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SBB > Molecular Immunohematology > Flashcards

Molecular Immunohematology Flashcards

1
Q

Antithetical

A

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

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2
Q

C and c a.a. differences

A

differ by 4 a.a. most important (extracellular) Serine103Proline

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3
Q

E and e a.a. difference

A

Proline226Alanine

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4
Q

“Rhesus boxes”

A

boxes that sit on the outside of the RhD gene.

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5
Q

RhD deletion

A

can cross or recombine onto other chromosome- causes D deletion

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6
Q

Blood groups location on red cells

A

blood group antigens are carried on molecules expressed on the RBC membrane

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7
Q

Most common type of genetic variation

A

Single Nucleotide Polymorphism

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8
Q

How can molecular mehthods be used to predict blood group antigens

A

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

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9
Q

Problems with serologic methods

A

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

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10
Q

Why are some proteins expressed on certain surfaces (red cells) but not others (tissues)

A

ALL have the same DNA in every cell, depending on the cell type, different genes are either turned on or turned off

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11
Q

Steps to PCR

A

denaturation, primer annealing, primer extension and repeat

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12
Q

Low resolution Gel based molecular methods

A

SSP (sequence specific primers) PCR for known SNPs, PCR-RFLP restriction fragment length polymorphism for known SNPs

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13
Q

Medium resolution molecular methods

A

Immucor Bead chip and Grifols IDCore (arrays), Taqman genotypling, SBE single base extension- using MALDI-TOF (mass spectrophotometer)

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14
Q

High Resolution molecular methods

A

exon scanning, cDNA analysis, NexGen Sequencing.

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15
Q

PCR RFLP

A

restriction fragment length polymorphism PCR- can be used when a SNP creates or abolishes a restriction enzyme site

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16
Q

Restriction Enzymes

A

proteins that recognize and cleave double-stranded DNA segment, often palindromic, Highly specific recognize a unique sequence, typically ranging from 3-6 bases.

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17
Q

Gel based

A

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.

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18
Q

Sequence specific primer PCR

A

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’

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19
Q

Internal control for gel based

A

human growth hormone

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20
Q

Bead chip assay- genotyping

A

Multiple beads looking for specific sequence, different colors fluoresce when bound. if there is not florescence it’s not positive

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21
Q

Sanger sequencing

A

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.

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22
Q

downside of sanger sequencing

A

more time consuming and expensive but good for rare blood types, need to understand specific allele/mutations

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23
Q

Home brew or lab developed tests

A

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’

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24
Q

Molecular Testing validations

A

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)

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25
Q

Novel test method

A

At least 20 samples, at least one wild-type and heterozygous sample (bringing up new instrument)

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26
Q

New test, existing test method

A

at least a wild-type and heterozygous sample. (validation platform performing okay after serious maintenance

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27
Q

Ala

A

alanine-A

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28
Q

arg

A

arginine-R

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29
Q

asn

A

asparagine-N

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30
Q

asp

A

aspartic acid-D

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31
Q

cys

A

cystein-C

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32
Q

gln

A

glutamine-Q

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33
Q

glu

A

glutamic acid-E

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34
Q

gly

A

glycine-g

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35
Q

his

A

histidine-h

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36
Q

ile

A

isoleucine-i

37
Q

leu

A

leucine-L

38
Q

met

A

methionine-M

39
Q

phe

A

phenylalanine-F

40
Q

pro

A

proline-P

41
Q

ser

A

serine-S

42
Q

thr

A

threonine-T

43
Q

trp

A

trptophan-W

44
Q

tyr

A

tyrosine-Y

45
Q

val

A

valine-V

46
Q

Lys

A

Lysine -K

47
Q

DARC and Fy blood group system

A

ACKRI gene expression is impacted by SNP in gene promoter region (GATA box- usually bound by transcription factor)

48
Q

GATA

A

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.

49
Q

Fy null due to homozygosity- disease association

A

resistance to malaria

50
Q

Can GATA box mutation patients make anti-Fyb

A

no, still produced on epithileal cells not recognized as foreign

51
Q

Phenotype of GATA box mutation

A

(Fya-Fyb+w)

52
Q

DARC and new name.

A

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

53
Q

Universal antigen frequency

A

U, around 1% a.a. S-s-U- , U- phenotype is common (37%) in west africans and rare (0.001%) in Caucasians.

54
Q

U-S-s- causation

A

large deletion within the GYPB gene

55
Q

Closed platform

A

Can only use reagent that can be purchased from manufacturer

56
Q

Open platform

A

(Luminex, Sanger sequencing) not limited by manufacturer for what you can use on these platforms

57
Q

anti-U

A

very clinically significant, HTR and HDFN

58
Q

S antigen

A

Met48

59
Q

s antigen

A

Thr48

60
Q

indel

A

this means insertional deletion!!

61
Q

Genetic determinant for S-s-U-

A

entire deletion of GYPB gene

62
Q

Genetic determinant of U+var

A

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

63
Q

IVS

A

intervening sequence

64
Q

What is the clinical manifestation of U+var

A

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.

65
Q

How was U+var detected prior to molecular

A

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

66
Q

What percentage of caucasians serologically test as weak D, what is definition of serological weak D

A

0.2-1.2%. Testing no or weak <2+ reaction at is but agglutinating moderately or strongly with AHG

67
Q

mutation in Weak D 1

A

c. 809G (270G)

68
Q

mutation in weak D 2

A

c. 1154 C (70Q)

69
Q

mutation in weak D 3

A

c. 8G (3G)

70
Q

Weak D types associated with allo anti-D

A

Type 4.2, 11, 15, 21 and 57

71
Q

Weak D Variants DIII, DIV, DAR and weak partial 4.0 carry what variation?

A

p. Phe223Val (P=protein)

72
Q

6 main reasons for DNA analysis in blood banking

A
  1. 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
73
Q

Common RHCE alleles

A

CE, ce, cE, Ce, *all are negative for V and VS antigens

74
Q

RHCE variants can be associated with

A
  1. 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
75
Q

what is the predicted phenotype of someone with DIIIa for one allele, and DIII-CE(4-7)-D on the other allele

A

partial D (DIIIA). altered C, partial e, E-, partial c, V+ VS+ hrb-

76
Q

HPA inheritance

A

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.

77
Q

Difference between FNAIT and HDFN

A

FNAIT can occur during first pregnancy, typically HDFN become exposed during first pregnancy and causes problems during second

78
Q

How common is FNAIT to HPA-1a

A

1 in 1000 to 1 in 2000 infants born to HPA-1a negative moms,

79
Q

Most common antigen implicated in FNAIT

A

HPA-1a=80% of the cases, the rest are a combination of 2,3,4,5 and 15

80
Q

PCR vs TMA

A

PCR- DNA, TMA-RNA

81
Q

SSP

A

Sequence specific primer, looking for presence of specific trait or allele, not full picture

82
Q

SSOP

A

sequence specific oligonucleotide probes

83
Q

CREG example, cannot be differentiated serologically

A

B15 and B16 SSP and SSOP distinguishes between these

84
Q

SBT

A

Sequence based typing- Sanger sequenzies, cannot tell cis/trans interactions because it evaluates both alleles (paternal and maternal) simultaneously

85
Q

NGS

A

Next generation sequencing sequencing of whole gene- single strand no ambiguities like in sanger sequencies because each strand done independently

86
Q

microlymphotoxicity assay

A

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

87
Q

When is molecular testing required by regulatory or accrediting agency

A

Solid organ and hematopoietic stem cell transplant

88
Q

Limitations of microlymphotoxicity assay

A

Cannot differentiate some allele families, low reproducibility for HLA class, C, DP, DQ

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