1. University of Tabuk Blood bank, MLT 309
Faculty of Applied Medical Sciences
Department Of Medical Lab. Technology
3rd Year – Level 6 – AY
Blood bank, MLT 309

2. Rh BLOOD GROUP SYSTEM

3. Objectives
Compare the three theories of inheritance of the Rh antigens.
List the antigens and antibodies of the system using both Wiener and Fisher-Race nomenclature.
Convert haplotype from Fisher-Race nomenclature into Wiener, and vice versa.
Discuss key characteristics of antigens and antibodies in the Rh system.
Compare major characteristics of the Rh system to the ABO system.
List the three theories of the weak D antigen.
Evaluate reactions of Rh typing, using conventional reagents.
Explain the principle of the weak D test.
Discuss situations when weak D testing would be appropriate.
BB. MLT Lec.5.Mr. Waggas

4. Introduction
Rh is the most important blood group system after ABO in transfusion medicine.
One of the most complex of all RBC blood group systems with more than 50 different Rh antigens.
Discovered in 1940 after work on Rhesus monkeys.
The genes that control the system are autosomal codominant located on the short arm of chromosome 1.
Only the most clinically significant Ags will be discussed.
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5. HISTORY
Levine and Stetson (1939) described a hemolytic transfusion reaction in an obstetric patient following delivery of stillborn infant. The women required transfusion. Her husband, who had the same ABO type, was selected as her donor, after transfusion the recipient, demonstrated the classic symptoms of acute hemo]ytic transfusion reaction.
Subsequently an antibody was isolated from the mother’s serum that react both at 37°C and 20 °C with the father’s red cells. It was postulated that the fetus and the father possessed a common factor that the mother lacked. While the mother carry the fetus, she was exposed to this factor and subsequently built up an antibody that reacted against the transfused red cells from the father and resulted in hemolytic transfusion reaction.
Landsteiner and Wiener (1940) reported on an antibody by guinea pigs and rabbits when they were transfused with rhesus monkey red cells.
This antibody which agglutinated 85% of human red cells was named Rh.
The name Rh was retained for the human produced antibody.
Anti-rhesus formed by the animals was renamed anti-LW (Landsteiner and Wiener).
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6. Antigens of Rh System
Terms “D positive” and “D negative” refer only to presence or absence of the Rh antigen D on the red blood cell.
Terms “Rh pos” and “Rh neg” are old terms, although blood products still labeled as such.
Four additional antigens: C, c, E, e.
Named by Fisher for next letters of alphabet according to precedent set by naming A and B blood groups.
Major alleles are C/c and E/e.
MANY variations and combinations of the 5 principle genes and their products, antigens, have been recognized.
The Rh antigens and corresponding antibodies account for majority of unexpected antibodies encountered.
Rh antibodies stimulated as a result of transfusion or pregnancy, they are immune.
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7. Clinical Significance
D antigen, after A and B, is the most important RBC antigen in transfusion practice.
Individuals who lack D antigen DO NOT have anti-D.
Antibody produced through exposure to D antigen through transfusion or pregnancy.
Has been reported that > 80% of D neg individuals who receive single unit of D pos blood can be expected to develop immune anti-D.
Testing for D is routinely performed so D neg will be transfused only with D neg.
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8. Nomenclature Fisher Race : CDE Terminology
Suggested that antigens are determined by 3 pairs of genes which occupy closely linked loci.
Each gene complex carries D or its absence (d), C or c, E or e.
Each gene (except d, which is an amorph) causes production of an antigen.
The order of loci on the gene appears to be “DCE” but many authors prefer to use “CDE” to follow alphabet.
Inherited from parents in linked fashion as haplotypes
The gene d is assumed to be present when D is absent.
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9. Fisher-Race
Three loci carry the Rh genes are so closely linked that they never separate but are passed from generation to generation as a unit or gene complex.
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10. Fisher-Race
With the exception of (d); each allelic gene controls presence of respective antigen on RBC.
The gene complex DCe would cause production of the D, C and e antigens on the red cells.
If the same gene complex were on both paired chromsomes (DCe/DCe) then only D, C and e would be present on the cells.
If one chromsome carried DCe and the other was DcE this would cause D, C, c, E and e antigens to be present on red blood cells.
Each antigen except d is recognizable by testing red cells with specific antiserum.
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11. Wiener
Postulated that TWO genes, one on each chromosome pair, controls the entire express of Rh system.
Each gene produces a structure on the red cell called an agglutinogen (antigen).
Eight (8) major alleles (agglutinogens): R0, R1, R2, Rz, r, r’, r” and ry.
Each agglutinogen has 3 factors (antigens or epitopes)
The three factors are the antigens expressed on the cell.
For example the agglutinogen R0= Rh0 (D), hr’ (c), hr’ (e)
Each agglutinogen can be identified by its parts or factors that react with specific antibodies (antiserums).
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12. Weiner’s Theory
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13. Weiner 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 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.
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14. Weiner and Fisher-Race
D = R
1 ( C)
2 ( E )
0 (neither C or E )
Z (both C & E )
D C
D c E
D c e
D C E
d = r
‘( C)
‘’ ( E )
(neither C or E )
y (both C & E )
d C e
d cE
d c e
d C E
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15. Comparison of Weiner and Fisher-Race
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16. Produces D antigen on RBC
The main difference between the Fisher-Race and Wiener theories is that the:
Fisher-Race theory has three closely linked loci,
the Wiener theory has only one gene locus at which multiple alleles occur.
Wiener theory:
Single gene at Rh locus R’ r’ R0 r” R”
Produces D antigen on RBC
Produces C antigen on RBC
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17. Differentiating Superscript from Subscript
Superscripts (Rh1) refer to genes
Subscripts (Rh1) refer to the agglutinogen (complex of antigens)
For example, the Rh1 gene codes for the Rh1 agglutinogen made of D, C, e
Usually, this can be written in shorthand, leaving out the “h”
DCe is written as R1
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18. Converting Wiener into Fisher-Race or Vice Versa
R  D
r  no D
1 and ‘  C
2 and “  E
Example: DcE  R2
r”  dcE
Written in shorthand
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19. Rosenfield Each antigen assigned a number Rh 1 = D Rh 2 = C Rh 3 = E
In writing the phenotype, the prefix “Rh” is followed by colon, then number (if negative, number is preceded by -)
e.g. D+, C+, E-, c+, e+ is written as
Rh:1,2,-3,4,5
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20. Comparison of Three Systems
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21. International Society of Blood Transfusion
Abbreviated ISBT
International organization created to standardize blood group system nomenclature.
Assigned 6 digit number for each antigen.
First 3 numbers indicate the blood group system, eg., 004 = Rh
Last 3 numbers indicates the specific antigen, eg., = D antigen.
For recording of phenotypes, the system adopts the Rosenfield approach
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22. Genotype Frequencies Refer to textbook.
Genotypes are listed as “presumptive” or “most probable”.
Genotypes will vary in frequency in different racial groups.
Gene Complex
Shorthand
% Caucasians
% Blacks
Dce R0 2 46
DCe R1 40 16
DcE R2 14 9
dce r 38 25
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23. Weak Expression of D
Not all D positive cells react equally well with anti-D.
RBCs not immediately agglutinated by anti-D must be tested for weak D.
Incubate cells with anti-D at 37C, coating of D antigens will occur if present.
Wash X3 add AHG
AHG will bind to anti-D coating cells if present.
If negative, individual is D negative (By AHG method).
If positive, individual is D positive w
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24. Three Mechanisms for Weak D
Genetic
Position effect
Mosaic
Results in differences from normal D expression
Quantitative (inherited weak D or position effects)
Qualitative (mosaic D; could produce Anti-D)
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25. Weak D - Genetic
Inheritance of D genes which result in lowered densities of D Antigens on RBC membranes, gene codes for less D.
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26. RBC with normal amounts of D antigen
Weak D - Genetic
RBC with normal amounts of D antigen
Weak D (Du)
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27. Position Effect C trans - position effect;
The D gene is in trans to the C gene, eg., C and D are on OPPOSITE sides: Dce/dCe
C and D antigen arrangement causes steric hindrance which results in weakening or suppression of D expression.
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28. Position Effect D c e / d C e D C e / d c e C in trans position to D:
Weak D
C in cis position to D:
D C e / d c e
NO weak D
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29. Partial D (“D mosaic”).
Absence of a portion or portions of the total material that comprises the D antigen.
Known as “partial D” (old term “D mosaic”).
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30. D Mosaic/Partial D
If the patient is transfused with D positive red cells, they may develop an anti-D alloantibody to the part of the antigen (epitope) that is missing
Missing portion
RBC
RBC
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31. Significance of Weak D Donors Patients
Labeled as D positive
Weak D substantially less immunogenic than normal D
Weak D has caused severe HTR in patient with anti-D
Patients
If weak D due to partial D can make antibody to portion they lack.
If weak D due to suppression or genetic expression theoretically could give D positive
Standard practice to transfuse with D negative
Weak D testing on donors by transfusion service not required.
Weak D testing on patients not required except in certain situations.
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32. Rh Null Red cells have no Rh antigen sites Genotype written ---/---
The lack of antigens causes the red cell membrane to appear abnormal leading to:
Stomatocytosis
Hemolytic anemia
2 Rh null phenotypes:
Regulator type – gene inherited, but not expressed
Amorph type – RHD gene is absent, no expression of RHCE gene
Complex antibodies may be produced requiring use of rare, autologous or compatible blood from siblings.
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33. Rh Antibodies
Except for rare examples of anti-E and anti-Cw which may be naturally occurring, most occur from immunization due to transfusion or pregnancy.
Associated with HTR and HDFN.
Characteristics
IgG but may have MINOR IgM component so will NOT react in saline suspended cells (IS).
May be detected at 37C but most frequently detected by IAT.
Enhanced by testing with enzyme treated cells.
Order of immunogenicity: D > c > E > C > e
Do not bind complement, extravascular destruction.
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34. Rh Antibodies
Anti-E most frequently encountered antibody followed by anti-c.
Anti-C rare as single antibody.
Anti-e rarely encountered as only 2% of the population is antigen negative.
Detectable antibody persists for many years and sometimes for life.
Anti-D may react more strongly with R2R2 cells than R1R1 due to higher density of D antigen on cells.
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35. Hemolytic disease of the Newborn (HDN)
Usually related to D antigen exposure and the formation of anti-D
Usually results from D negative female and D positive male producing and offspring.
The baby will probably be D positive.
1st pregnancy not effected, the 2nd pregnancy and on will be effected-results in still birth, severe jaundice, anemia related to HDN.
To prevent this occurrence the female is administered RH- IG.
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36. Rh factor First pregnancy Placenta Rh+ antigens
Rh factor can cause complications in some pregnancies.
Placenta
Rh+ antigens
Mother is exposed to Rh antigens at the birth of her Rh+ baby.
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37. Mother makes anti-Rh+ antibodies. Possible subsequent pregnancies
During the mother’s next pregnancy, Rh antibodies can cross the placenta and endanger the fetus.
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38.  Thanks 
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