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