Molecular Immunohematology Flashcards
(88 cards)
Antithetical
a pair (or more) of antigens coded by different alleles of a SINGLE GENE (get one from one parent either M or N but not both on one gene) therefore M is antithetical to N
C and c a.a. differences
differ by 4 a.a. most important (extracellular) Serine103Proline
E and e a.a. difference
Proline226Alanine
“Rhesus boxes”
boxes that sit on the outside of the RhD gene.
RhD deletion
can cross or recombine onto other chromosome- causes D deletion
Blood groups location on red cells
blood group antigens are carried on molecules expressed on the RBC membrane
Most common type of genetic variation
Single Nucleotide Polymorphism
How can molecular mehthods be used to predict blood group antigens
Known genetic variants that code for specific antigens- ‘predict’ is the appropriate word because you can’t see the whole picture- there could be an Inhibitor in action
Problems with serologic methods
variety of different methods with variable reactions: Tube, gel, solid phase, simple vs complex epitopes (Fy very straight forward, RhD can be very complex) variants can be missed, expression level can cause problems with detection ability (weak D) Cross reactivity- ceHar and ceCF
Why are some proteins expressed on certain surfaces (red cells) but not others (tissues)
ALL have the same DNA in every cell, depending on the cell type, different genes are either turned on or turned off
Steps to PCR
denaturation, primer annealing, primer extension and repeat
Low resolution Gel based molecular methods
SSP (sequence specific primers) PCR for known SNPs, PCR-RFLP restriction fragment length polymorphism for known SNPs
Medium resolution molecular methods
Immucor Bead chip and Grifols IDCore (arrays), Taqman genotypling, SBE single base extension- using MALDI-TOF (mass spectrophotometer)
High Resolution molecular methods
exon scanning, cDNA analysis, NexGen Sequencing.
PCR RFLP
restriction fragment length polymorphism PCR- can be used when a SNP creates or abolishes a restriction enzyme site
Restriction Enzymes
proteins that recognize and cleave double-stranded DNA segment, often palindromic, Highly specific recognize a unique sequence, typically ranging from 3-6 bases.
Gel based
electrophoresis, with negative charge, DNA is repelled from negative charge and moves towards cathode end. - this is then analyzed because smaller DNA moves farther down the gel, can predict the size, if there is a mutation and it’s affecting the restriction enzyme site then the DNA doesn’t get cleaved and therefore it doesn’t travel as far. These are known locations on the gel to compare them to, not unknown.
Sequence specific primer PCR
same idea as RFLP, DNA primer attaches, if there is a SNP in the primer region, it won’t bind therefore when running gel it won’t be present- can conclude that it has a SNP doesn’t have ‘wild type’
Internal control for gel based
human growth hormone
Bead chip assay- genotyping
Multiple beads looking for specific sequence, different colors fluoresce when bound. if there is not florescence it’s not positive
Sanger sequencing
primers bind, within the ‘master mix’ dntp’s and ddntp’s once ddntp’s are bound no more elongation can take place. obviously every nucleotide added is a slight increase in the length of the strand. Each nucleotide has it’s own fluorescence as well and when through a gel the shortest fragments travel the farthest with the last nucleotide fluorescent on the ddNTP’s (dideoxy) you can track the fluorescence and the distance traveled to determine the exact sequence. on the graph if there are two colors at same location =heterozygous, if homozygous only one color usually taller peak.
downside of sanger sequencing
more time consuming and expensive but good for rare blood types, need to understand specific allele/mutations
Home brew or lab developed tests
using research only reagents (LDT=lab developed tests) may be designed and optimized and validated by the laboratory, must be validated by laboratory and have disclaimer ‘not approved by FDA’
Molecular Testing validations
design: repeatability and reproducablitliy, validation sample set: sample type, heterozygous and wild type (need a wide range of samples to test, not all wild type) Method type (Novel test method or new test existing test method)
