MODULE 4: RH BLOOD GROUP SYSTEM

Lesson 1: Discovery and Inheritance

Discovery:

 In 1939, ______________________ described a hemolytic transfusion reaction in an

Levine After
obstetrical patient. and stetson
delivering a stillborn infant, the woman required transfusions. Her
husband, who had the same ABO type, was selected as her donor. After transfusion, the
recipient demonstrated classic symptoms of an acute hemolytic transfusion reaction
(AHTR).
 In 1940, ______________________ described an antibody made by guinea pigs and
Landsteiner
rabbits when&they were transfused with ____________________________ RBCs. The
Weiner Rhesus
antibody agglutinated 85% of human RBCs and was named anti-Rh.
macaque
 However, the name Rh was retained for the human produced antibody, and anti-rhesus
formed by the animals was renamed anti-LW in honor of those first reporting it.

By the mid-1940s, five antigens were defined in the Rh system : ___________________
D, C, E, c, e
Inheritance:
Rh Genes
 RHD and RHCE: Chromosome 1

 The gene RHD codes for the presence or absence of the RhD protein, and the
second gene RHCE codes for either RhCe, RhcE, Rhce, or RhCE proteins.
 RHD and RHCE are codominant

 RHAG: Chromosome 6

 The product of this gene is Rh-associated glycoprotein

 RhAG is termed a coexpressor
 Responsible for the formation of Rh antigen-like polypeptides in RBC surfaces.
 When mutations in the RHAG gene occur, it can result in missing or significantly
altered RhD and RhCE proteins: Rh Null phenotype

Rh-Positive Phenotypes

 RH genes are inherited as codominant alleles.

 In addition to the RHD gene(s), two RHCE genes are inherited, one from
each parent.

Rh-Negative Phenotypes

 Rh-negative phenotypes are so called because the RBCs lack detectable D antigen (d)
 The most common Rh-negative phenotype results from the complete deletion of the
RHD gene

Rh Antigens

 Five major antigens: D, C, E, c, e

 "d" = denotes absence of D antigen
 85% of the population = Rh (+); 15% of the pop. = Rh (-)
 Rh phenotypes in Asian =
70% DCe
21% DcE


 3% Dce
3% dce
2% dCe
1% DCE

Weak D: Variation of D antigen Expression: [Du Variant/Du Phenotype]

Position effect: C in trans to RHD (Gene Interaction Effect)

 The Rh antigen on the RBC is normal, but the steric arrangement of the C antigen in
relationship to the D antigen appears to interfere with the expression of D antigen.

 C in trans position to D: Dce/ dCe = weak D antigen (Du variant)

 C in cis position to D: DCe/ DCe = No weak D, Normal Rh(+)

 Molecular studies would differentiate the two types.

Weak D: Quantitative Changes Due to Fewer D Antigen Sites

 Weakened expression of D antigen

 The D antigens expressed appear to be complete but fewer in number.

Partial D or D Mosaic

 One or more D epitopes within the entire D protein is either missing or altered, termed
partial D
 Partial-D antigens can be classified on a molecular level and are attributed to hybrid
genes resulting from portions of the RHD gene being replaced by portions of the RHCE
gene.
 If the patient is transfused with D+ red cells, they may develop an Anti-D alloantibody
to the part of the antigen (epitope) that is missing.

Del

 Del is a phenotype occurring in individuals whose red blood cells possess an extremely
low number of D antigen sites that most reagent anti-D are unable to detect
 Adsorption- elution test
 Common in Asian population (10-30%)

Lesson 2: Nomenclature

Breaking the Rh Secret Code:

If you see… Think…

R/r before h (rh) “Big Letter” antigen To Fisher Race- DcE

h before r (hr) “Small letter” antigen To Wiener- R1r

R Presence of D To Blood factor- Rhorh’rh”

To Rosenfield- Rh 1 -2 -3 4 5
BIG LETTER


 SMALL
r
LETTER
Absence of D (d)

1 or ‘ C or c

2 or “ E or e

0 c+e

z or y C+E

Rh-positive (+) Rh-negative (-)

R0 = Dce r = dce
R1 = DCe r’ = dCe
R2 = DcE r” = dcE
RZ = DCE ry = dCE

Fisher-Race: DCE Terminology

 In the mid-1940s Fisher and Race defined the five common Rh antigens and postulated
that the antigens of the system were produced by three closely linked genes (D/d, C/c,
and E/e).
 Each gene was responsible for producing one antigen.
 The combination of genes inherited from one parent is called haplotype
 The pairing of paternal and maternal haplotypes determines the offspring’s genotype. The
genotype is written as two haplotypes separately.
 If one chromosome carried DCe and the other was DcE (DCe/DcE) this would cause D, C,
c, E, and e antigens to be present on red blood cells.

Gene Frequency of Rh Antigens:

D = 85% d = 15%
C = 70% c = 80%
E = 30% e = 98%
NOTE: Most common Rh phenotype in White and Asian: DCe ; in Black: Dce
CV CV
BIG LETTER
BIG LETTER

Wiener: Rh-Hr Terminology

 Wiener believed there was one gene responsible for defining Rh that produced an
agglutinogen containing three Rh factors.
 Each factor is an antigen recognized by an antibody.
 The original Wiener nomenclature named the five common Rh antigens as Rhₒ, rh’, rh”,
hr’, and hr”, but these terms are no longer used in favour of a modified form of Wiener
notation.
 Modified Wiener terminology allows one to convey Rh antigens inherited on one
chromosome or haplotype and makes it easier to discuss genotype.
 Fisher-Race nomenclature may be converted to Wiener nomenclature and vice versa.


 Rosenfield and Coworkers: Alphanumeric Terminology

 Early 1960’s- Rosenfield and associates proposed a system that assigns a number to
each antigen of the Rh system in order of its discovery/ recognized relationship to the Rh
system.
 Demonstrates the presence or absence of the antigen on the RBC.
 A minus sign preceding a number designates the absence of the antigen.
 For the five major antigens:

 D is assigned as Rh1
 C is assigned as Rh2
 E is assigned as Rh3
 c is assigned as Rh4
 e is assigned as Rh5
SMALL
 For RBCs that type D+ C+ E+ c+ e-
 The Rosenfield designation is Rh: 1, 2, 3, -4, -5
 If the sample was not tested for e, the designation would be: 1, 2, 3, -4
 Adapts well to computer-entry

International Society of Blood Transfusion (ISBT) Committee: Updated Numeric

Terminology

 When there was a need for a universal language in the world of blood transfusion,the
ISBT formed the Committee on Terminology of Red Cell Surface Antigens.
 The ISBT adopted a six- digit number for each authenticated antigen belonging to a blood
group system.
 International organization created to standardize blood group system nomenclature.
 Assigned 6 digit number for each antigen.

 First 3 numbers – indicate or refers to the Blood Group System

 Last 3 numbers – indicate or refers to Specific Antigen

 For recording phenotypes, the system adopts the Rosenfield approach.


 Lesson 3: Rh Antibodies and Its Characteristics
Immunogenicity of Common Rh Blood Group:

 Immunogenecity – Means reactivity or ability of a particular substance such as antigen

of epitope to provoke an immune response to a body of an animal or humans.

 Unlike the ABO antibodies that are mainly IgM, the Rh antibodies are commonly
IgG. They are NOT naturally occurring and therefore are formed by immune stimulus
due to transfusions or baby's red blood cells during pregnancy.
 Rh antibodies are IgG in nature, they bind best at 37 degree Celcius and their reactions
will be observed with the indirect antiglobulin technique (IAT).
 Exposure to less than 0.1mL of Rh (+) RBCs = stimulates antibody production in an
Rh(-) individual.
 Can cross the placenta and cause hemolytic disease of the newborn (HDN)
 Rh-mediated hemolytic transfusion reactions usually result in extravascular hemolysis
 Both Hemolytic Disease of the Newborn and Hemolytic Transfusion Reactions can occur
due the various Rh antibodies. Anti-D has been the biggest concern since it was
recognized in the 1940's as being the most common cause of hemolytic disease of the
newborn.
 To prevent problems due to anti-D:

 Giving Rh-negative individuals Rh-negative blood

 Giving Rho immune globulin to Rh-negative mothers to prevent the formation of
anti-D during pregnancy

The incidence of Rh antibodies

 Anti-D most common antibody seen in Rh(D) negative people

 Anti-E most common antibody seen in Rh (+) people since only 30% of the
population have the antigen
 Anti-C or Anti-c less common - most people have the antigen
 Anti-e often seen as autoantibody and will make it difficult to find compatible blood since
98% of the population have the e antigen

Rh antibodies in Pregnancy

 Rh antibodies formed by pregnant women cross the placenta and may coat fetal RBCs
that carry the corresponding antigen. This results in the fetal cells having a HDN
Direct antiglobulin test; in HDFN, the coated fetal cells are removed prematurely from the
fetal circulation which can result in anemia. HDFN due to anti-D can be prevented with the
use of Rh-Immune Globulin, but complications due to other Rh antibodies cannot be
prevented.

Hemolytic Disease of the Newborn

 is the destruction of the red blood cells (RBCs) of a fetus and neonate by antibodies
produced by the mother.
 IgG of mothers can cross the placenta and destroys the fetal antigens.
 In Rh(D) HDN, the Rh positive first born infant is unaffected because the mother is
not yet immunized. After delivery, variable fetal antigens enter the circulation of the
mother and stimulate the production of anti-D that will affect future offspring
 Rh HDN occurs in Rh (-) mother who gave birth to Rh (+) baby


Pathogenesis of HDN
1. Blood incompatibility between mother and baby
2. TPH occurs
3. Mother is immunized
4. Antibody Production
5. _____ crosses the placenta
6. Hemolysis of fetal cells

 Clinical Features: Anemia of the Fetus (Earliest), Hyperbilirubinemia (Prehepatic),

Hydrops Fetalis

 ______________ is the most severe form of hyperbilirubinemia and results from the
buildup of bilirubin in the brain.

 ABO HDN – Characterized by a weakly positive or negative DAT. Anemia is absent or

very mild, but spherocytes and reticulocytes are increased. Jaundice (Hyperbilirubinemia),
if present, appears 24-48 hours after delivery. Seen most often in Group A or B infants
who have Group O mothers.

Comparison of ABO HDN & Rh HDN

Point of
ABO HDN Rh HDN
Difference

Type of
Naturally Occuring IgG Immune IgG
Antibody

Obstetric Firstborn is unaffected, Disease is

Firstborn may be affected
History more severe in subsequent offspring

Anemia Absent or Mild

Bilirubinemia Peak at _____ days Rapid Rise


 DAT Weakly Positive or Negative

Blood
No Microspherocytes
Picture

Prevention None

Rhogam (RhIg)

 It is administered to a Rh ___ mother within ______ hours after the delivery of a Rh

______ baby.
 It is used to prevent the mother from forming _________
 Purpose:_______________________________________________________

50 ug ___________ 300 ug

D negative females for

D negative females for
abortion or miscarriage during
first ______ weeks of pregnancy
abortion or miscarriage after 12
weeks of pregnancy
If newborn is D positive or Rh
After 34 weeks of gestation cannot be determined or
when amniocentesis is previous immunization with D
performed antigen
Negative for screening test
It can neutralize the effects of up
to _____mL of Rh positive
PRBCs/ _____ mL of whole blood

When is Rhogam given?

 ____________ - during the early 3rd trimester or at about 28 weeks of gestation

 Postpartum - within _________ hours after delivery of an Rh ____________ infant
 Can be given after amniocentesis, abortion, miscarriage or ectopic pregancy of an
unsensitized D-negative woman

Standard Criteria for Administration of Rhogam

1. The mother must be Rh _____________

2. The baby must be Rh _________________
3. The mother must not be immunized to ______________

Fetomaternal hemorrhage (FMH) can be assessed by:

1. Rosette Test

 Qualitative screening test that detects fetal D+red cells in maternal Rh negative blood
 If positive, proceed with the quantitative test


2. Kleihauer Betke stain (Acid Elution Technique)

 A quantitative test used to distinguish hemoglobin F containing fetal RBCs from those of
adult cells (Hemoglobin A)
 Principle: Hgb F are resistant to acid whereas Hgb A are eluted by acid. Therefore, Hgb F
containing cells takes up the stain and Hgb A containing cells
appear_________________

Simpler formula:
FMH = % of fetal cells x 50

Calculating the number of Rhogam vials to be given:

To determine the vials, divide the FMH volume by 30

General rules:

 For decimals <5, round down and add one dose

 For decimals >5, round up and add one dose

Example:

A Kleihauer Betke stain for post partum fetomaternal hemorrhage is reported a percent fetal
cells of 2.7%. What is the total volume of fetomaternal hemorrhage?

 Computation:

 Interpretation: The fetomaternal hemorrhage is estimated to be 135 mL. Meaning, we

need to neutralize approximately 135mL of fetal blood that has entered the mother’s
circulation.

Calculate the number of Rhogam vials to be given:

 Computation:

 Interpretation: 6 Vials of Rhogam should be administered to a mother who has an

estimated fetal hemorrhage of 135 mL


Diagnosis/Treatment Management:
1. Intrauterine Transfusion

 Blood must be compatible with maternal antibodies capable of crossing the placenta
 If blood type is not known, Group O Rh (-) should be given
 Blood should be fresh and no older than 7 days
 The goal of intrauterine transfusion is to maintain fetal hemoglobin above 10 g/dL

2. Exchange Transfusion

 Simultaneous withdrawal of blood and transfusion of compatible donor blood to infants

with HDN
 Preference of Specimen for Compatibility Testing: Mother’s serum, An Eluate Prepared
from the Infant’s Red cells, Infant’s Serum.
 Donor Blood Characteristics:

 Must be Group-Specific or Must be Negative for the Antigen against which the
Mother’s antibodies are directed.
 Must not have any unexpected antibodies.
 Must be less than 7 days old.
 Should be negative for CMV, should be negative for HbS.
 Recommended to be irradicated to prevent GVHD.

 Goals:
Remove sensitized cells.
Reduce level of maternal antibody.
Removes bilirubin and corrects anemia.

Lesson 4: Rh variants

Rhnull

 Expresses no Rh antigen on RBC and the phenotype is expressed as “”” / “””

 Rhnull syndrome is inherited in one of two ways—amorphic and regulator.

 Regulator type– Mutation of RHAG gene. No Rh antigen on RBC but normal

complement of RHD and RHCE genes. These individuals can pass normal genes to
children
 Amorphic Type – mutation in RHCE genes and deletion of RHD gene. RHAG gene is
normal.

 The Rhnull phenotype is usually written in Fisher-Race as “”” / “””, in Wiener as

Rhnull, and in Rosenfield nomenclature as (RH: -1, -2, -3, -4, -5)
 Symptoms: Stomatocytosis, Reticulocytosis, Compensated Hemolytic Anemia, Increased
HbF & Bilirubin, Decreased Haptolglobin & OFT, Slight decreased Hemoglobin &
Hematocrit.
 Anti-Rh:29- Antibody of Broadest specificity

Cw

 Cw was originally considered an allele at the C/c locus.

 Combination with both C and c and in the absence of either allele.


f (ce)

 The f antigen is expressed on the RBC when both ___ and ____ are present on the
same haplotype.
 __________ antigen
 Known to cause HDN and transfusion reaction

rhi (Ce)

 Only present if C and e are in ______ configuration

 G is an antigen present on most D-positive and all C-positive RBCs.

 The antigen results from the amino acid ________ at position 103 on the RhD, RhCe, and
RhCE proteins.

Rh17 (Hr0)

 Rh17, also known as Hr0, is an antigen present on all RBCs with the “common” Rh
phenotypes (e.g., R1R1, R2R2, rr).

Rh23, Rh30, Rh40, and Rh52

 Rh23, Rh30, and Rh40 are all low-prevalence antigens associated with a specific
category of partial D.
 Rh23 is an antigenic marker for category Va partial-D.
 Rh30 is a marker for partial DIVa.
 Rh40 is a marker for partial DVII.
 Rh52 is associated with some partial-DVI types.

Rh33 (Har)

 The low-prevalence antigen Rh33 is most often found in whites and is associated with the
rare variant haplotype called __________
 The D reactions are frequently so weak that the cells are often typed as _____________

Rh32

 Rh:32 is a low-prevalence antigen associated with a variant of the R1[D(C)(e)] haplotype

called = RN 61

Rh43 (Crawford)

 also known as the Crawford antigen, is a low-prevalence antigen on a variant _________

protein.
 Crawford (ceCF) antigen is of very low prevalence found in individuals of African descent.


 Lesson 5: Techniques in Rh Grouping
Rh Typing (Rh Phenotyping):

 Uses of Rh Typing:

 Parentage testing
 Predicting Hemolytic Disease of the Fetus and Newborn (HDFN & Erythroblastosis
Fetalis
 Confirmation of Rh antibody specificit
 Locating compatibles blood for recipients with Rh antibodies.

 Rh (+) means__________________
 Rh (-). “d”, means__________________, may be presence of C or c or E or e antigens.

2 Methods:

 __________- More convenient method, Specimen: ____________ RCS.

 Results interpretation should be done ________ minutes but if negative, maximize

reading up to 5 minutes.

 Tube Method- Specimen: 2% RCS.

 Modified Tube Method- ____________________________

 Saline Tube Method- ______________________________

 Reagents: ____________________________

Anti-D Reagent Variants: Saline-Reacting Anti-D, Low Protein or High Protein Base Reagent,
Chemically-Modified Anti-D Reagent, Monoclonal Anti-D Reagent, S Monoclonal Anti-D
Reagent.

Test for Du Variant- Confirmatory test for Rh negative individuals.

 Specimen: __________ RCS.

 Reagent: Anti-D Reagent, AHG Reagent

Point of Difference between Test and Control

Test Control
Anti-D 1 drop ---
Cell Suspension 2 drops
Incubation 37C (15-30 mins) No incubation
Cell washing 3x 3x
Antiglobulin rgt 2 drops

Mix+Centri yes yes

Result
T(+), C(-) = Rh(+) Du(+) = Report as Rh __________
T(-), C (-) = Rh(-) Du(-) = Report as Rh ______
IgG Sensitized Cell maybe added to ensure that the AHG is working


False Reactions with Rh Typing:

 False Positive Reactions may be caused by:

 Positive DAT , Rouleaux, Cold Autoagglutinins.

 False Negative Reactions may be caused by:

 Incorrect cell suspension (Too heavy or Too light)

 Pro Zone Effect
 Post Zone Effect



BB QUIZ MOD 3-4

1) Convert the following: Rhorh’rh”/hr’rh” to Modified Wiener = DcE/dcE

2) DCe/dcE to MODIFIED WIENER? R1r”

3) DCe/DCE to MODIFIED WIENER? R1rz

4) Dce/DcE to ROSENFIELD? Rh: 1 -2 3 4 5

5) R2r’ to FISHER RACE? DcE/dCe

6) Rory to FISHER RACE? Dce/dCE

Improper Procedure. 7) DCE/DcE to BLOOD FACTOR? Rhorh’rh”/Rhohr’rh”

8) dce/dce to BLOOD FACTOR? hr’hr”/hr’hr”

9) Why is type O considered as Universal Donor for whole blood?

- because it does not contain antigens on the surface of its RBC  (A and B) that will react with the
antibodies in the serum of individuals with A, B and AB blood types.
10)What is the phenotype of a Rh null individual? - - - / - - -

11)What is the secondary purpose of Cell typing? To detect antigens in the surface of RBC

12)What is the lectin used for confirming A2 individual? (Scientific name of the plant)

- Dolichos biflorus

13) Antibody associated with severe HDN? Anti- D

14) What blood type is agglutinated by both A and B anti-sera? AB

15) The agglutinin of this blood group can agglutinate the A cell and the antigen of this blood group can
agglutinate Anti-B. What is the blood type?

16) In what chromosome number RHD gene found? Chromosome 1

17) What subgroup uses saliva as specimen? Bel

18) Rh nomenclature can be based on genetic assumption. One example is the Fisher-Race terminology?

19) What specimen is used in Tube method of Rh typing?

20) Rh43 is also known as _____________

21) Rh antibodies are IgG antibodies. They are naturally occurring antibodies. They react best at less than
25C?
- First sentence is correct. Second sentence is incorrect. Third sentence is incorrect
22) Which alloantibody is most likely to be produced if a patient that has the Rh genotype of DCe/DCe is
transfused with red blood cells that have an Rh genotype of R0R0?

23) ABO antibodies are immune antibodies that is produce upon exposure to pregnancy and transfusion?
False - They are naturally-occurring antibodies
 24) Why is RhAG is termed as Co-expressor?

- It is termed as Co-expressor because RhAG is needed to be expressed together with RHD and
RHCE gene to produce a specific Rh antigen.

25) Subgroup where secretors contain only H substance and no A substance in saliva? Ael

26) Identify the cause of discrepancy (COD) and resolution (RES)

Blood group Anti-A Anti-B A1 cells B cells O cells Auto-control

4+ 2+ 0 4+ 0 0

[ Select ]
COD:              ["", "", "", ""]           
RES: Test with Anti-B lectin

Blood group Anti-A Anti-B A1 cells B cells O cells Auto-control

4+ 4+ 2+ 2+ 2+ 2+

COD:              ["", "", "", ""]           

RES:              ["", "", "", ""]           

Answer 1:
Group B with cold autoantibody

Answer 2:
Test with Anti-B lectin

Answer 3:
Group A with Acquired B Antigen

Answer 4:
React patients cells with Anti-A1 lectin

27) What condition is the destruction of fetal RBCs by antibodies produced by the mother? Hemolytic
Disease of the Newborn

28) What would developed if a mosaic D patient was transfused with Rh (+) cells?

29) What is the confirmatory test used for weak subgroups of A and B? Adsorption-Elution

30) What does ‘d’ stands for in the Rh nomenclature? Absence of D antigen
31) It is the confirmatory test for Rh negative individuals? Du Variant Test

32) How many vials of RhIg is needed to be given to a mother who has a fetomaternal hemorrhage of
205mL? 6.8- 7vials

33) What is the reagent used for Serum typing? Patient’s serum

34) What is the formula of Fetomaternal hemorrhage in a simpler form? FMH = % Fetal cells x 50

35) It contains purified Anti-D which is administered within 72 hours after delivery of first born? Rhogam

36) What type of HDN causes the severe form of HDN? Rh HDN

37) What blood group is mistaken as bombay phenotype? O blood Group

38) Blood group that has the least amount of H antigen? Bombay Blood Group

39) ISBT assigned a six digit number for each blood group and antigen to standardize blood group system.
What does the last 3 numbers indicates in the six digit number assigned by ISBT?
- Antigen specificity

40) Blood group that has the greatest amount of H antigen? O Blood Group

41) Subgroup where it demonstrates agglutination with anti-B and most anti-AB? B3

42) Mother is considered Universal Donor for O blood and Father is homozygous B. What are the possible
blood type/s of the offspring? 50% OB, 50% oB

43) What is the most preferred specimen for Compatibility testing in Exchange transfusion?

44) What condition results from the buildup of bilirubin in the brain?

45) Genes needed to inherited to expressed Rh antigen? RHD, RHCE, RHAG