BB - THE RH BLOOD GROUP SYSTEM HISTORY

 Key observation by Levine and Stetson in

INTRODUCTION 1939 that deliveries of stillborn fetus and
 Rh is the most important blood group system adverse reaction in mom to blood
after ABO in transfusion medicine. transfusion from father were related.
 One of the most complex of all RBC blood group - Syndrome in fetus is now referred to as
systems with more than 50 different Rh hemolytic disease of the fetus and newborn
antigens. (HDFN).
 Stetson and Levine (1939) described hemolytic - Syndrome had complicated pregnancies for
transfusion reaction with obstetrical patient decades causing severe jaundice and fetal
 1940- Landsteiner & Weiner reported on an death. “erythroblastosis fetalis”.
antibody made by guinea pigs & rabbits when - Erythroblastosis fetalis (HDN) linked with
they were transfused with Rhesus monkey red anti-Rh by Levine in 1941.
cells  Rh system IDENTIFIED by Landsteiner and
 The antibody discovered which agglutinate 85%
Wiener in 1940.
red blood cells was named Rh
- Immunized animals to Rhesus macaque
 Commonly naturally occurring (IgG antibody)
monkey RBCs.
and therefore are formed by immune stimulus
- Antibody agglutinated 100% of Rhesus and
due to transfusions or baby’s red blood cells
85% of human RBCs.
during pregnancy
- Reactivity paralleled reactivity of sera in
 The genetics nomenclature and antigenic women who delivered infant suffering from
interactions are unsettled.
hemolytic disease.
 This unit will concentrate on the most - Later antigen detected by rhesus antibody
COMMONLY encountered observations, and human antibody established to be
problems and solutions. dissimilar but system already named.
 Since Rh antibodies are IgG they bind best at 37
C and their reactions will be observed with the CLINICAL SIGNIFICANCE
indirect antiglobulin technique
 D antigen after A and B, is the most
 Agglutination reactions are enhanced by high
important RBC antigen in transfusion
protein (albumin), low-ionic strength saline
(LISS), proteolytic enzymes (ficin) and practice.
polyethylene glycol (PEG) - Individuals who lack D antigen DO NOT have
 TERMS “D Positive” and “D Negative” refer only anti-D.
to presence or absence of the Rh antigen D on - Antibody produced through exposure to D
the red blood cell antigen through transfusion or pregnancy.
- Terms”Rh pos” and “Rh neg” are old terms, - Immunogenicity of D greater than that of all
although blood products still labeled as other RBC antigens studied.
such  Has been reported that 80%> of D neg
- Early name “Rho” less frequently used individuals who receive single unit of D pos
 Four additional antigens: C, c, E, e. blood can be expected to develop immune anti-
- Named by Fisher for next letters of alphabet D.
according to precedent set by naming A and  Testing for D is routinely performed so D neg
B blood groups. will be transfused with D neg.
- Major alleles are C/c and E/e
 MANY variations and combinations of the 5 FISHER-RACE: CDE TERMINOLOGY
principal genes and their products, antigens,  Suggested that antigens are determined by 3
have been recognized pairs of genes which occupy closely linked loci
 The Rh antigens and corresponding antibodies  Each gene complex carries D or its absence (d),
account for majority of unexpected antibodies C or c, E or e
encountered  Each gene (except d, which is an amorph)
 Rh antibodies stimulated as a result of causes production of an antigen
transfusion or pregnancy, they are immune
  The order of loci on the gene appears to be  With the exception of d each allelic gene
“DCE” but many authors prefer to use “CDE” to controls presence of respective antigen on RBC.
follow alphabet  The gene complex DCe would cause production
 Inherited from parents in linked fashion as of the D, C and e antigens on the red cells.
haplotypes  If the same gene complex were on both paired
 The gene d is assumed to be present when D is chromosomes (DCe/DCe) then only D, C and e
absent would be present on the cells.
 If one chromosome carried DCe and the other
INHERITANCE AND NOMENCLATURE was DcE this would cause D, C, c, E and e
 Two systems of nomenclature developed prior antigens to be present on red blood cells.
to advances in molecular genetics.  Each antigen except d is recognizable by testing
 Reflect serologic observations and inheritance red cells with specific antiserum.
theories based on family studies.
 Because these are used interchangeably it is Rhesus Genotypes
necessary to understand the theories well
enough to translate from one to the other. Genotype Symbol Rh (D) status
 Two additional systems developed so universal cde/cde rr Negative
language available for use with computer CDe/cde R1r Positive
 Derived from 4 sets of investigators CDe/CDe R1R1 Positive
 Two based on the postulated genetic cDe/cde R2r Positive
mechanism of Rh CDe/Cde R1R2 Positive
 Third describes the presence or absence of a cDE/cDE R2R2 Positive
given antigen
 Fourth based on ISBT terminology WIENER’S THEORY

Gene Frequency in %
D 85%
D 15%
C 70%
C 30%
E 80%
e 98%

FISHER-RACE
 Three loci carry the Rh genes are so closely  Postulated that two genes, one on each
linked that they never separate but are passed chromosome pair, controls the entire express of
from generation to generation as a unit or gene Rh system
complex.  Each gene produces a structure on the red cell
 Below an offspring of the Dce/dce individual will called an agglutinogen (antigen)
inherit EITHER Dce or dce from the parent,  Eight major alleles (agglutinogens) RO, R1, R2, Rz,
never dCe as this would indicate crossing over r, r’, r” and ry
which does not occur in Rh system in man.  Each agglutinogen has 3 factors (antigens or
epitopes)
- The three factors are the antigens
expressed on the cell
 Each agglutinoen can be identified by its parts
or factors that react with specific antibodies
(antiserums)
WIENER AND FISHER-RACE
 The two theories are the basis for the two
notations currently used for the Rh system.
  Immunohematologists use combinations of
both systems when recording most probable Converting Wiener into Fisher-Race or Vice-Versa
genotypes.
 You MUST be able to convert a Fisher-Race
notation into Wiener shorthand, i.e., Dce
(Fisher-Race) is written R0.
 Given an individual’s phenotype you MUST
determine all probable genotypes and write
them in both Fisher-Race and Wiener notations.
 R1r is the most common D positive genotype.
 rr is the most common D negative genotype

COMPARISON of WIENER and FISHER RACE

ROSENFIELD
 In 1962 proposed a nomenclature based ONLY
on serologic (agglutination) reactions.
 Antigens are numbered in the order of their
discovery and recognition as belonging to the
Rh system.
 No genetic assumptions made
 The phenotype of a given cell is expressed by
the based symbol of “Rh” followed by a colon
and a list of the numbers of the specific antisera
used.
 If listed alone, the Antigen is present
(Rh:1 = D Ag)
 If listed with a “-“, antigen is not
present (Rh:1, -2, 3 = DcE)
WIENER AND FISHER RACE  If not listed, the antigen status was not
determined
 Adapts well to computer entry
Rosenfield and co-workers

Major antigen Numeric representation

D Rh1
C Rh2
E Rh3
c Rh4
e Rh5

International Society of Blood Transfusion

 Abbreviated ISBT
DIFFERENTIATING SUPERSCRIPT FROM SUBSCRIPT
 International organization created to
 Superscripts (Rh1) refer to genes
standardized blood group system
 Subscripts (Rh1) refer to the agglutinogen
nomenclature.
(complex of antigens)
 Assigned 6 digit numbers for each antigen.
 For example the Rh1 gene codes for the Rh1
 First 3 numbers indicate the blood group
agglutinogen made of D,C,e
system eg., 004 = Rh
 Usually, this can be written in shorthand,
 Last 3 numbers indicates the specific
leaving out the “h”
antigen. e.g., 004001 = D antigen
 DCe is written as R1
  For recording of phenotypes, the system adopts  Typing sera continue to be the “gold standard”
the Rosenfield approach but this will change in the future.

Phenotyping and Genotyping GENOTYPING FREQUENCIES

 The phenotype is the result of the reaction  Refer to textbook
between the red cells and antisera  Genotypes are listed as “presumptive” or “most
 The genotype is the genetic makeup and can probable”.
be predicted using the phenotype and by  Genotypes will vary in frequency in different
considering the race of an individual racial groups.
 Only family studies can determine the true
genotype Gene Shorthand %Caucasians % Blacks
 Five reagent antisera available. Complex
 Only anti-D required for routine testing. Dce R0 2 46
 Other typing sera used for typing rbcs to DCe R1 40 16
resolve antibody problems or conduct DcE R2 14 9
family studies. dce r 35 25
 Agglutination reactions (positive and negative)
will represent the phenotype. WEAK EXPRESSION OF D
 No anti-d since d is an amorph.  Not all D positive cells react equally well with
 Use statistical probability to determine most anti-D
probable genotype.  RBCs not immediately agglutinated by anti-D
must be tested for weak D.
RH PHENOTYPING 
Incubate cells with anti-D at 37C, coating of
Uses D antigens will occur if present.
 Parentage testing 
Wash X3 add AHG
 Predicting hemolytic disease of the fetus and 
AHG will bind to anti-D coating cells if
newborn (HDFN). present.
 Confirmation of Rh antibody specificity 
If negative, individual is D negative
 Locating compatible blood for recipients with 
If positive, individual is D positive W
Rh antibodies.
Three Mechanisms for Weak D
Protocol  Genetic
 Mix unknown RBCs with Rh antisera  Position effect
 Agglutination indicates presence of antigen on  Mosaic
cell and determines phenotype
 Use published frequencies and subject o Results in differences from normal D expression
information to determine genotype  Quantitative (inherited weak D or
position effects)
PHENOTYPIG AND GENOTYPING  Qualitative (mosaic D; could produce
 Molecular testing becoming more popular Anti-D)
 Cannot use anti-sera on recently used Weak D-Genetic
individuals, molecular testing can  Inheritance of D genes which result in lowered
differentiate densities of D Antigens on RBC membranes,
 Anti-sera not available for some antigens, gene codes for less D.
molecular testing being developed for all Position Effect
blood group genes.  C trans – position effect;
 D zygosity van be determined.  The D gene is in trans to the C gene, eg.. C and
 Fetal genotyping for D can be done on fetal D are on OPPOSITE sides: Dce/dCe
DNA present in material plasma.  C and D antigen arrangement causes steric
 Monoclonal reagents from different hindrance which results in weakening or
manufacturers react differently with variant suppression of D expression
D antigens, molecular test specific.
 POSITION EFFECT  R(cde) gene makes c and e but also
mskes f(ce)
C in trans position to D:  ONLY OCCURS when c and e are the CIS
position
D c e / d C e Weak D  F antigen will NOT be present in trans
position
C in cis position to D:  Rh1 or Ce antigen occur when C and e are in cis
(ex. dCE/dce)
D C e/ d c e NO weak D  Ab rarely encountered but if individual had anti-
f would only react with f positive cells, not cells
PARTIAL D positive for c or e in trans only
 Absence of a portion or portions of the total  F cells clearly marked on antigram of screen and
material that comprises the D antigen. panel cells
 Known as “partial D” (old term “D mosaic”).
G ANTIGENS
D Mosaic/ Partial D  Genes that codes for C or D code for G
 If the patient is transfused with D positive red  G almost invariably present on RBCs processing
cells, they may develop an anti-D alloantibody* C or D
to the part of the antigen (epitope) that is  Anti-G mimics anti-C and anti-D
missing  Anti-G activity cannot be separated into anti-C
*alloantibody – antibody produced with specificity and anti-D
other than self D DELETION
 Very rare
SIGNIFICANCE OF WEAK D  Individuals inherit Rh gene complex lacking
alleles
 Donors  May be at Ee or Ce
- Labeled as D positive  Must be homozygous for rare deletion to be
- Weak D substantially less immunogenic detected
than normal D  No reaction when RBCs are tested with anti-E,
- Weak D has caused severe HTR in patient anti-e, anti-C or anti-c
with ant-D  Requires transfusion of other D-deletion red
 Patients cells because these individuals may produce Ab
- If weak D due to partial D can make with single or separate specificities
antibody to portion they lack  Written as D - - or -D-
- If weak D due to suppression or genetic RH NULL
expression theoretically could give D  Red cells have no Rh antigen sites
positive  Genotype written - - -/- - -
- Standard practice to transfuse with D  The lack of Ag causes the red cell membrane to
negative appear abnormal leading to:
 Weak D testing on donors by transfusion service  Stomatocytosis
not required  Hemolytic anemia
 Weak D testing on patients not required except  2 Rh null phenotypes:
in certain situations  Regular type –gene inherited, but not
exposed
Compound Antigens  Amorph type –RHD gene is absent, no
 Compound antigens are epitopes which occur expression of RCHE gene
due to presence of two Rh genes on the same  Complex AB may be produced requiring use of
chromosome, cis position rare, autologous or compatible blood from
 Gene products include not only products of siblings
single gene but also a combined gene that is LW
also antigenic (f, rh1, etc.)
 F antigen occur when c and e are found in cis  Discovered at same time as Rh antigen
(example: dce/dce)
  LW detected on cells of Rhesus monkeys and  Chemically modified –“relaxed” from
human RBCs in same proportion as D antigen IgG anti-D in low protein medium; false
- Thought was the same antigen but positive; saline control performed;
discovered differences converts to weak D testing
- Named LW in honor of Landsteiner and  Monoclonal source, low protein, blends
Wiener of mAbs
 Rare individuals lack LW yet have normal Rh  Must know the preparation, use, advantages
antigens and limitations of each.
 Can form allo anti-LW
- Reacts mre strongly with D pos than D neg HIGH PROTEIN ANTI-D
cells  IgG anti-D potentiated with high protein and
- Keep in mind when D pos individual appears other macromolecules to ensure agglutination
to have anti-D at IS.
 May cause false positive with rbcs coated with
RH ANTIBODIES Ab
 Diluent control REQUIRED
 Except for rare examples of anti-E and anti-Cw  False positive due to autoagglutininins,
which may be naturally occurring, most occur abnormal serum proteins, antibodies to
from immunization due to transfusion or additives and using unwashed rbcs
pregnancy  Can be used for weak D test.
 Associated with HTR and HDFN
 Characteristics IgM ANTI-D (low protein/saline)
- IgG but may have MINOR IgM component  Prepared from predominantly IgM Ab, scarce
so will not react in saline suspended cells due to difficulty obtaining raw material
(IS)  Reserved for individuals giving false positive
- May be detected at 37 C but most with high protein anti-sera
frequently detected by IAT  Newer saline anti-sera require incubation at 37
- Enhanced by testing with enzyme treated  No negative control required unless AB positive
cells  CANNOT be used by slide test OR weak D test
 Order of immunogenicity: D > c > E > C > c
 Do not bind complement, extravascular Chemically modified
destruction  IgG converted to saline agglutinin by weakening
 Anti-E most frequently encountered antibody disulfide bonds at hinge region, greater
followed by anti-c flexibility, increases span distance
 Anti- C rare as single antibody  Stronger reactivity than IgM antibodies
 Anti-e rarely encountered as only 2% of the  Can be used for slide, tube and weak D test
population is antigen negative  Negative control unnecessary unless AB
 Detectable antibody persists for many years and positive
sometimes for life
 Anti-D may react more strongly with R2R2 cells MONOCLONAL ANTI-D
than R1R1 due to higher density of D antigen on  Prepared from blend of monoclonal IgM and
cells polyclonal IgG
 IgM reacts at IS
DETECTION OF D ANTIGENS  IgG reacts at AHG (weak D test)
 Four types of anti-D reagents  Most frequently utilized reagent
 High protein –faster, increased  Used for tube, slide and weak D test
frequency of false positive; requires use  Negative control unnecessary unless AB positive
of Rh control tube, converts to weak D
testing CONTROL FOR LOW PROTEIN REAGENTS
 IgM (low protein/saline reacting) –low
protein (fewer false positive); long  Diluent used has protein concentration equaling
incubation times; cannot convert to human serum
weak D testing
  False positives due to immunoglobulin coating RH SYSTEM CONTINUES TO GROW
of test RBCs occurs no more frequently than
with other saline reactive anti-sera  Last decade has led to abundance of
 False positives do occur, patient will appear to information detailing genetic diversity of the RH
be AB positive on forward type locus
 Must run saline or manufacturer’s control to  Has exceeded all estimates predicted by
verify serology
 Well over 100 RHD and more than 50 different
PRECAUTION FOR RH TYPING RHCE have been documents
 MUST follow manufacturer’s instructions as  New alleles are still being discovered
testing protocols vary
 Cannot use LAT unless explicity instructed by
manufacturer RH BLOOD GROUP SYSTEM
 Positive and negative controls must be tested in  Discovered in 1940 by Landsteiner & Wiener
parallel with test rbc  Most complex erythrocyte antigen system;
 QC performed daily for anti-D located on chromosome 1
 QC for other anti-seras performed in  Found exclusively on surface of the rbc
parallel with test since these usually not integral part of red cell membrane
tested each day, only when necessary  Primary antigen if present, consider Rh (+)
 Lack corresponding naturally-occurring
SOURCES OF ERROR – FALSE POSITIVE antibodies in serum
 Spontaneous agglutination
 Contaminated reagents CLASSIFICATION/NOMENCLATURE SYSTEM
 Use of wrong typing sera
 Autoagglutinins or abnormal serum proteins Wiener
coating rbcs  Multiple allele hypothesis
 Using anti-sera in a test method other than that  5 antigens: Rh0, rh’,rh”,hr’,hr”
required by the manufacturer  Single locus inheritance system with 8 alternate
common alleles coding for agglutinogen 1
SOURCES OF ERROR – FALSE NEGATIVE individual produces 2 agglutinogens inherited
 Use of wrong anti-serum from both parents
 Failure to add anti-serum to test
 Incorrect cell suspension Rh Antigens
 Incorrect anti-serum to cell ratio  With three integral membrane proteins
 Shaking tube to hard 1. RhD
 Reagent deterioration 2. RhCcEe
 Failure of anti-serum to react with variant 3. Rh-associated glycoproteins (Rh50,
antigen RhAG)
 Anti-serum in which the antibody is directed  D antigen – resides in RhD protein – most
against compound antigen, often problems with immunogenic followed by c, E, C and e
anti-C
Fischer & Race
SUMMARY  Three alleles : D/d, C/c and E/e
 Rh system second to ABO in transfusion  Five antigens: D, C, E, c, e
medicine  D – no D locus – no antigenic products
 Correct interpretation of D is essential to Rosenfeld
prevent immunization of D negative which may  Numerical system
result in HDFN  Rh1 to Rh5
 Most polymorphic of all blood group systems
 Of the five antigens only D testing is required

HEMOLYTIC DISEASE OF THE NEWBORN

  Kidd blood group (e.g. anti Jka and anti-
 Hemolytic –braking of red blood cells Jkb)
 Also called erythroblast fetalis  Duffy blood group (e.g. anti-Fya) and
 Erythroblastosis –refers to making of  MNS and s blood group antibodies
immature red blood cells  To date, antibodies directed against the P and
 Fetalis –refers to fetus Lewis blood groups have not been associated
 Condition occurs when there is an with HDN
incompatibility between the blood types of the WHO IS AFFECTED
mother and baby  Babies affected by HDN are usually in a
 An alloimmune condition that develops in a mother’s second or higher pregnancy, after she
fetus, when the IgG antibodies that have been has become sensitized with a first baby. HDN
produced by the mother and have passed due to Rh incompatibility is about three times
through the placenta more likely in Caucasian babies than African-
 Include ones which attack the red bloos cells in American babies
the fetal circulation SYMPTOMS
During pregnancy symptoms may include:
HOW DOES IT HAPPEN  With amniocentesis, the amniotic fluid
may have a yellow coloring and contain
 Rh incompatibility occurs when the bilirubin
mother’s blood type is Rh negative and her  Ultrasound of the fetus shows enlarged
fetus’s blood type is Rh positive liver, spleen, or heart and fluid buildup
 If some of the fetal blood gets into your in the fetus’ abdomen
blood stream, in your body wil produce After birth, symptoms may include:
antibodies. These antibodies could pass  A pale coloring may be evident due to
back through the placenta and harm the anemia
developing baby’s red blood cells, causing  Jaundice, or yellow coloring of amniotic
very mild to very serious anemia in the fluid, umbilical cord, skin and eyes may
fetus. Your first baby is usually safe because be present. The baby may not look
fetal and maternal blood usually do not mix yellow immediately after birth, but
until delivery jaundice can develop quickly usually
 If your second baby is also Rh positive, within 24 to 36 hours
there’s a risk that your antibodies will attack  The newborn may have an enlarged
her blood cells and cause problems liver and spleen. Babies with hydrops
fetalis have severe edema (swelling) of
WHAT ARE THE OTHER CAUSES the entire body and are extremely pale.
 It may also happen anytime blood cells of the They often have difficulty breathing
two circulations mix such as during a
miscarriage or abortion, or during an invasive DIAGNOSTIC
prenatal testing procedure The diagnosis of HDN is based on history and
(i.e. an amniocentesis or chronic villus sampling) laboratory findings:
 Incompatibility of the ABO blood group  Blood test done on the newborn baby:
 It arises when a mother with blood type o Biochemistry tests for jaundice
O become pregnant with a fetus with a o Peripheral blood morphology
different blood type (type A, B or AB). shows increased reticulocytes
The mother’s serum contains naturally o Erythroblast (also known
occurring anti-A and anti-B, which tend nucleated red blood cells) in
to be of the IgG class and can therefore moderate and severe disease
cross the placenta and hemolyse fetal o Positive direct Coombs test
RBCs. (might be negative after fetal
 Less common causes of HDN include antibodies interuterine blood transfusion)
directed against antigens of the:
 Kell blood group (e.g. anti-K and anti-k)  Blood tests done on the mother:
 o Positive indirect Coombs test, AFTER BIRTH TREATMENT MAY INCLUDE:
testing the presence of Rh  Blood transfusions (for severe anemia)
positive antibodies in the  Intravenous fluids (for low blood pressure)
mother’s blood  Help for respiratory distress using oxygen or a
o Amniocentesis – to measure mechanical breathing machine
the amount of bilirubin in the  Exchange transfusion to replace the baby’s
amniotic fluid damaged blood with fresh blood
o Sampling of some of the blood
from the fetal umbilical cord PREVENTION
during pregnancy to check for  Rh immunoglobulin (RhIg) also known as
antibodies, bilirubin, and RhoGAM
anemia in the fetus  This specially developed blood product
that can prevent an Rh negative
COMPLICATION mother’s antibodies from being able to
 KERNICTERUS –the most severe form of react to Rh positive cells.
hyperbilirubinemia and results from the  Given around the 28th week of
buildup of bilirubin in the brain. This can cause pregnancy. After the baby is born, a
seizures, brain damage, deafness and death woman should receive a second dose of
 HEPATOSPLENOMEGALY the drug within 72 hours.
 INSPISSTAED (thickened or dried) BILE
SYNDROME and/or GREENISH STAINING OF THE
TEETH
 HEMOLYTIC ANEMIA
 DAMAGE OF THE LIVER due to excess bilirubin

TREATMENT
 Once HDN is diagnosed, treatment may be
needed. Specific treatment for hemolytic
disease of the newborn will be determined by
your baby’s physician based on:
 Your baby’s gestational age, overall
health and medical history
 Your baby’s tolerance for specific
medications, procedures or therapies
 Expectation for the course of the
disease your opinion or preference

DURING PREGNANCY, TREATMENT FOR HDN MAY

INCLUDE:
 Intrauterine blood transfusion of red blood cells
into the baby’s circulation

 This is done by placing a needle through

the mother’s uterus and into the
abdominal cavity of the fetus or directly
into the vein in the umbilical cord

 Early delivery if the fetus develops

complications
 If the fetus has mature lungs, labor and
delivery may be induced to prevent
worsening of HDN.