RH BLOOD

GROUP SYSTEM
Part 3

MLS 404
RH BLOOD GROUP
SYSTEM
HISTORY OF THE RH SYSTEM
 Levine and Stetson described a hemolytic transfusion
reaction in an obstetric patient following delivery of
stillborn infant. Her husband, who had the same ABO
type, was selected as her donor. After transfusion she
demonstrated the classic symptoms of acute hemolytic
transfusion reaction.

 A year later, Landsteiner and Wiener reported on an

antibody by guinea pigs & rabbits when they were
transfused with rhesus monkey red cells. This antibody
which agglutinated 85% of human red cells was named
Anti-Rh.

 It was found out that the two antibodies were different.

The name Anti-Rh was retained for the human produced
antibody while the Anti-rhesus formed by the animals was
renamed anti-LW (Landsteiner and Wiener) – ISBT 016
RH SYSTEM
 ISBT 004
 Discovered by Levine and Stetson
 SECOND MOST IMPORTANT blood group in
terms of transfusion
 Cause of MOST SEVERE HDFN
 Phenotypically related to Anti-LW
 Antibody produced after transfusion with
red cells from Rhesus monkey
(Macaca mulatta)
 In the mid 1940s, it was made up of 5
antigens nut now it has 50 antigenic
specificities
RH GENETIC INHERITANCE THEORY
1. Fisher Race/ UK Theory
 Theory of pairs of linked genes
 3 alleles
 D and d
 C and c
 E and e
2. Wiener Theory/ US theory
 Theory of multiple allelomorphic genes
 Multiple allelic genes occuring at a single
chromosomal loci
3. Molecular Studies
 RHD gene
 RHCE gene
GENE FREQUENCY OF RH ANTIGENS
GENE FREQUENCY (%)
D 85
d (absence of D) 15
C 70
E 30
c 80
e 98
MUST KNOW!
 Frequency:

 e>D>c>C>E>d
 Immunogenecity:

 D>c>E>C>e
 D antigen is the MOST CLINICALLY
SIGNIFICANT OF ALL NON-ABO ANTIGENS
 It is so highly immunogenic that a single
exposure to D-positive blood
(even <0.1mL)results in the formation of
anti-D antibodies in more than 50% of D-
negative individuals.
RH SYSTEM NOMENCLATURE
1. Fisher-Race (DCE)
 Based on the theory that antigen of the systems were
produced by 3 closely-linked set of alleles, each gene was
responsible for producing a product or antigen on
erythrocyte surface
2. Wiener (Rh-hr)
 Postulated that the gene responsible for defining Rh
actually produced an agglutinogen that contained a
series of blood factors, in which each factor is an antigen
recognized by an antibody
3. Rosenfield (Alpha numeric)
 Number is assigned to each antigen of the Rh system in
order of its discovery; has no genetic basis, it only indicates
presence or absence of the Rh antigen
4. International Society of Blood Transfusion
 Employs the use of six-digit number for each blood group
specificity which has an advantage of being eye and
machine readable
 First 3 digits represent the system and the last 3 for the
antigenic specificity
RH NOMENCLATURE
FISHER- WIENER ROSENFIELD ISBT
RACE
D Rho Rh1 004001
C rh’ Rh2 004002
E rh’’ Rh3 004003
c hr’ Rh4 004004
e hr’’ Rh5 004005
 FISCHER-RACE WIENER
Blood
Gene Antigen Gene Agglutinogen
Factors
Dce Dce R0 Rh0 Rh0hr'hr''
DCe DCe R1 Rh1 Rh0rh'hr''
DcE DcE R2 Rh2 Rh0hr'rh''
DCE DCE Rz Rhz Rh0rh'rh''
dce ce r rh hr'hr''
dCe Ce r' rh' rh'hr''
dcE cE r'' rh'' hr'rh''
dCE CE ry rhy rh'rh''
TIPPET GENETIC MODEL
GENE RHD RHCE SHORTHAND
COMPLEX GENE GENE NOMENCLATURE
Dce D ce R0
DCe D Ce R1
DcE D cE R2
DCE D CE Rz
dce d ce r
dCe d Ce r’
dcE d cE r’’
dCE d CE ry
NOMENCLATURE
NOMENCLATURE
MUST KNOW!
 ABO antigens on red cells: glycolipids,
glycoproteins, glycosphingolipids

 ABO antigens on secretions: glycoproteins

 Rh antigens: non-glycosylated protein ;

transmembrane polypeptide (integral part
of the red cell membrane)

 Rh null genotype → Stomatocyte

MUST KNOW!
 Dce genotype
 MOST COMMMONLY SEEN IN BLACKS
 DCe genotype
 MOST COMMONLY SEEN IN WHITES
THE NUMBER OF D AG SITES, MEASURED
ON A VARIETY OF RH PHENOTYPES
RH PHENOTYPE # OF D ANTIGEN SITES
R1r 9,900-14,600
R0r 12,000-20,000
R2r 14,000-16600
R1R1 14,500-19300
R1R2 23,000-31000
R2R2 15,800-33300
D-- 110,000-202,000
WEAK D ANTIGEN EXPRESSION
1. C Trans
 Position effect or gene interaction effect which results in
steric hindrance
 The allele carrying D is in trans or opposite haplotype to
the allele carrying C (e.g. Dce/dCe)
 Expression of D is affected by the position effect
 Individuals can receive D positive red cells
2. Weak D
 Weakened expression of D antigen
 D antigens are expressed appear to be complete but
fewer in number
3. Partial D or D Mosaic
 One or more parts of the D antigen is missing, can result
to weakened antigen expression
TESTING FOR D ANTIGEN
 Note: an Rh viewbox may be
employed (45-50 degrees Celsius) in
reading agglutination (within 2 mins)
using slide method to enhance the
reaction (Turgeon)
WEAK D TESTING
1. If negative at IS, patient cells + anti-D →
incubated at 37 degrees Celsius for 20
minutes → centrifuged
2. If still negative → wash x3 → add AHG
3. If negative → add Check cells
→ report as Rh negative if check cells
agglutinate
4. If positive → report as Weak D Positive
Du or the a weakened form of the
D antigen
 D-negative donors and obstetric patients must
be tested for Du using IAT
 Du-positive individuals are classified as D
positive in blood donor testing.
 Rh negative recipients of the blood may develop
alloantibodies against the weak D antigens
 Du-positive
recipients should always receive
D-negative blood.
 Individuals with weak D expression may develop
anti-D in their body which will pose a problem upon
receiving an Rh positive blood unit
A D-negative, Du-positive infant born to a
mother with anti-D in her serum can suffer from
severe hemolytic disease of the newborn (HDN)
RH NEGATIVE PHENOTYPES
1. European ethnicity
 Inheritance of one or two RHCE genes and
deletion of RHD genes → dCe/dCE
2. African ethnicity
 RHDψ / RHD pseudogene “psi”
 Does NOT produce RhD protein
 66% incidence
3. Asian ethnicity
 Termed as Del
 Individuals possess extremely low number of D antigen
sites that most reagent Anti-D are unable to detect
 Adsorbing and eluting anti-D from the red cells is the
only way to detect the D antigen
 10-30% incidence
RH ANTIBODIES
 IgG
 react optimally at 37 oC or following the addition
of antiglobulin reagent
 Clinically significant
 Primary response = 120 days
 Secondary response = 3-7 days
 Often persists in the circulation for years
 Do not bind complement
 For complement to be fixed, two IgG molecules
must attach in close proximity on the red cell
surface.
 Causes extravascular hemolysis (liver or spleen)
 classically characterizes a delayed hemolytic
transfusion reaction
RH REAGENTS
1. Saline reactive reagents
2. High protein reagents
3. Chemically-modified reagents
4. Monoclonal reagents
RH REAGENTS
1. SALINE REACTIVE REAGENTS
 uses IgM antibodies
 Advantages: low-protein based,
can be used to test cells coated
with IgG
 Disadvantages: limited availability,
cost of production, lengthy
incubation time, cannot be used
for Weak-D typing
RH REAGENTS
2. HIGH PROTEIN REAGENTS
 Has Anti-D added with potentiators of
bovine albumin and macromolecular
additives such as dextran or
polyvinylpyrrollidone
 Advantages: reduced incubation
time, can be used for both slide
typing and weak-D testing
 Disadvantages: prone to false-
positives due to high protein content,
requires a saline negative control
RH REAGENTS

3. MONOCLONAL ANTIBODY REAGENTS

 Derived from single clones of antibody-
producing cells hybridized with
myeloma cells
 Advantages: very specific and sensitive,
can be used for slide, tube, microwell
and automated Rh typing methods,
does not transmit infectious diseases (as
they are not human-derived)
 Disadvantages: expensive
RH REAGENTS
4. CHEMICALLY MODIFIED REAGENTS
 Anti-D IgG molecule with broken
disulfide bonds at the hinge region to
increase antibody span
 Advantages: more sensitive, does not
require a negative control if patient is
Type A, B or O, fewer false-positives,
can be used for slide and tube typing
 Disadvantages: requires a negative
control if patient is Type AB
RH REAGENTS
 Coomb’s cells

 Control Cell
A group O Rh positive donor’s sample is
mixed with 1:10 dilution of reagent anti-D
and allowed to incubate.
After sensitization of RBC, they are washed
with saline and suspended to a 5% RBC
suspension
These sensitized RBCs then used to confirm
the anti-IgG activity of the AHG.
 UNUSUAL RH PHENOTYPES
1. D-DELETION (D-- or -D- )
 No reaction when RBCs are tested with
anti-E, anti-e, anti-C or anti-c
 Requires transfusion of other D-deletion red cells,
because these individuals may produce antibodies
with single or separate specificities
2. RH NULL
 red cells have no Rh antigen sites leading to:
 Stomatocytosis and hemolytic anemia

 2 Rh null phenotypes:
 Regulator type – mutation in the RHAG gene, RHD
and RHCE genes normal but unable to express
 Amorph type –RHAG gene normal, mutated or

deleted RHD and RHCE genes

MUST KNOW!
 Require weak D testing on all donor red
blood cells that do not agglutinate at IS
 DO NOT require weak D testing on recipient
blood
 each facility has their own protocol
 If only IS is performed and patient is negative,
they will receive negative units
 However, some labs don’t like to waste D-
negative units, so they take the test to AHG
 If the patient is positive, they may receive D-
positive units (it would be rare that the patient is
a Partial D)
OTHER RH ANTIGENS
1. COMPOUND ANTIGENS
 cis-product antigens
a) f antigens occur when c and e are
found in cis (e.g.dce/dce)
b) rh1 or Ce antigens occur when C
and e are in cis (e.g. dCe/dCe )
2. G ANTIGENS
 Genes that code for C or D also code
for G;
anti-G mimics anti-C and anti-D
 G antigen present when either C or D
antigen is present
OTHER RH ANTIGENS
3. CW ANTIGEN
 low frequency antigen found in
only 2% of Whites and rare in Blacks
 Most individuals who are C+ are Cw+
 Antibodies to these antigens can be
naturally occuring and may play a role
in HDN and HTR
4. OTHERS: more antigens can be found in
page 164
HEMOLYTIC
DISEASE OF THE
FETUS & NEWBORN
PATHOPHYSIOLOGY
CONDITIONS FOR HDFN TO OCCUR:
❑ The woman must be exposed, either thru
pregnancy or transfusion to a red cell antigen she
lacks
❑ The antigenic exposure must result in
immunization (active) and production of an
antibody
❑ The antibody must be able to cross the placenta
and of sufficient concentration to cause red cell
destruction
❑ The infant must possess and antigen
corresponding to the maternal antibody
MECHANISM OF HDFN
 Immunization (active) and
Production of Antibody
 Fetomaternal Bleed
 During subsequent pregnancies
CAUSES OF HDFN
oCommon Causes:
oABO system antibodies: MOST COMMON
o Rh system antibodies: MOST SEVERE
oUncommon Causes:
oKell system antibodies
oRare Causes:
oDuffy system antibodies
oMNS and s system antibodies
oNo occurrence in HDFN:
oLewis system antibodies
oP1Pk system antibodies
RH HDFN
Conditions:
❑Father must be Rh positive
❑ Mother must be Rh negative
❑ Offspring must be Rh positive
❑The first born is usually not affected
❑HDFN usually occurs in the 3rd child
 Pathogenesis

Fetomaternal Hemorrhage

Maternal Antibodies formed against Paternally

derived antigens

During subsequent pregnancy, placental

passage of maternal IgG antibodies

Maternal antibody attaches to fetal red blood

cells

Fetal red blood cell hemolysis

HDFN EFFECTS
 BEFORE BIRTH
 Anemia (destruction of red cells)
 Heart failure
 Fetal death
 AFTER BIRTH
 Anemia (destruction of red cells)
 Heart failure
 Bilirubinemia
 Kernicterus / Icterus gravis neonatorum
(>20mg/dL)
 Severe growth retardation
DIAGNOSIS
•CBC
•Anemia
•Increased nRBC
•Neutropenia
•Thrombocytopenia
•AMNIOCENTESIS
•Liley’s Method
•SEROLOGIC STUDIES
•IAT- DAT
•Prenatal Antibody detection
•Antibody Titration
•Neonatal Studies
• ULTRASOUND IMAGING
PRENATAL TESTING
 Test Mom for ABO, Rh (Weak D), and
Antibody Screen

 Group O Mom
 Not a problem until baby is born OR is
also Group O.

 Rh Negative Mom
 If she is Rh negative, has she been
administered antenatal RhIg?
 Is this her first or second pregnancy?
PRENATAL TESTING
 Positive Antibody Screen?
 Identify antibody and perform Titration if
antibody is clinically significant (anti-D, -K,
etc.)
 FREEZE the serum sample. If a subsequent
titer is requested you need to compare the
first titer results with the second titer. Run both
titers in parallel and compare endpoints.
 Has the titer increased? Two tube increase

is clinically significant. May lead to more

sensitive testing (Amniocentesis, etc.) to
determine severity of disease.
PRENATAL SEROLOGICAL STUDIES
• AMNIOCENTESIS
• A good indicator of intrauterine hemolysis
and fetal well-being is the level of
bilirubin pigment found in the amniotic
fluid.
• Usually performed on women with
alloantibody or have an antibody titer at
or greater than critical level.
• A change in optical density (OD450)
value of the amniotic fluid in the upper
mid zone of a Liley graph indicates the
need for fetal blood sampling.
LILEY GRAPH
 The amniotic fluid is subjected to a spectrophoto-
metric scan at steadily increasing wavelengths so
that the change in the optical density at 450 nm
(OD450) can be calculated.
 Liley graph plots the change in OD at 450nm
versus gestational age in weeks.
 Zone 1
 Observe fetus for stress-repeat 2-4 weeks
 Zone 2
 Moderate disease: May require treatment
 Zone 3
 Severe problems - Deliver/treat
 Zone 3

Zone 2

Zone 1
PERCUTANEOUS UMBILICAL BLOOD
SAMPLING

 Insertionof a needle into umbilicus

vein and withdrawal of fetal blood.
 Allows direct measurement of
fetal hemoglobin and hematocrit
which gives a better assessment of
fetal anemia.
TREATMENT
❑ INTRAUTERINE TRANSFUSION
▪Correct fetal anemia: <10 g/dl hgb
▪24-26 week gestation
▪Frozen, deglycerolized blood with normal electrolytes,
no anticoagulant or plasma (washed out during
deglycerolization), and low platelets/WBCs.

▪Group O Negative
▪75 to 80% Hematocrit
▪Hemoglobin S negative
▪CMV negative (leukoreduced)
▪Irradiated red blood cells
INTRAUTERINE TRANSFUSION (IUT)
 Methods
 Intraperitoneal: Red cells are infused into fetal
abdomen and absorbed into circulation.
 Intravascular: Red cells are infused directly into
umbilical vein using ultrasound guidance. Quicker
resolution of anemia.
 A combination of methods may be used to avoid
peaks and troughs of fetal hematocrit.
 Once began, the procedure is repeated
every 2 to 4 weeks until delivery.
TREATMENT
◼ EXCHANGE TRANSFUSION
◼ A process where you exchange
baby red cells with transfused red
cells.
Accomplishes the following:
1. Remove antibody coated RBCs:
Not all but many.
2. Removal of maternal antibody.
3. Removal or reduction of bilirubin
4. Replacement of RBCs
COMPATIBILITY TESTING FOR
EXCHANGE TRANSFUSION
 BESTindication of red cell survival is
to crossmatch with the MOTHER’S
SERUM. Remember the source of the
antibody is Mom!

 Can crossmatch with BABY’S SERUM

OR ELUATE if mom is not available.
EXCHANGE TRANSFUSION
 Group O red cells (<7 days old) suspended
in Group AB plasma are commonly used.
 If Mom & Baby are ABO identical, group
specific RBCs or whole blood may be used.
 The blood should also be irradiated to
prevent TA-GVHD
 A volume of twice the infants blood volume
is used.
 CMV-negative, Rh-negative units given for
undetermined types
 For IUT, Hct > 70%
TREATMENT
 PHOTOTHERAPY
 460-490 nm
 Lumirubin is the predominant isomer
formed during high-intensity
phototherapy.
 Decrease in bilirubin is mainly the
result of excretion of these
photoproducts in bile and removal
via stool.
HDFN
RHIG
 The mechanism of
action of RhIG is
uncertain.
 Administered antibodies
will bind the fetal Rh-
positive cells
 Spleen captured these
cells by Fc-receptors
 Spleen remove anti-D
coated red cells prior to
contact with antigen
presenting cells
“antigen deviation”
preventing priming of B
cells to produce Anti-D
WHEN TO GIVE RHIG?
 Antenatal administration
 Given at 28 (to 32) weeks gestation to Rh
negative pregnant women as long as the
antibody screen is negative for anti-D.
 Amniocentesis
 When an amniocentesis is preformed at 16-
18 weeks gestation, mom should receive
full dose.
 Postpartum Administration
 When Mother is Rh negative (and is
negative for allo anti-D) and Baby is Rh
positive.
RH IMMUNE GLOBULIN (RHOGAM)
 FULL DOSE: 300 micrograms of anti-D
 For 15 mL D-positive RBCs or
30 mL whole blood
 100 ug for UK standard

 MINI/MICRO DOSE: 50 g
 For 2.5mL D-positive blood
 For first trimester abortion or
miscarriage
 NOT commonly used
DOSING FOR RHOGAM
•Dosing
•USA/WHO: 300 ug
•UK: 100 ug
•Standard RhoGam Dosage
•Before 12th week of gestation: 1 microdose
•After 12th week of gestation: 1 regular dose
• First Trimester Bleeding or Late Pregnancy Bleeding
•dosage based on Kleihauer-Betke Test
LAB TESTS:
ROSETTE TEST
◼ Purpose: Screening test to detect the
presence of Rh positive RBCs in the
circulation of Rh negative person.
◼ Qualitative: Tells us that there are Rh
Positive cells in an Rh Negative person.
◼ Sensitivity: 10ml of FMH
◼ Procedure:
◼ anti-D + Mother’s washed Post Partum
(EDTA) red cells → incubate at 37oC →
wash + R2R2 indicator cells → Centrifuge
and resuspend the suspension and
read microscopically looking for
rosettes
LAB TESTS:
KLEIHAUER BETKE (ACID ELUTION)
 Purpose: To detect the presence of
Hemoglobin F. If a fetomaternal bleed has
occurred then fetal red cells will be
present in the maternal circulation.
 Quantitative: Can determine the extent of
the fetomaternal bleed. How much fetal
blood entered the maternal circulation.
(And thus how much RhIg to administer!)
 Principle: The hemoglobin of adult red
cells is washed out by the acid solution
while red cells with Hgb F are not. Cells
with Hgb F stain red while the adult red
cells remain transparent
LAB TESTS:
KLEIHAUER BETKE (ACID ELUTION)
 Procedure:

 Mother’s post partum EDTA sample

→ smear and fix → flood with an
acid solution → Rinse slide and
counter stain (Safranin) → Count
number of stained Hgb F red cells
within 2000 adult (Hgb A) red cells
KLIEHAUER BETKE STAIN CALCULATIONS
% of Fetal cells present in the maternal circulation
= No. of fetal cells / 2000 adult cells x 100

% of Fetal cells X 50 = number of mL of Fetal

bleed

# of mL of fetal bleed / 30 = No. of vials of RhIg

required Plus 1

NOTE: We always add one additional dose of

RhIg to insure adequate suppression of immune
production of allo anti-D.
CORD BLOOD STUDIES
• Required testing on the Cord Blood of Newborn’s
with Rh Negative Moms
(suggested on Group O Moms)
• ABO group: If Mom is Group O and Baby is Group
A or B baby may have ABO HDN.
• Rh typing:
• If baby is Rh Negative Mom is NOT a candidate
for RhIg.
• If baby is Rh Positive then she is a candidate for
RhIg.
• Direct Antiglobulin Test: If DAT is positive perform
eluate to identify antibody that is coating the
babies red blood cells.
ABO HDFN
Conditions:
❑ Father is either Type A or B
❑ Mother is Type O
❑ Offspring is Type A or B
PATHOPHYSIOLOGY

o The most common Type of HDN usually

present in about 1 out of 5 pregnancies
o Presents mild or and subclinical
symptoms
o Usually manifest on the firstborn
o Often does not require treatment
COMPARISON
CHARACTERISTIC RH ABO
Clinical Aspect Moderate to Mild
Severe
First Born 5% 50%
Late Pregnancies More Severe No increased
severity
Stillborn/ hydrops Frequent Rare
Severe Anemia Frequent Rare
Jaundice Moderate to Mild
Severe
Frequent
Late Anemia Frequent Rare
 LAB WORKS
LABORATORY RH ABO
FINDINGS
DAT Positive Weakly Positive
IAT Positive Usually Positive
Spherocytosis Rare Frequent
Bilirubin at Birth Increased Normal
Anemia at Birth Yes No
MODES OF TREATMENT
INTERVENTION RH ABO
Phototherapy Yes Yes
Exchange Common Rare
Transfusion
Intrauterine Sometimes None
Transfusion
WHICH MOTHERS ARE CANDIDATES FOR RHIG?

 Mother Rh negative with anti-D (prev RhIg)

 Baby Rh positive with negative DAT

 Mother O negative with anti-D (titer 32)

 Baby A positive

 Mother A negative with positive IAT (anti-D)

 Baby O positive with positive DAT
WHICH MOTHERS ARE CANDIDATES FOR RHIG?

 Mother: A negative with anti-D, C, K

 Baby: B positive, +DAT eluate showed D, C

 Mother:A negative with positive IAT (RhIg)

 Baby: O positive with pos DAT, eluate anti-E

 Mother: AB negative with anti-D

 Baby: A positive with negative DAT
MUST KNOW!
 Rh negative mothers who have
received antenatal RhIg will have a
positive antibody screen for the
presence of anti-D.
 However, they are still eligible for
postpartum RhIg administration as long
as there are no evidence that the
Rh positive baby have already been
affected by HDFN.