CLS 306 - Rh Blood Group System

Rh BLOOD GROUP SYSTEM

Introduction

• The Rh blood group system is the second most important blood group system next to ABO, especially concerning blood transfusions.
• When expressing someone's blood type, the terms "Pos" or "Neg" refer to the presence or absence of the D antigen in the Rh system.
• Unlike the ABO system, there are no naturally occurring antibodies in the Rh system.

Antigens

• There are 5 significant Rh antigens routinely tested in blood banks:
  • D, C, E, c, and e antigens
  • The D antigen is the most routinely tested and the most immunogenic of all blood group antigens.
  • C, E, c, and e antigens are considered when a corresponding antibody is detected and identified in the patient's serum before a transfusion.
• The Rh system is complex, comprising 49 antigens, variants, and rare genes.

Genetics

• The Rh blood group system follows Mendelian inheritance patterns.
• Genes are inherited in a linked fashion as haplotypes.
• Each gene is responsible for the production of an antigen.

Genes Involved

• Two closely linked genes (RHD & RHCE) are found on chromosome 1.
  • RHD codes for the presence or absence of the D protein.
  • RHCE codes for the presence of other proteins (C, c, E, e).
• Another gene (RHAG) located on chromosome 6 is a co-expressor gene, and it must be present for successful expression of Rh antigens.
• Inheritance occurs via codominant alleles, where offspring inherit one haplotype from each parent.

Immunogenicity Order of Rh Antigens

• The immunogenicity order from most to least is: D > c > E > C > e (See Table 7-6)

Nomenclatures

• Four types of nomenclatures are used to designate each Rh antigen:
  • Fisher-Race
  • Wiener
  • Rosenfield
  • ISBT (International Society of Blood Transfusion)

Fisher-Race

• Three separate, closely linked genes produce the antigens.
• Antibody production can result from exposure to an antigen not present in the patient.
• Developed antibodies recognize a single antigen.
• The Fisher-Race nomenclature includes:
  • D, d (absence of D)
  • C, c
  • E, e

Wiener

• Multiple alleles exist at one complex locus.
• One locus encodes for the production of an "agglutinogen", which has 3 factors (antigens/epitopes).
• Antibodies can recognize single or multiple factors.

Rosenfield

• This nomenclature makes no genetic assumptions; it is a numerical system:
  • If a number is listed, the antigen is present (e.g., Rh:1 = D antigen).
  • If a number is listed with a "-" (minus sign), the antigen is not present.
  • If not listed, the antigen was not determined.
• Adapts well to computer entry.
• Antigens are designated by number:
  • Rh1: D
  • Rh2: C
  • Rh3: E
  • Rh4: c
  • Rh5: e
• Example: D+, C+, E-, c+, e+ is written as Rh:1,2,-3,4,5.

ISBT (International Society of Blood Transfusion)

• Uses a 6-digit number for each antigen specificity (e.g., 004001 for Rho(D)).
  • The first 3 digits indicate the blood group system (e.g., 004 = Rh; 001 = ABO).
  • The last 3 digits indicate the blood group antigen specificity (e.g., 004001 = D antigen of the Rh system; 001001 = A antigen of the ABO system).
• For recording phenotypes, the system adopts the Rosenfield approach.

Rh Antigens Functional Role

• Unlike the extended carbohydrate antigens in the ABO system, Rh antigens play an important functional role on the RBC membrane, including maintaining integrity and intracellular cation transport.
• The absence of some (or missing) Rh antigens is called "Deletion" for the missing antigen.
• The absence of all Rh system antigens, called "Rh null", causes a membrane abnormality that shortens the normal RBC survival.

Genetics / Haplotype Inheritance Example

• Mom - R1R2, Father - rr
• DCe / DcE + dce / dce
• Possible offspring genotypes: R1r (DCe / dce), R2r (DcE / dce)

Rh Phenotyping Uses

• Parental or paternity testing
• Predicting HDFN (Hemolytic Disease of the Fetus / Newborn)
• Confirmation of Rh antibody specificity
• Finding compatible blood for transfusion recipients with Rh antibodies
• Using published frequencies & subject information to help determine possible genotype(s) & availability of antigen-negative blood

% Rh0(D) Pos by Population

• 99-100%: Japanese, Chinese, American Indians, Melanesians
• 95%: Black Africans
• 85%: Europeans, Caucasian North Americans
• 60-75%: Basques

D Antigen Variations

Weak Rho(D)

• Most D antigen-positive cells will hemagglutinate at the Immediate Spin (IS) test phase using normal Anti-D antisera.
• However, weak D antigen expression requires further testing.
• To demonstrate hemagglutination to weak D positive cells, testing continues to the 37°C incubation phase, followed by the AHG testing phase.

Weak D Antigen Categories

• Three mechanisms/categories of weak D antigen expression:
  1. Quantitative (genetic)
  2. Position Effect
  3. Partial / Mosaic D antigen (missing parts)
• Immune response within those categories can be inconsistent/variable.

Quantitative D Variants

• The RHD gene codes for weak expression of the D antigen.
• The D antigen is complete (all epitopes of the D antigen are present), but there are fewer D Ag sites on RBC.
• Normal D sites: 15,000 - 33,000 D sites/cell
• Weak D: 70- 5200 D sites/cell

Weak D Antigen - Position Effect

• The D-antigen expression will be weaker when the D and C genes are inherited on the opposite chromosome (trans position).

Weak D Antigen - Partial / Mosaic

• 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.

D Antigen Expression Variations

• When C is in the trans position to D, C creates a limiting effect on the expression of D, resulting in a weak D expression.
• Deletions of Cc and/or Ee (D-- or D··) represent partial locus deletions.
• "Exalted D" refers to unusually strong D antigen expression and is commonly seen in consanguineous marriages/situations.

Cis-Product Antigens

• Antigen products are formed when 2 genes are inherited on the same chromosome (cis position).
  • Rh6 (cis ce or f): c & e inherited as a haplotype (cde or cDe)
  • Rh7 (cis Ce or rhi) C & e inherited as a haplotype
  • Rh27 (cis cE)
  • Rh22 (cis CE)

Rh Compound Antigens

Cw

• Not allelic to C & c
• C & Cw usually seen together, as in R1wR1 phenotype
• 2% of Caucasians; very rare in Afro-Americans
• Anti-Cw may be naturally occurring & shows dosage

f (ce)

• When c & e are in cis position, e.g., dce or Dce haplotype
• Compound antigen
• Anti-f antisera may be helpful in phenotyping

Other Rh Alleles & Antigens

Ce

• When C & e in cis position.
• Compound antigen.
• Antibody helpful in phenotyping.

G

• Antigen always found with D- & C-positive RBCs.
• Anti-G appears to bind to D, C, & G antigens & it looks like anti-C & anti-D are present but cannot be separated.
• Anti-G is commonly found in an Rh negative patient who was exposed to the C antigen only.

Rare (But Significant) Rh Phenotypes

Deleted Rh Phenotypes

• Deleted Rh phenotypes exist, lacking many Rh antigens, and may be caused by the rearrangement of some of the Rh genetic material.
• Occurs from replacement of large portions of the RHCE gene with the normal RHD gene.
• RBCs that lack C/c and/or E/e result in enhanced D activity.
• D--, Dc-, DCw- = fail to produce some or all Rh CE antigens.

Rh Deficiency Syndromes - Allelic Deletions

• Rhmod (weakened expression of Rh antigens)
• Rhnull (no Rh antigens expressed at all)

Rhmod Syndrome Genetics

• Rhmod characteristic: substantial decrease in all Rh antigen expression.
  • Mutation in RHAG gene.
  • Weakened expression of Rh antigens.
  • RBC abnormalities similar to those of Rh null.
  • Clinical symptoms less severe & rarely remarkable.
  • Expression varies per individual.

Rhnull Syndrome Genetics

• Rhnull characteristic: no Rh antigens are expressed; there are 2 genetic mechanisms:

  1. Regulator Type:
     • More common
     • Mutation in RHAG gene, where no RHAG protein is expressed
     • Can transmit normal RHD & RHCE genes
  2. Amorphic Type:
     • Mutation in each RHCE gene & a deletion in the RHD gene
     • RHAG gene is normal

• Fisher Race expressed as "--- / ---" or Wiener "Rhnull “

• Mild hemolytic anemia & reticulocytosis [reticulo - immature RBC; cyto - cell; osis - abnormal condition of]

• Increased level of Hgb F

• A potent anti-Rh29 can be produced if normal Rh cells are transfused; transfuse only using another Rhnull RBCs

Lab Testing

Serologic Testing

• ABO Forward Typing & Reverse Typing
  • Add 1 drop of reagent antisera + 1 drop of patient cells; mix; spin* & read
  • Add 2 drops of patient serum + 1 drop of red cell reagent; mix; spin* & read
• Rh Typing (same process as ABO Forward Typing)
  • Add 1 drop of reagent antisera + 1 drop of patient cells; mix; spin* & read.
  • No Reverse Typing performed.
  • The above test phase description is identified as "Immediate Spin" or IS.
• Accurate Rh typing is the second of the primary foundations in pretransfusion testing & a safe transfusion
• TJC & CAP PT: Rh typing test must be 100% correct / accurate at ALL TIMES!

Serologic Testing - Reagent Order

• Always add the "clear" substance(s) FIRST…this ensures that you've added the reagent or serum to the test system.
• Add 1 drop of reagent antisera + 1 drop of patient cells; mix; spin & read
• Add 2 drop of patient serum + 1 drop of red cell reagent; mix; spin & read

Serologic Testing - Cells

• Always add the "cells" LAST…this accomplishes 3 things:
  1. The reagent / serum was added to the test.
  2. The test is properly setup for serological testing.
  3. A "false" negative test result due to the lack of adding reagent or serum can be avoided.

Types of Rh Antisera Reagents

• Saline reactive: limited availability; high cost of production; lengthy incubation time; cannot be used for weak-D typing.
• High protein: potentiators increase the likelihood of false positive reactions, so must run manufacturer's control; Can be used for weak-D typing.
• Chemically modified: relaxes antibody molecule by breaking disulfide bonds; This allows antibody to span distance between RBCs in a low protein medium; No required manufactured control (only AB+ uses saline control); Fewer false positives; can be used for weak-D typing.
• Monoclonal: hybridized to increase antibody production & antibody strength; Generally combine several clones to ensure reaction with all mosaic parts. Also blend anti-IgM & IgG to enhance visualization.

RH0(D) Test

• Add Anti-D reagent to “D” labeled tube; add Rh control reagent* to “C” tube (when applicable)
  • This is a "negative" control and must remain negative throughout testing.
• Add patient's RBCs (3-5%); mix
• Spin & read, grade reaction:
  • If “D” tube reaction is positive & Rh control is negative, cells are considered Rh positive
  • If “D” tube reaction is negative & Rh control is negative, continue testing - perform "Du Test"

Du Test

• Incubate both tubes at 37°C for 15 mins
• Spin, read, & grade reaction
  • If “D” tube reaction is positive, cells are considered Rh positive
  • If “D” tube reaction is negative, continue testing
• Wash both tubes 3x in saline
• Add AHG, spin & read, grade reaction

Du Test Result Interpretation

• Some reagent manufacturers may not require the use of an Rh control reagent. If you are using such a reagent, ensure that you run a "control" to correctly interpret the Du positive test.
• A "control" can be performing a:
  • DAT on the patient's cells
  • Perform a Rh control by using the manufacturer's Rh control reagent, if available
• Some reagent manufacturer's suggest that you use 2 different anti-D reagents to "confirm" a patient's Du status, as per the anti-D reagent insert. Must follow this suggestion when the reagent manufacturer suggests this.

Du Test Result Interpretations

• If “D” tube is positive & the Rh control tube is negative, cells are considered Du Positive, therefore, the Rh phenotype is interpreted as / considered Rh positive

• If “D” tube is negative & Rh control is negative, cells are considered Rh negative

• If “D” is positive & the Rh control is positive, cells are considered Rh undetermined =& must perform additional tests to determine Rh phenotype; do DAT & elution

• If “D” is negative & Rh control is positive = Rh ??

• Individuals with a weak D positive test result are considered Rh+.

  • Weak D+ RBCs are NOT given to Rh negative recipients
  • Weak D+ recipients may receive: 1) normal D positive RBCs (dependent on hospital policy) OR 2) D negative RBCs (especially for child-bearing aged females)

• Expectant mothers must be tested for weak D

• However, in the case of a transfusion recipient, the weak D test may not be included in routine testing. Therefore, the weak D / Du status can be missed… as some hospital policies state that, if the initial Rh test is negative, a Du test is not required and Rh negative blood is to be given.

False Reactions with Rh Typing Reagents

• False-Positives

  • Likely Cause
    1. Cell suspension is too heavy
    2. Cold agglutinins
    3. Test incubated too long or drying (slide)
    4. Rouleaux
    5. Fibrin interference
    6. Contaminating low-incidence antibody in reagent
    7. Polyagglutination
    8. Bacterial contamination of reagent vial
    9. Incorrect reagent selected
  • Corrective Action
    1. Adjust suspension, retype
    2. Wash with warm saline, retype
    3. Follow manufacturer's instructions precisely
    4. Use saline-washed cells, retype
    5. Use saline-washed cells, retype
    6. Try another manufacturer's reagent or use a known serum antibody
    7. See chapter on polyagglutination
    8. Open a new vial of reagent, retype
    9. Repeat test; read vial label carefully

• False-Negatives

  • Likely Cause
    1. Immunoglobulin-coated cells (in vivo)
    2. Saline-suspended cells (slide)
    3. Failure to follow manufacturer's directions precisely
    4. Omission of reagent
    5. Resuspension too vigorous
    6. Incorrect reagent selected
    7. Variant antigen
    8. Reagent deterioration
  • Corrective Action
    1. Use saline-active typing reagent
    2. Use unwashed cells
    3. Review directions; repeat test
    4. Always add reagent first and check before adding cells
    5. Resuspend all tube tests gently
    6. Read vial label carefully; repeat
    7. Refer to a sample for further investigation
    8. Open a new vial

ABO & Rh Blood Types (US Data)

• O Rh Positive: ~37.4% (1 person in 3)
• O Rh Negative: ~6.6% (1 person in 15)
• A Rh Positive: ~35.7% (1 person in 3)
• A Rh Negative: ~6.3% (1 person in 16)
• B Rh Positive: ~8.5% (1 person in 12)
• B Rh Negative: ~1.5% (1 person in 67)
• AB Rh Positive: ~3.4% (1 person in 29)
• AB Rh Negative: ~0.6% (1 person in 167)

Rh Antibodies

General Characteristics

• Rh antibodies are "immune" IgG; therefore, they are considered "clinically significant."
• They react optimally at 37°C and/or at AHG test phases.
• Usually does not bind C', RBC destruction by Rh antibodies is commonly extravascular.

Clinical Significance

• The D antigen is considered very antigenic, because as little as a 0.5 ml exposure to the D antigen in an Rh-negative patient could elicit anti-D antibody production.
• When larger volumes of Rh-positive cells are transfused, anti-D antibody production will occur in approximately 85% of Rh-negative recipients.
• Severe HDFN (Hemolytic Disease of the Fetus & Newborn) - conditions called, Erythroblastosis Fetalis or Hydrops Fetalis.
• Because of HDFN & its clinical significance, it is known ( & may be common practice in some hospitals) that child-bearing women who are Du + are to receive Rh-negative blood, instead of Rh-positive blood.
• Commonly causes an extravascular HTR (Hemolytic Transfusion Reaction), however, could also cause intravascular hemolysis if Rh antibody binds C'.
• For transfusion purposes, antigen-negative RBCs must be found for the corresponding antibody