Designer genes
The Molecular face of red cells
What is it?
Refers to the detection of the molecular basis of an antigen rather than the antigen itself
Prerequisites: Knowledge of the molecular
structure Appropriate genotyping methods
A buzzzzz word??
Is there a need?
What are the potential advantages?
Is it sustainable?
Service / research mode?
Some real life scenarios
25 year old - aplastic anemia
O pos few years ago – transfused 25 units of red cells / PRCs etc
Now requires a transfusion
New sample for crossmatch O Rh Negative
Done on 3 different platforms, using different antisera
History – No transplant
Patient is convinced we have gooooofed – wants an
explanation
Request from OG
27 year old
4th pregnancy at 10 weeks of gestation
1 miscarriage, 2 deaths with severe hydrops
Blood group – O Rh negative, ICT , antibody screen Pos. Anti D – titre 1:256
A question…….
What will the fate of this pregnancy be ?
Indications for DNA typing Fetal DNA analysis
Typing multiply transfused
Serological discrepancies – weak D / ABO Subgroups / AIHA with pos DAT ……
QC of antibody screening /ID RBCs
Routine phenotyping of red cells
RBC Antigens
Products of Genes
Antigens carried by proteins – direct products of genes- Rh / Kell
Antigens carried by Carbohydrates – under the control of genes coding for the glycosyl transferases – ABO / H
Nature of differences
Mostly SNPs
Single amino acid differences
However……….
Multiple alleles can code for a single same antigen!!!!!
Events other than SNPs in the same region – can affect antigen expression
Hence incomplete genotyping may not correlate with phenotyping
The D antigen Greatest Contribution to health care – in
pregnancy
To assess if a D negative Mother is carrying a D positive baby
If mother unsensitised – Anti D given only if baby is Rh positive
If mother sensitised – impact on clinical follow up
Fetal DNA in maternal serum Previously fetal DNA testing done on
Amniocentesis samples
Now - found that sufficient amount of fetal DNA is present in sera of mothers
About 3-10% of free plasma DNA in pregnant mothers is fetal. Clears rapidly post pregnancy
Cell free fetal DNA
Occurs due to apoptosis and necrosis etc of placental tissue
Part of a process of physiological remodelling
Results in ffDNA getting released into maternal plasma
From when?
1st trimester
Average 17 weeks
When can it be done? Akolekar et al – 11 – 13 weeks of pregnancy High throughput robotic technique 100% pos predictive value 96.5% negative predictive value
Cardo et al - sensitivity of 100% and a specificity of 93%, with a 97% diagnostic accuracy for RhD genotyping
first trimester of pregnancy using a quantitative PCR
Sensitivity -----
Compared to post natal serology
Muller et al – 2008
>1000 typings
25 weeks of pregnancy
>99.6% concordance
Platforms available
Real time PCR
The luminex platform
Automated High throughput analysers using these technologies available
Blood chip technology
The Multiply transfused
Cannot be typed by serology – mixed field reaction seen
DNA from WBC – Accurate type
Epithelia also can be used
Helps select blood for transfusion
Multiply transfused - contd Likely to be sensitised
Worth antigen matching
If sensitised – can attempt to antigen match henceforth – to prevent further sensitisation