & add AHG, Centrifuge
If NEGATIVE add CC cells & report as Rh Neg if CC
cells agglutinate
If POSITIVE report as Weak D Positive
Weak D Mechanisms
1. Genetically Transmissible
2. Position Effect
3. Partial D (D Mosaic)
Genetically Transmissible
RHD gene codes for weakened expression of
D Ag
D Ag is complete but fewer D Ag sites on rbc
(Quantitative!)
Common in Blacks (usually Dce haplotype)
Rare in Whites
Agglutinate weakly or not at all at IS

Agglutinate strongly at AHG phase
Can safely transfuse D+ blood
components
Position Effect
(Gene interaction effect)
Normally: C is in cis position (DCe/dce)
Defect: C allele converts to trans position
(Dce/dCe, DcE/dCE)
Result: C Ag crowds D Ag (Steric hindrance)
Can safely transfuse D+ blood components
Partial D (D Mosaic)
Missing 1 or more PARTS of D Ag
D Ag comprises many epitopes
PROBLEM
Person types D+ but forms alloanti-D that reacts
w/ all D+ RBCs except their OWN
Partial D: Multiple epitopes make up D Ag. Each color

represents a different epitope of D Ag
A.
B.
Patient B lacks
1 D epitope.
The difference between Patient A & B is a single epitope of the D

Ag. The problem is that Patient B can make an Ab to Patient A
even though both appear to have the entire D Ag present on their
rbcs using routine anti-D typing rgts
No Differentiation In
Weak-D Status Is Made Serologically
In Routine BB
In routine BB we cannot differentiate
w/c mechanism accounts for patients
Weak D status
Weak-D Determination:
Donor Blood
When testing Donor Blood for D Ag, testing
required through all phases
Weak-D testing REQUIRED
We need to know D Status of all Donor Blood

Why?
Main problem is Rh Negative women of child
bearing age & pediatric patients
Donor RBCs are labelled Rh+ if any part of

the D Ag is present on RBC membrane
Recipient Blood
Controversy
AABB Standards: you do NOT have to perform
complete D typing of recipient blood
Most weak-D patients can receive D+ blood w/o
forming anti-D
Partial D is very rare, BUT these patients are
capable of making alloanti-D even though they are
Weak D+
So, some BBs ONLY perform IS D & if negative NO

further D testing & the label recipient Rh (D) neg &
transfuse Rh Neg blood components
Some consider it wasteful to transfuse Rh Neg

blood into Weak-D recipient. The testing
policy is up to each individual facility
Recipients who need complete testing:
Obstetric patients: Weak D status MUST be
determined on all obstetric patients. Why? What
will you transfuse?
Newborn: Need to determine D status on all
newborns. Why?
Rh Antibodies
RBC Immune: IgG (anti-D, anti-C, anti-c, etc.)
Rh Abs do NOT bind completely
Only in extremely rare cases
Cause extravascular hemolysis
Cross the placenta

Cause HDN
Rh Ags are well developed at birth
Rh Ab reactivity is ENHANCED using

enzyme treated rbcs
Rh System Antibodies
1. React optimally
1. 37oC & AHG Phases
2. RBC Immune
2. Transfusion or pregnancy,
IgG, HDN, HTR, etc.
3. Clinically
Significant
3. Result in shortened
RBC survival - need to
transfuse Ag neg
blood
Rh Antigen: Typing Reagents

Routine Rh typing for donors & patients
involves typing for only the D Ag. We dont
routinely type for E, e, C or c.
Historically speaking:
Original D typing tests require long saline
incubation times because it is IgG Ab
The goal was to produce an antisera that reacts at
IS
Rh Antigen: Typing Reagents

Saline Anti-D (IgM) Reagent
Reacts strong at IS
One of the 1st IS anti-D rgts
Low protein rgt
Advantage: Can be used to test Ab (IgG) coated
cells
Disadvantages:
Very expensive!! Cost prohibitive
Lengthy incubation
Not used for Du test
D Antigen: Typing Reagents

High protein anti-D
w/ macromolecular additives
Protein enhanced reactivity of IgG anti-D rgt so it would
react at IS
Disadvantages:
False positives because of high protein content

Must run an Rh Control!! Why?
Control rgt is the suspending media in w/c anti-D Abs swim
Advantages:
Reduced incubation time
Both slide & tube testing can be performed
Used for Du test

Chemically Modified Anti-D
Rgt Abs w/ broken disulfide bonds so IgG
anti-D can span distance between RBCs
Advantages:
Few false positives because of Low protein

suspending media
Slide & tube method testing
No need for Rh Control when patient is A, B
or O positive
Need control for AB Pos, Why?
This applies to all the remaining anti-D rgts

Monoclonal Polyclonal Blend Anti-D
(Hybridoma)
Monoclonal anti-D rgts are too specific & may miss
some partial D categories so
Mix monoclonal IgM & polyclonal IgG into one
anti-D reagent:
Increase rxn strength at RT
Able to test Weak-D at AHG phase
Low protein suspending media: No control

necessary
Non human derived, less transmission of infection

Monoclonal Blend
Blend monoclonal IgM w/ monoclonal
IgG anti-D
Added multiple clones to increase reactivity
w/ Partial D patients
Low protein rgt: No need for a control
unless patient is what ABO group?
High-Protein
Anti-D
Chemically
Modified
Anti-D
Anti-D
Monoclanal/Polyclonal
Blend
Saline
Anti-D
Description
Polyclonal IgG
anti-D in 2024% protein
IgG anti-D
converted to direct
agglutinin. In 6%
protein
concentration.
Blend of IgM monoclonal

anti-D and IgG polyclonal
anti-D. Low protein content.
IgM anti-D
suspended in
saline
Protein
concentration
High
Low
Use
Routine typing
When Rh control is
positive with high
protein reagent
Low
When Rh control is positive

with high protein reagent
Low
When Rh control
is positive with
high protein
reagent
High-Protein
Anti-D
Chemically Modified
Anti-D
Anti-D
Monoclanal/Polyclonal Blend
Saline
Anti-D
Control
Same
ingredients as
reagent, except
no anti-D.
Should be
purchased
from same
manufacturer
as anti-D.
ABO forward
grouping serves as
control for all
except type AB. For
type AB, an
autocontrol, 6%
albumin, or saline
may be
recommended.
Follow the
manufacturers
instructions.
ABO forward grouping

serves as control for all
except type AB. For type
AB, an auto-control, 6%
albumin, or saline may be
recommended. Follow the
manufacturers instructions.
6%bovine
albumin
Used for weak D

testing
Yes
Yes
Yes
No
High-Protein
Anti-D
Comments
High protein
content
enables reagent
to react in
immediate
spin. Falsepositive results
if RBCs have a
positive DAT.
Chemically
Modified
Anti-D
Anti-D
Monoclanal/Polyclonal
Blend
Saline
Anti-D
Reacts in immediate
spin without high
protein
concentration.
Lower incidence of
false-positives with
antibodu-coated
RBCs.
IgM anti-D reacts with D

antigen in immediate spin.
IgG anti-D enables reagent to
be used to detect weak D.
Lower incidence of falsepositives with antibodycoated RBCs.
Lower incidence
of false-positives
with antibody
coated RBCs
Selection of Rh Type
Recipient Type
Rh Type Patient Can Receive
Rh positive
Rh positive or Rh negative
Weak D
Rh positive or Rh negative
Rh negative
Rh negative only, especially women of childbearing age. (If Rh

positive must be given in an emergency, Rh immune globulin can be
given to prevent immunization.)
FALSE POSITIVES
CAUSE
ACTION
RBC SUSPENSION TOO HEAVY

COLD AGGLUTININ
INC T TOO LONG, DRYING
(SLIDE)
ROULEAUX
FIBRIN INTERFERENCE
CONTAMINATING LOW
INCIDENCE Ab IN RGT OR
KNOWN SERUM Ab
POLYAGGLUTINATION
BACTERIAL CONTAMINATION
INCORRECT RGT SELECTED
ADJUST, RETYPE
WASH W/ WARM SALINE,
RETYPE
FF INSTRUCTIONS
SALINE WASH CELLS, RETYPE
SALINE WASH CELLS, RETYPE
USE OTHER RGT
INCIDENCE Ab IN RGT OR
KNOWN SERUM Ab
USE MONOCLONAL Abs
USE NEW RGT, OF RGT
RETYPE
REPEAT TEST, READ VIAL
LABEL CAREFULLY
FALSE NEGATIVES
CAUSE
ACTION
Ig COATED CELLS IN VIVO

SALINE SUSP CELLS
(SLIDE)
FAILURE TO FF
INSTRUCTIONS
RESUSPENSION TOO
VIGOROUS
INCORRECT RGT
SELECTED
VARIANT Ag
REAGENT DETERIORATION
SALINE BASED RGT

UNWASHED CELLS
REVIEW DIRECTIONS,
RETYPE
GENTLE RESUSP
READ VIAL LABEL
REF
NEW RGT
Rh Null Phenotype
Persons lack ALL Rh Ags
Lack both RHD & RHCE genes
No D, C, c, E, e Ags present on RBC membrane
Demonstrate mild hemolytic anemia (Rh Ags are

integral part of RBC membrane & absence
results in loss of membrane integrity)
Reticulocytosis, stomatocytosis, slight decrease in
hgb & hct, etc.
ONLY Rh Null blood can be used for

transfusion
Other Rh Antigens
Cw Ag
Usually found in combination w/ C or c Ags
2% whites, rare in blacks
Anti-Cw seen in BOTH RBC Immune (Transfusion &
pregnancy) & NON RBC Immune situations
f (ce) Ag
c & e in cis position, same haplotype
Compound Ag (ce), however f is a single Ag
anti-f : test w/ R1R2 (f negative) & R1r (f positive)
rbcs
Other Rh Antigens
rhi (Ce) Ag
Also a compound Ag
C & e in the cis position
R1R2 is positive for the rhi Ag
R0Rz is negative for the rhi Ag
G Ag
G Ag is generally weakly expressed & is associated w/
the presence of the VS Ag
Almost invariably present on RBCs possessing either C
or D Ags
Abs to G appear to be anti-C+D, but anti-G activity
CANNOT be separated into anti-C & Anti-D
Other Rh Antigens
V, VS antigens
Deletion Phenotype: D-- or -D Both designations indicate the same phenotype
C, c, E, e Ags are absent from RBC membrane
in this phenotype
Very strong D Ag expression: STRONGEST
CAN make Abs to all missing Ags. Usually
make anti-Rh17 Ab
Kliehauer Betke (Acid Elution)

Principle: Draw a Post Partum EDTA sample
from the mother and make and fix a blood smear
on a glass slide. Flood the smear with an acid
solution. The Hgb of adult red cells is washed out
by the acid solution while red cells with Hgb F are
not. Counter stain (Safranin) the smear. Cells with
Hgb F stain red while the adult red cells remain
transparent.
Count number of stained Hgb F red cells within
2000 adult (Hgb A) red cells.
Kliehauer Betke Stain

Calculations
# Fetal cells / 2000 adult cells x 100 = % of
Fetal cells present in the maternal circulation.
% of Fetal cells X 50 = number of mls of Fetal
bleed
# of mls of fetal bleed / 30 = # vials of RhIg
required

RH Blood Group System

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RH Blood Group System

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Rh Blood Group System

Rules for converting

R1, R2, R0, and r. (~97% of blacks

Further test for Weak D Status

Agglutinate weakly or not at all at IS

Partial D: Multiple epitopes make up D Ag. Each color

The difference between Patient A & B is a single epitope of the D

We need to know D Status of all Donor Blood

Donor RBCs are labelled Rh+ if any part of

So, some BBs ONLY perform IS D & if negative NO

Some consider it wasteful to transfuse Rh Neg

Cross the placenta

Rh Ab reactivity is ENHANCED using

1. 37oC & AHG Phases

Rh Antigen: Typing Reagents

Rh Antigen: Typing Reagents

D Antigen: Typing Reagents

False positives because of high protein content

D Antigen: Typing Reagents

Few false positives because of Low protein

D Antigen: Typing Reagents

Low protein suspending media: No control

D Antigen: Typing Reagents

Blend of IgM monoclonal

When Rh control is positive

ABO forward grouping

Used for weak D

IgM anti-D reacts with D

Rh Type Patient Can Receive

Rh negative only, especially women of childbearing age. (If Rh

RBC SUSPENSION TOO HEAVY

Ig COATED CELLS IN VIVO

SALINE BASED RGT

Demonstrate mild hemolytic anemia (Rh Ags are

ONLY Rh Null blood can be used for

Kliehauer Betke (Acid Elution)

Kliehauer Betke Stain

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