Molecular Immunohematology Flashcards
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)
Novel test method
At least 20 samples, at least one wild-type and heterozygous sample (bringing up new instrument)
New test, existing test method
at least a wild-type and heterozygous sample. (validation platform performing okay after serious maintenance
Ala
alanine-A
arg
arginine-R
asn
asparagine-N
asp
aspartic acid-D
cys
cystein-C
gln
glutamine-Q
glu
glutamic acid-E
gly
glycine-g
his
histidine-h
ile
isoleucine-i
leu
leucine-L
met
methionine-M
phe
phenylalanine-F
pro
proline-P
ser
serine-S
thr
threonine-T
trp
trptophan-W
tyr
tyrosine-Y
val
valine-V
Lys
Lysine -K
DARC and Fy blood group system
ACKRI gene expression is impacted by SNP in gene promoter region (GATA box- usually bound by transcription factor)
GATA
SNP usually found in african americans, SNP disrupts the binding site for the erythroid* GATA-1 transcription factr resutling in loss of Fyb antigen on RBCs - expression on endothelial cells not affected.
Fy null due to homozygosity- disease association
resistance to malaria
Can GATA box mutation patients make anti-Fyb
no, still produced on epithileal cells not recognized as foreign
Phenotype of GATA box mutation
(Fya-Fyb+w)
DARC and new name.
duffy antigen receptor for chemokines. - may play a role as a scavenger on the red blood cell surface to eliminate excess of toxic chemokines produced in some pathologic situations. Duffy=DARC duffy encoded by DARC gene. DARC gene is now called ACKR1
Universal antigen frequency
U, around 1% a.a. S-s-U- , U- phenotype is common (37%) in west africans and rare (0.001%) in Caucasians.
U-S-s- causation
large deletion within the GYPB gene
Closed platform
Can only use reagent that can be purchased from manufacturer
Open platform
(Luminex, Sanger sequencing) not limited by manufacturer for what you can use on these platforms
anti-U
very clinically significant, HTR and HDFN
S antigen
Met48
s antigen
Thr48
indel
this means insertional deletion!!
Genetic determinant for S-s-U-
entire deletion of GYPB gene
Genetic determinant of U+var
IVS5+8G>T, termination codon is moved up further and exon 5 not longer included in final product, nearly undetectable surface-expression, two different types of U variable. The second one is a partial deletion of exon 5, stop codon still moved up due to mutation. this one is much more rare
IVS
intervening sequence
What is the clinical manifestation of U+var
U antigen can exist in variant form, it’s expressed very weakly but often not detected. S-s-U- individuals can make anti-U to U variant cells.
How was U+var detected prior to molecular
serology: adsorption elution with anti-U, PEG enhancement, limited by the anti-U specificity. (anti-U vs. anti-GYPB.U). now molecular methods can distinguish U- from U+var
What percentage of caucasians serologically test as weak D, what is definition of serological weak D
0.2-1.2%. Testing no or weak <2+ reaction at is but agglutinating moderately or strongly with AHG
mutation in Weak D 1
c. 809G (270G)
mutation in weak D 2
c. 1154 C (70Q)
mutation in weak D 3
c. 8G (3G)
Weak D types associated with allo anti-D
Type 4.2, 11, 15, 21 and 57
Weak D Variants DIII, DIV, DAR and weak partial 4.0 carry what variation?
p. Phe223Val (P=protein)
6 main reasons for DNA analysis in blood banking
- analysis of HDFN potential in testing child and father for gene/heterozygosity 2. WAA patients to know molecular phenotype prior to transfusion 3. In patients with partial/weak D to determine necessity for Rhig 4. getting phenotype of a patient that was recently transfused 5. When positive DAT in patient(coated with IgG) 6. distinguishing alloantibodies from autoantibodies
Common RHCE alleles
CE, ce, cE, Ce, *all are negative for V and VS antigens
RHCE variants can be associated with
- altered e antigens- patients can make anti-e 2. variable reactivity with monoclonal anti-e reagents 3. loss of high prevalence antigens hrs and hrb or both 4. gain of low incidence antigens V and VS or both
what is the predicted phenotype of someone with DIIIa for one allele, and DIII-CE(4-7)-D on the other allele
partial D (DIIIA). altered C, partial e, E-, partial c, V+ VS+ hrb-
HPA inheritance
Human Platelet antigens, bi-allelic SNP determines antigen status. autosommal codominance, “a” more common “b” less common. 29 human platelet antigen pairs, can result in alloimmunization if exposed to different antigen.
Difference between FNAIT and HDFN
FNAIT can occur during first pregnancy, typically HDFN become exposed during first pregnancy and causes problems during second
How common is FNAIT to HPA-1a
1 in 1000 to 1 in 2000 infants born to HPA-1a negative moms,
Most common antigen implicated in FNAIT
HPA-1a=80% of the cases, the rest are a combination of 2,3,4,5 and 15
PCR vs TMA
PCR- DNA, TMA-RNA
SSP
Sequence specific primer, looking for presence of specific trait or allele, not full picture
SSOP
sequence specific oligonucleotide probes
CREG example, cannot be differentiated serologically
B15 and B16 SSP and SSOP distinguishes between these
SBT
Sequence based typing- Sanger sequenzies, cannot tell cis/trans interactions because it evaluates both alleles (paternal and maternal) simultaneously
NGS
Next generation sequencing sequencing of whole gene- single strand no ambiguities like in sanger sequencies because each strand done independently
microlymphotoxicity assay
CDC- complement dependent cytotoxicity. HLA antibody is on a platelet, lymphocytes from a patient are added. Addition of rabbit complement, activation of complement occurs and if cell is present there is cell death. If cells do not die they exclude dye and appear refractile. If cells diee they incorporate the dye- HLA type is deined when antibody causes 50% of patients cells to die. Graded apprpriatel 81-100% of death is strong positive
When is molecular testing required by regulatory or accrediting agency
Solid organ and hematopoietic stem cell transplant
Limitations of microlymphotoxicity assay
Cannot differentiate some allele families, low reproducibility for HLA class, C, DP, DQ
