Week 11 Part 2 - Molecular Aspects of Blood Grouping

Question 1

Molecular Genotyping

Answer 1

Indirect method for predicting a blood group phenotype
Genotyping for most groups is straightforward
ABO, Rh, Le, and MNS blood groups can be more difficult
Simple PCR, RFLP, sequencing, qPCR, bead-based high- throughput methods

Question 2

Immucor Bioarray Beadchip

Answer 2

High throughput method for determining antigen expression
RHD chip for Rh(D) antigen variants
- D negative, weak D, partial D
HEA chip for the major blood group variants

Question 3

Immucor Bioarray Beadchip - How are SNPs Identified?

Answer 3

Coloured beads labelled with specific probe
≈ 100 different bead colours, each labelled with a different probe → ≈ 100 different antigens interrogated in a single assay

Question 4

Immucor Bioarray Beadchip - Method

Answer 4

1. Beads are applied to a silicon wafer, which is then cut and assembled onto a slide or plate
2. Isolate genomic DNA from blood
3. Multiplex PCR to amplify region containing the SNP that accounts for antigenic difference
4. One primer in each primer pair is 5’ phosphorylated
5. Remove primers and dNTPs using ExoSAPIT
6. Treat PCR product with λ-exonuclease
   - degrades phosphorylated DNA strand to give ssDNA
7. To ssDNA add, thermo sequenase, fluorescently-labelled dCTP, unlabelled dNTPs
8. Perform elongation reaction
9. Add mixture to Beadchip array, incubate
10. Array is washed and imaged
11. Assay image is overlaid onto decoding image to determine which beads/probes have bound amplicon

Question 5

Immucor Bioarray Beadchip - Principle

Answer 5

ssDNA binds to probe
3’ end of probe binds at the SNP site that differentiates the antigens
If there is a match at 3’ end of probe, elongation reaction will occur and labelled dCTP is incorporated into second strand

Question 6

Applications of Immucor Bioarray Beadchip - Recent Transfusion

Answer 6

Patient blood contains both their own and donor RBCs
If typing patient blood by serology, you will phenotype both the patient and donor RBCs
Result - inaccurate patient phenotype
Genotyping overcomes this issue
- uses DNA isolated from WBCs, so there is no interference from donor blood

Question 7

Applications of Immucor Bioarray Beadchip - Chronic Transfusion

Answer 7

E.g. Sickle cell disease
Genotyping interrogates more antigens than possible with serological techniques

Question 8

Applications of Immucor Bioarray Beadchip - Haemolytic Disease of the Newborn (HDNB) Paternity Testing

Answer 8

In pregnant women with an alloantibody that can cause HDNB (i.e. anti-D), paternal expression of the antigen is determined
However, RHD zygosity (i.e. homozygous vs heterozygous) can’t be determined serologically
Molecular methods can be used to determine zygosity
Used to guide monitoring/treatment options
- if father is homozygous, foetus will be heterozygous
- if father is heterozygous, 50% chance that the foetus will be heterozygous

Question 9

Applications of Immucor Bioarray Beadchip - Haemolytic Disease of the Newborn (HDNB) Foetal Testing

Answer 9

Determine foetal antigen expression
Amniocentesis to isolate foetal DNA (invasive)
Or Cell-free DNA
- foetal DNA is present in maternal plasma from ≈5 weeks gestation
- can be isolated from maternal plasma and amplified via PCR
- non-invasive
- can be used to determine foetal RHD genotype

Question 10

Applications of Immucor Bioarray Beadchip - Guiding Administration of Prophylactic Anti-D

Answer 10

Prophylactic anti-D is administered to pregnant Rh(D) neg individuals w/o prior knowledge of foetal genotype
Foetal RHD genotype can be determined from cell-free DNA and administered only to women whose babies are Rh(D)pos

Question 11

Applications of Immucor Bioarray Beadchip - Weak vs Partial D Antigen Expression

Answer 11

Weak D
- decreased expression of the entire D antigen
- considered D positive
Partial D
- express only part of the D antigen
- considered D positive donors, D neg recipients
Genotyping can be used to discriminate the two

Question 12

Weak vs Partial D Antigen Expression - Useful for Pregnancy

Answer 12

Pregnant women who are partial D and are carrying a Rh(D) pos baby may benefit from prophylactic anti-D
The majority of weak D individuals fall into three types (1, 2, 3)
Are considered D positive
Pregnant females who are weak D type 1, 2, or 3 don’t need to receive prophylactic anti-D

Question 13

Weak vs Partial D Antigen Expression - Useful in Transfusion

Answer 13

Those who test serologically as weak D are usually given D neg blood
Genotyping would result in D neg blood going to only those expressing partial D

Question 14

Weak vs Partial D Antigen Expression - Useful in Blood Donors

Answer 14

Donors who type as D neg are tested for weak D to ensure that units are correctly labelled
Weak D can present as D neg with certain serological reagents
Genotyping identifies these, resulting in correct labelling on units

Question 15

Applications of Immucor Bioarray Beadchip - Autoimmune Haemolytic Anaemias

Answer 15

IgG-coated RBCs complicate phenotyping
Elution to remove IgG is not always successful, and can destroy or weaken antigen
Genotyping allows you to predict extended phenotype of patient RBCs
Determine which alloantibodies can be made
Better match donor phenotype to that of the recipient

Question 16

Applications of Immucor Bioarray Beadchip - Alloantibody vs Autoantibody

Answer 16

Patient presents with an apparent autoantibody
Sequencing may reveal a novel mutation resulting in new epitopes or partial antigen expression
E.g. Sickle cell disease
- 23% of patients have hybrid RHD gene where D expression has been lost and instead encodes a C epitope (i.e. partial C)
- phenotype serologically C pos, but can make anti-C (should be transfused w/ C neg RBCs

Question 17

Applications of Immucor Bioarray Beadchip - Typing for Antigens to which there are No Reagents

Answer 17

E.g. Dombrock (Doa/Dob)
Anti-Doa and –Dob are weak, disappear over time, and often occur with multiple antibody specificities
Anti-Doa,-Dob phenotyping reagents are not widely available
Instead, DNA analysis is performed

Question 18

Applications of Immucor Bioarray Beadchip - High Throughput Screening for Rare Antigens

Answer 18

Used by donor centres to screen donor units for expression of rare antigens
Provide antigen-negative units
Provide antigen-matched units
Identification of rare donors

Question 19

Differences between Genotyping and Serological Testing

Answer 19

Variant alleles encoding weak antigen expression
E.g. Fyx in Duffy blood group
- results in weak expression of Fyb [Fy(b+w)]
- types Fy(b-) with most antibodies
Fy(a-b-) phenotype due to GATA1 mutation
- homozygous carriers do not express Fyb on their RBCs, but do on other tissues
In both examples, individual don’t make anti-Fyb and therefore can receive Fy(b+) cells