The Molecular face of red cells

 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

 Is there a need?  What are the potential advantages?  Is it sustainable?  Service / research mode?

 25 year old - aplastic anemia  O pos few years ago – transfused 25 units of red cells / PRCs etc  Now requires a transfusion

 O Rh Negative  Done on 3 different platforms, using different antisera  History – No transplant

 Patient is convinced we have gooooofed – wants an explanation

 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

 What will the fate of this pregnancy be ?

 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

 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

 Mostly SNPs  Single amino acid differences

 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

 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

 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

 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

 1 st trimester  Average 17 weeks

 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

 Compared to post natal serology  Muller et al – 2008  >1000 typings  25 weeks of pregnancy  >99.6% concordance

 Real time PCR  The luminex platform  Automated High throughput analysers using these technologies available  Blood chip technology

 Cannot be typed by serology – mixed field reaction seen  DNA from WBC – Accurate type  Epithelia also can be used  Helps select blood for transfusion

 Likely to be sensitised  Worth antigen matching  If sensitised – can attempt to antigen match henceforth – to prevent further sensitisation