Rh GROUPING
Red – Blaney’s
Blue - Harmening
Rh Grouping
 more complex than ABO blood group
system
 2nd most important blood group system
 Group of red cell antigen with the
greatest clinical significance because of
its role in Hemolytic Disease of Newborn
(HDN) & Transfusion
 there are 50 Rh Ag (other books: 57 Rh
Ag)
 Five principle antigens: D, C, E, c, and e
 “Rh positive” means presence of D red
cell antigen
 “Rh negative” means absence of D red
cell antigen
 Absence of D or other Rh antigens does
not correspond to presence of antibody
in plasma
 Type A has Anti-B in their serum
but not Anti-D unless an immune
red cell stimulation from red cells
from D antigen has occurred
Levine and Stetson (1939)
 Reported the first human example of
antibody directed to D antigen
 They found out an antibody in the sera
of a woman whose fetus has fatal HDN.
 After delivering a stillborn infant, this
woman required transfusions. Her
husband, who had the same ABO type,
was selected as her donor.
Wife = Group O, Rh (-)
Husband = Group O, Rh (+)
o *Foreign substance introduce to the
woman: D antigen*
 The discovery was reported as
“Transfusion Reaction” not HDN.
 After transfusion, she suffered from
Severe Hemolytic Reaction aside from
having a stillbirth.
Group 6 | 3A-MT (2016-2017)
o In Rh HDN, the succeeding
pregnancies are affected (first child
not affected because during the first
pregnancy the red cells of the
mother are still being sensitized).
 The mother produced an antibody
against the fetal antigen that the
offspring inherited the antigen from the
father.
 The antibody produced by the mother
causing the hemolytic transfusion
reaction was not identified (not given a
name).
Karl Landsteiner and Wiener (1940)
 discovered the Rh blood group system
 Rh was derived from the Rhesus
monkey whose red cells were injected
into rabbit/guinea pig  antibody
produced by the test animals
reactedwith human RBCs
Red cells w/ agglutination = Rh (+)
Red cells w/o agglutination = Rh (-)
o 80% of us are Rh (+)
o 20% are considered Rh (-)
 At first, they thought that the antibody
produced by the woman and the test
animals were identical. They were
different and were named as:
Anti-LW: antibody produced by the test
animals
Anti-Rh/Anti-D: antibody produced by
the woman/wife.
 Anti-Rh was also found in the sera of
several women who had children who
suffered from:
o Erythroblastosis fetalis
o Hydrops fetalis
o Icterus gravis
Weiner and Peter
 Also observed Anti-Rh in Rh(-) patients
who had received ABO compatible
blood group transfusions of Rh(+) blood
5 Antigens in Rh Blood Group System:
 D, C, E, c, e
o D – most antigenic/immunogenic
(when introduced to an individual
who lacks the antigen, it will
stimulate the production of
antibody); inherited as autosomal
dominant; most important antigen
o D > c > E > C >e
 No problem if Rh (+) – report it
immediately
 If Rh (-), do not report it immediately,
proceed to confirmatory test – Indirect
Antihuman Globulin Test
Rh Antigens
 human-produced antibody
 non glycosylated and no sugar moiety
(compare them to ABO antigens:
precursor is compose of CHOs)
 protein in nature
 Rh locus reside on chromosome 1 along
with the genes for(1) elliptocytosis, (2)
6-phosphogluconate dehydrogenase,
(3) phosphoglucomutase, and (4)
phosphopyruvatehydratase,
 found on RBC membrane
 The structure of Rh antigens are difficult
to describe generally. (But for weakened
exporession of D antigen, it is described
as mosaic or partial)
 Low MW compound–174k
 All 5 Rh antgiens are inherited as
autosomal dominant (D antigen-pinaka
acting as autosomal dominant)
E.g.
Anti – D + D (Rh (+))
Anti – C + C
Anti – E + E
Anti – c` + c
Anti – e + e
Antigens detected: D, C, E, e, c
 Regardless of antigens detected, as
long as you are positive for the D
antigen  Rh (+)
 Theoretically, if C is present, e si also
present. If E is present, c is also
present.
Group 6 | 3A-MT (2016-2017)
 IgG (immune antibody) is acquired
through (1) incompatible transfusion, (2)
incompatible pregnancy, (3) injection of
purified blood group substance, and (4)
incoulation with viral or bacterial
products containing blood group
substances
 There’s no reverse method in Rh typing
because majority of them have IgG
(most can cross the placenta which can
lead to HDN and Hemolytic Transfusion
Reaction).
NOMENCLATURE
1. Fisher Race: DCE Terminology
o 3 closely-linked loci (carrying the Rh
genes) that can be inherited from
parents
o Each parent contributes one haploype
or set of Rh genes
o C, c, E, and e are now considered to be
a part of only 1 gene; hence, 2 closely-
linked genes encode for Rh antigens on
chromosome 1: RHD and RHCE
o RHD determines D antigen expression
on surface of red cells and so D-
negative people would have no genetic
material at this site;
o RHCE determines C, E, c and e antigen
o identified the 5 most important antigens
o “d” = amorph/silent gene (does not
produce a detectable antigen); when
written, it only indicates that D antigen is
absents
o DCE never separate since they are
“closely-linked” (lie close along the
chromosome). When transmitted to an
offspring, they are always in groups of 3
– GENE COMPLEX
o In dCE, crossing over took place
o Rhnull
 no Rh antigens on the RBC
 phenotype —/—
o Rhmod phenotype
 weakened expression of all Rh
antigens
2. Wiener: Rh-Hr Terminology o no genetic basis, nor was it proposed
o proposed that one gene at a single based on a theory of Rh inheritance, but
locus of each chromosome controls the simply demonstrates presence or
entire Rh system absence of the antigen on the RBC
o Each parent contributes one Rh gene  (+) = presence of Ag
o Can be identical (homozygous) or  (-) = absence of Ag
different (heterozygous) from each o each antigenic determinant is given a
parent number in a chronological order of
o 8 alleles exist at Rh gene locus: R0, R1, discovery
R2, R3, r, r’, r”, ry o If red cell is negative for an Rh antigen,
o you only have one gene which a minus sign is written before the
produces an antigen which is called an number
agglutinogen o Advantage : RBC phenotype is thus
o each agglutinogen is made up of 3 succinctly described
blood factors o Disadvantage -there is a similar
o Rh gene produced at least three factors nomenclature for numerous other blood
within an agglutinogen groups such as Kell, Duffy, Kidd,
o R indicates D antigen, r indicates no D Lutheran, Scianna and etc
antigen, 1 and ‘ denote C, 2 and “
denote E (numbers for R, and ‘/” for r), 4. ISBT (International Soceity of Blood
y denotes ce and z denotes CE Transfusion): Numeric Terminology
o Agglutinogen in Wiener nomenclature o Made up of 6 numbers
represents the presence of a single  First 3 = blood group system
haplotype expressing three different  Last 3 = antigen
antigens  ISBT symbol is used to refer to a
specific antigen and is written in

Shorthand designantion: RO uppercase letters and antigen

Some books: RhO (gene) will produce the number immediately follows
agglutinogen Rh0 which is made up of 3
blood factors Fisher
Weiner Rosenfield ISBT
Race
3. Rosenfield: Alphanumeric Terminology
o proposed the system that described the RhO D Rh1 004001
reaction with a particular anti-sera rhI C Rh2 004002
o assigns a number to each antigen of the rhII E Rh3 004003
Rh system in order of its discovery or hrI c Rh4 004004
recognized relationship to the Rh hrII e Rh5 004005
system

FISHER RACE – 8 possible combinations

Fisher Weiner Blood
Antigens Gene Agglutinogen
Race (shorthand desgnation) Factors
Dce D, c, e R0 Rh0 Rh0 Rho, hrI, hrII
DCe D, C, e R1 Rh1 Rh1 Rho, rhI, hrII
Rh (+)
DcE D, c, E R2 Rh2 Rh2 Rho,rhII, hrI
DCE D,C,E Rz Rhz Rhz Rho, rhI, rhII
dce c, e r rh rh hrI, hrII
dCe C, e rI RhI rhI rhI, hrII
Rh (-)
dcE c, E rII rhII rhII rhII, hrI
dCE C, E ry rhy rhy rhI, rhII
Group 6 | 3A-MT (2016-2017)
EXERCISE
Anti – D (+) Anti – D (+)
Anti – C (+) Anti – E (+)
Anti – E (+) Anti – c (+)
Anti – c (-) Anti – e (+)
Anti – e (-) Rh:1,3,4,5 (Rosenfield)
Rh:1,2,3,-4,-5 (Rosenfield) Anti C was not used

Group 6 | 3A-MT (2016-2017)

Variants of Rh
1. CU e.g. Depressed D
2. CX
3. CW(most important variant of C) Genotype: Dce/dCE
4. EU
*C is trans position to D*
5. DU (most important variant of D)

DU
 red cell sample carrying D antigen
 weak form of the D antigen
 has only few antigenic sites
 gives a negative or weak reaction in Rh
typing
 phenotype: D+w.
 not a different antigen but just a different
expression of the D antigen
 rare in Caucasians but more common in
Blacks
 When D antigen is weakly expressed on
red cell, its detection requires the
Indirect Anti-Human Globulin Test
(IAT)
o Red cells that are positive for D
only by IAT is referred to as
Weak D or Du
 Weaker expression results from different
genetic circumstances; only first type of
Weak D needs detection of D antigen by
IAT
 Advantage: Rh (-) individuals that are DU
(+) when exposed to Rh (+) blood do not
produce Anti-D because of the steric
arrangement of C in relation to D
o If C is in cis position to D, DU phenotype DOES
NOT occur thus, normal expression of thantigen.
2. Partial or Mosaic
 There is a difficulty in describing the
structure of Rh antigens
 Molecular Weight: 174,000 Da
 only about the structure of D antigen
which is described as “mosaic,” not the
entire Rh antigens
 one or more epitopes are missing from
the D protein
 due to hybrid genes by the replacement
of the RhD with RhCE

3 Mechanisms D Antigen is Weakly D antigen has 4 fragments

Expressed: or epitopes.

1. C trans
 refers to the position effect of C on the
chromosome
 Presence of steric arrange of the C gene
to the D antigen e.g. Patient: Rh (-)
 Can also be referred to as “Weak
D:Position Effect” When transfused with a D
 Weaker expression of D antigen can be (+) sample, patient will
found when Ce (r’) gene is inherited in produce Anti-D (alloantibody).
trans (inherited on opposite
chromosomes) to the RHD gene
 If paired with R0 and R1, D antigen o Alloantibody is produced in response to a
foreign antigen of the same species.
expression will weaken

e.g. Patient will produce

Anti-C

Group 6 | 3A-MT (2016-2017)

  When exposed to “whole D” antigen,
they can make an antibody to the part
they are missing
 It was termed “D variant” or “D mosaic”
 9 phenotypes classified by part and
epitope
 Partial D should be suspected if D-
positive individuals make an antibody
that reacts with D positive cells but
nonreactive with his or her own cells
3. Genetic Weak D
 all epitopes (antigenic sites) are present
but few in number, D is weakly
expressed on the red blood cells
 IAT using anti-D is usually required
 often part of Dce (R0) haplotype
 more common in blacks
 D antigen is present but few in number
 Due to mutation in the gene
Mutations
 Type 1
 Type 2
2 Types of DU:
A. Low-grade DU
 direct product of inherited gene
 can be passed on to future generation
 detectable only by Indirect Antiglobulin
Test (IAT)
B. High-grade DU
 cannot be passed on to future
generations
 rarely uses IAT for detection
Rh Typing Procedure:
 Red cells + Anti-D
Group 6 | 3A-MT (2016-2017)
 Centrifuge
With agglutination Without agglutination
Rh (+) Rh (-) [preliminary report]
 If no agglutination is observed, incubate
at 37*C for 10-15 minutes.
 Centrifuge at 1500 rpm at 15 seconds.
 Dislodge and observe.
 If no agglutination, wash 3x with NSS.
After last wash, aspirate supernatant.
Make sure to have a cell button.
 Add 2 drops of AHG reagent.
o There is no agglutination observed because red
cells are already sensitized with IgG.
o Anti Human Globulin Reagent – acts as a
bridge inducing agglutination with RBCs coated
with human IgG or complement.
 Centrifuge
 With agglutination: DUor weak D
antigenis detected on red cells
 As recipient: Rh (-)
 As donor: Rh (+)
 Without agglutination:
 As recipient /donor: Rh (-)
[final result]
 Confirm final result (Rh (-)) by adding
the Check Cells.
o Agglutination must be observed upon adding the
check cells because the AHG reagent contains
Anti-IgG while the check cells are group O cells
coated/sensitized with IgG. (anti-IgG + IgG =
agglutination)
Characteristics of Rh Antibodies: 2. RH CE
 Not considered as natural antibodies but - responsible for the expression of CC
rather IMMUNE antibodies and EE antigens
 Usually made by exposure to Rh
antigens through transfusion or ** 3. RH AG
pregnancy - also important
 Agglutination observed by IAT - located on chromosome 6
 Enhancements (for ID) with: High - produces the Rh Associated
Protein, Low-ionic Strength Saline Glycoprotein
(LISS) -called as “CO-EXPRESSOR” because
 Majority are IgG this must be present for the expression
 IgG is associated with Hemolytic of Rh antigens. It doesn’t express any of
Disease of the Newborn (HDN) the Rh antigens by itself.
because it is capable of passing
through the placenta. o A mutation in Rh AG affects the Rh
antigens resulting to:
 IgG 1 and 3 are the most  missing Rh antigens (all Rh
clinically significant (implicated on antigens or just 2 are missing)
severe transfusion reactions) o Rh Null – all Rh antigens
among the other IgGs (IgG 2 and are missing
IgG 4).
 alteration on Rh D or Rh CE
 Other Rh Antibodies:
 IgM (Anti-E) Unusual Rh Phenotypes:
 Anti-Cw (never underwent  Rare
transfusion or were never  Cells that have diminished or
pregnant) undetectable Rh antigen expression
 Stronger reactivity with
homozygous Ag expression 1. Rh deletion/deleted
(dosage) of Antibody to C, c, E, 2. Rh null
and e, but not typical of anti-D 3. Rh mod (moderation)
 Anti-D is typically stronger with
R2R2 red cells because these
Rh deletion Rh null
cells have more D antigen sites (D_ _/D _ _) (_ _ _/_ __)
 Rh antibodies are not associated *missing C & E* *all Ag missing*
with complement activation
 Anti-D and Anti-G are the most Impaired Na and K
important antibodies in the Rh system. ion transport resulting
RED CELL
These are commonly found in Normal to hemolytic anemia
MORPHOLOGY
(spherocytes &
immunized patients.
stomatocytes)
 Anti-G can be Anti-C or Anti-D
 Do not bind complement Increased
number of D
No D, C, E, c, e
RH ANTIGEN antigens per
Antigens
RBC (strong
2 Closely Linked Genes That Control The
reaction)
Expression Of Rh: (found on Chromosome 1)
IN VIVO
Normal Reduced
SURVIVAL
1. RH D
- responsible for the presence or
absence of B antigen

Group 6 | 3A-MT (2016-2017)

  D-Deletion Phenotypes are rare Rh 1. Saline Reactive Anti-Sera
phenotypes where Genetic material has  Saline Anti-D”
been deleted or rendered nonfunctional  also known as low protein-based
at the RHCE site reagent
 Red cells that lack C/c or E/e antigens  contains IgM
may demonstrate stronger D antigen  can also be used to test red cells
activity coated with IgG
 Rhnull appears to have no antigen and  cannot be used for Weak D typing
have membrane abnormalities,  disadvantage: prolonged incubation
shortening their survival time because it’s saline

 Rh Null Syndrome is produced by 2 2. Chemically Modified IgG Sera

genetic systems:  also a low protein-based reagent
 for slide and tube methods
1. Regulator-type  doesn’t contain high molecular weight
 mutation in Rh AG potentiators
 normal comlement of Rh D  using this, antibody molecules have
and Rh CE genes been treated with a reducing agent
 RHAG (Rh associated
glycoprotein 3. Modified Tube Anti-D
 high protein-based reagent
2. Amorphic-type  contains IgG antibodies
 mutation in the Rh CE  contains high concentration of
 deletion in Rh D protein (POTENTIATORS)
 normal Rh AG  containsmolecular additives such as
dextran and polyvinylpyrrolidone
 Rhmod Phenotype  disadvantage: since it has a high
 Similar to regulator Rhnull protein concentration, it will give a false
 Red cells lack most of their positive reaction
antigen for puberty (caused by
o 22% Bovine Serum Albumin (20-30%) is
inheritance of a modified RHA the most commonly used potentiator.
Gene)
3 Varieties of Rh Antibodies:
 Anti-LW is produced by rabbits and
guinea pigs. This reacts strongly with Rh 1. First Order Rh Antibody
(+) blood, weakly with Rh (-), and never  “Saline Agglutinins”
reacts with Rh null.  “Bivalent Antibodies”
 “Complete Antibodies”
 Antibodies react with a specific Rh
Different Anti-Sera for Detection of Rh D antigen in a saline medium which will
and Other Rh Antigens can be: give a strong reaction.
 Antibodies react less strongly in a
 High Protein-based protein medium.
 Low Protein-based
 Saline-based
 Chemically Modified
 Monoclonal/Polyclonal Blended
Reagents

Group 6 | 3A-MT (2016-2017)

2. Second Order Rh Antibody
 “Albumin-Reacting Antibodies”
 “Monovalent Antibodies”
 “Incomplete Antibodies”
 Antibodies react strongly in a protein medium.
 Antibodies and antigens will just combine in a saline medium but will not produce a visible
reaction

3. Third Order Rh Antibody

 “Typical Rh Antibodies”
 “Anti-globulin Antibodies”
 Antibodies react visibly in anti-globulin medium only

A N A L Y S I S:
 Convert genotypes from Wiener to Fischer Race and Rosenfield:

Wiener Fischer Race Rosenfield

R1 r’’ DCe/dcE Rh: 1,2,3,4,5

R2 ry DCE/dCE Rh: 1,2,3,-4,-5

R0r Dce/dce Rh: 1,-2,-3,4,5

r’r’ dCe/dCe Rh: -1,2,-3,-4,5

R2r DcE/dce Rh: 1,-2,3,4,5

ryr dCE/dce Rh: -1,2,3,4,5

Group 6 | 3A-MT (2016-2017)