Rh Blood Group System History and Antibodies

History

• The Rh blood group system was discovered in 1940 by Levine and Stetson
• Discovery was linked to Hemolytic Disease of the Fetus and Newborn (HDFN)
• The Rh antigens were initially named after the Rhesus monkey cells used in the discovery
• The Rh blood group system was discovered due to an HDFN situation

Rh Antibodies

• Rh antibodies were further demonstrated by Landsteiner and Weiner
• Through experiments injecting Rhesus monkey red cells into guinea pigs and rabbits
• Researchers discovered another blood group system, named Rh
• The antibody produced by guinea pigs/rabbits infused with Rhesus monkey cells are similar, but not identical to the antibody from the HDFN mother
• Rh and Lw antigens are close in proximity on the same protein, but the name Rh was retained for human antigens
• Rh antibodies from guinea pigs and rabbits became known as Lw antibodies

Inheritance

Current Rh Genetic Theory

• The nomenclature is based on how Rh antigens are created
• RHD and RHCE genes are linked on Chromosome 1
• Genes responsible for Rh antigens are found on Chromosome 1
• Two genes produce Rh antigens: RHD and RHCE
  • They are located in close proximity on the same chromosome, considered a haplotype
  • RHD Alleles: D
  • RHD Antigens: D positive
  • RHCE Alleles: RHCE, RHCe, RHcE, Rhce
  • RHCE Antigens: CE, Ce, cE, ce
• The presence of the D gene and the CE gene allows for: DCE, DCe, DcE, Dce antigen combinations

RHAG - Chromosome 6

• RHAG is a Coexpressor in Chromosome 6
• Responsible for the production of RHAG glycoprotein
• It's not an antigen, but a glycoprotein related to the Rh glycoprotein
• The absence of RHAG gene can affect the expression of RHD and RHCE antigens
• The presence of products is dependent on RHAG, but RHAG is independent from product expression

Rh Glycoprotein

• Rh antigens are located in the Rh glycoprotein
• RH glycoprotein contains 416 amino acids that traverses to the plasma membrane 12 times RHD positive = no stomatocytosis
• Rh glycoprotein is vital for the plasma membrane stability of red cells
• Individuals who are Rh null most likely don’t have the Rh glycoprotein
• This can lead to a particular RBC morphology abnormality called Stomatocytosis

Differences in Amino Acid Sequence for the Antigens Produced by RHCE Gene

• Antigen C: Amino acid Serine at number 103Cc
• Antigen c: Amino acid Proline at number 103Cc
• Antigen E: Amino acid Proline at number 226Ee
• Antigen e: Amino acid Alanine at number 226Ee
• 103rd amino acid: C and c antigen based on the amino acid present
  • Serine: Capital C
  • Proline: small c
• 226th amino acid: Same goes with coding of E
  • Proline: Capital E
  • Alanine: Small e
• Slight difference
  • Different amino acid, different antigen

DCE Terminology

• 'd' Antigen does not exist
• 'd' Antigen denotes the absence of D antigen
• Only 'D' antigen is present
• Deletion phenotypes (particular gene deleted)
  • -De or -DE → C is deleted
  • CD- or cd- → E is deleted
  • -D- (Double deletion) → No C and E
• Rh null or (-/-)
  • No Rh antigens -For Rh-, D is absent, C and E present -For Rhnull (amorph), everything is absent
• Rhmod (modified)
  • Weakened expression: (D), (C), (e)
  • Modifications or modifications with the coexpressor → RHAG

Weiner Genetic Theory

• One gene codes for three combinations

Alleles:

• R0, R1, R2, RZ, r, r’, r’’, ry

Antigen Specificities

Dce, dcE

• dCE, DcE
• CE,
• DCe,
• dce e

Weiner: The Rh-Hr Terminology

• Rhnull is equivalent to rr
• Take note that in Weiner's theory: One gene is responsible for the creation of three antigenic combinations of D, C, and E
• In Weiner's notation, Genes are always in italic form and written in superscript
• Agglutinogen: synonym for immunogen, also known as Antigen
• Agglutinin: Antibody
• Immunogen: Ab (Immunoglobulin)
• Capital R becomes a subscript Blood Factor:
• If you have the gene, you have the agglutinogen It has its third form
• Agglutinogen has three antigens

Rosenfield Numeric Technology

• In order – Follow this arrangement: D C E c e (1 2 3 4 5)
• Write the word Rh: and remember to always write Rh when writing Rosenfield terminology
• D+, C+, E-, c+, e+ is equivalent to Rh: 1, 2, -3, 4, 5
• D = Rh1
• C = Rh2
• E = Rh3
• c = Rh4
• e = Rh5

ISBT Numeric Terminology

• Six digit number for every authenticated blood group specificity
• First three numbers: Blood Group System (004)
• Remaining three numbers: Antigenic specificity

Antigens:

• D>c> E >C>e is the order of antigen abundance
• Rh antigens are well developed early in fetal life

Antibodies

• Rh antigens are highly immunogenic leading to antibody formation
• Exposure to at least 1 mL of Rh positive cells would stimulate antibody production in Rh negative persons

Overall Designation Fisher-Race, Weiner, Rosenfield and ISBT

D = Rh0 = RH1 = 004001

• C = rh’ = RH2 = 004002
• E = rh’= Rh3 = 004003
• c = hr’ = RH4 = 004004
• e = hr’’ = RH5 = 004005
• Rh positive = only D antigen is present

Rh Negative and Rh null

• Rh negative = D is absent (C and E are present)
• Rh null = all are absent

Antigen Expression

• Anti D is used to detect the presence of the D antigen and D antigen is only one of the antigens of the Rh blood group system
• Hydrophilic heads and hydrophobic tails
• Rh glycoprotein is composed of 416 amino acids that traverse the plasma membrane 12 times.
• traverses 12 times to maintain the plasma membrane integrity of the red cell
• Rh null = stomatocyte appearance leading to an Absence of Rh glycoprotein = leading to integrity is weak

Variations of the Rh0 (D) Antigen Expression

• There is a decreased amount of D antigens also known as subgroup-like

Weak D expression:

Historically known as Du phenotype and Now referred as WEAK D POSITIVE

3 mechanisms:

• Genetic Weak D
• C TRANS
• Partial D (Mosaic D)

General Genetic Weak D

• has defective GENE
• RHD genes code for weaker expression of D antigen because it is mutated/modified
• reduced number of D antigens on the red cells

Weak D Testing IAT Procedure

1. Label 2 tubes as Anti-D and Rh control. Place one drop of the 2- 5 cell suspension of the sample to be tested
2. Add one-drop of the Anti-D into one test tube and 1 drop of the Rh control into the other test tube.
3. Incubate 15-30 minutes. Wash 3x with saline
4. Add 1-2 drops of AHG reagent.
5. Mix and centrifuge
6. Gently resuspend and examine agglutination, grade, and record
7. Add IgG-coated control cells to negative results, spin, and read.

RhD-positive & IAT procedure

If this is an individual with Partial D, the person still has D antigen making them a D positive

• Meaning if the person has a missing epitope that could make them reactive or non-reactive
• Meaning when tested the result should be positive

IAT for D-Negative testing

• the test requires someone to inject rhesus antigen in order to find missing epitopes
• IF THE PERSON HAS A MISSING EPITOPE BUT STILL REACTS WITH ANTI-D, THEY ARE STILL CONSIDERED TO BE D POSITIVE
• If the person has a requiring transfusion D POSITIVE RED CELL
• The person as with a missing RH group will develop antibodies against the one that has the full blood group epitopes
• Meaning a D POSITIVE as a DONOR and D NEGATIVE as a RECIPIENT

3 types of weak D that has antibody that can be made

1. Types of weak D:
2. Genetic, reduced D antigen is undetected and has low antibody
3. RhCe in trans to RHD antigen is undetected with anti-D
4. Partial with a weak D, it can make a Yes, has an Antibody to the missing epitope known as D antigen

Anti-D in Testing

• If having anti-antigen is unable to cause one not to be reactive meaning those epitopes have a marker
• Where Anti-D has nothing to attach to. causing a negative reaction. meaning to purpose of IAT isn’t achieved.

Results if IAT testing

1. Postive IAT- patient can be confirmed to be weak D and have an Anti D
2. Negative IAT- the patient can’t be confirmed as testing

How Anti Transfusion testing works

• Where the antigen is not reactive rather where there is Anti-d antigen has already in the patient. The patient is NOT the red cell
• Antibodies and antigens are present where the red cell needs further testing
• In order to make a transfusion need further testing where you are attempting to find antibodies with antigens where they bond
• Meaning in order to perform a testing there must be an exact location such that you can find antibody that can bind to that area

DAT Procedures

• The same pre process applied to the IAT test, but with more in vivo sensitizing.

1. Added the addition of D which allows on to see a lack of the immunoglobulin in the patient. This can indicate with lack sensitization of the red blood cells i . With lack and weak sensitization an Antigen can not be ruled out causing more potential issues

Procedure with in-vivo sensitizing

• Added to Anti Transfusion where other antibodies may apply but the patient can not be transfused due to unknown antigens/antibodies in their body
• Can be applied with another test with antibodies

Unusual Phenotypes and rare Allele Types

• Ce of f:
  • cis product is present in the red cells causing a rare antibody for the individual.
• CW: The individual is at high risk for HTR and HDFN because the individual is low in antigens and antibodies.

Clinical Considerations regarding HTR and HDFN

• Clinical implications are that most antibodies are at risk of HTR and HDFN. However is you give blood to someone that lacks those blood characteristics causes a risk for the antibodies to lack compatibility
• In other words, individuals with low antigen and antibody counts should not gain blood or donate blood.
• Certain conditions can regulate and effect the red cell that also effect the HA to a degree and affect the transfusion
• Partial suppression is also a factor that comes into play that can effect the HA that determines what an issue is

Lw Blood Group System

• the most common blood group is type (LW(a+b-) and has 3 alleles
• Alleles: LWª, LW, LW
• It has a rare amorph gene that is tested as a null testing the serum similarly from patient
• The red cells act very similar which are clinically not significant with transfusions.