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I&I SC2 > Molecular diagnostics III > Flashcards

Molecular diagnostics III Flashcards

1
Q

What are the three use cases of immunodiagnostics throughout the disease course of lymphoproliferations?

A
  • Diagnosis -> tumor clone vs. immune response
  • Prognosis -> heterogeneity - different outcomes
  • Monitoring -> evaluation of therapy effect
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2
Q

Which specific code involved in lymphoproliferative disease can be used to diagnose, prognose and monitor therapy effect?

A

Unique DNA-code of the receptors of lymphocytes

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3
Q

How does it come to be that every lymphocyte receptor has an unique receptor? (3)

A
  • VDJ-recombination
  • n-nucleotide addition/deletion
  • p-nucleotide addition/deletion
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4
Q

Where does the diversity in receptors occur?

A

Mainly in junction region = CDR3

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5
Q

In case of lymphoproliferation seen in lymph nodes or blood, lymph nodes can be investigated to determine whether this increase in lymphocytes is due to … (2)

A
  • Physiological immune response
  • Malignant process
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6
Q

Which kind of testing is used to determine the cause of lymphoproliferation?

A

Clonality testing

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7
Q

What are the results of clonality testing of lymphoid tumors/immune responses?

A

Lymphoid tumors: all cells have identical Ig/Tr-genes (monoclonal)
Immune responses: multiple different markers

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8
Q

How does a PCR-based analysis of Ig/TR rearrangement work? (2)

A
  • Filtering out non-lymphoid cells
  • PCR fragment analysis
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9
Q

How do you filter out non-lymphoid cells in a PCR-based analysis of Ig/TR rearrangement?

A

Ig/Tr genes are far apart -> no primers span this distance -> no PCR products

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10
Q

Lymphoproliferations: What does the PCR fragment analysis investigate? How?

A

Length diversity in coupling area -> using fluorescent primers

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11
Q

Lymphoproliferations: How do fluorescent primers determine length diversity in coupling area?

A

Different fluorescent intensities can be used to determine fragment length

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12
Q

Lymphoproliferations: monoclonal/polyclonal situations lead to a normally distributed curve of coupling area lengths (no signal fragment is dominant)

A

Polyclonal

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13
Q

Lymphoproliferations: monoclonal/polyclonal situations lead to a single peak because one fragment is dominant over the others?

A

Monoclonal

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14
Q

Lymphoproliferations: What makes fragment analysis complicated? (2)

A
  • VDJ recombinations -> grouped into 7 families -> multiplex settings needed
  • Somatic hypermutation
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15
Q

Lymphoproliferations: What is multiplexing?

A

Multiple primers at both sides

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16
Q

Lymphoproliferations: Why is it not necessary to use a primer for every different VDJ-combination?

A

7 families -> contain regions in which the DNA sequence is very similar

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17
Q

Lymphoproliferations: What ensures that all fragments will be covered by the assay?

A

Addition of probes for all IGVH-families

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18
Q

Lymphoproliferations: Why do B cells add extra complexity to PCR analyses?

A

Somatic hypermutation -> cause primer misannealing

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19
Q

Lymphoproliferations: How do you compensate for primer misannealing?

A

Extra primers at different locations -> reduce complete non-binding

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20
Q

Lymphoproliferations: How can we make immunodiagnostics on B cells more reliable? This leads to?

A

Investigate both heavy- and light chain –> higher sensitivity

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21
Q

Lymphoproliferations: PCR-based fragment analysis has a lower/higher resolution than NGS-based techniques

A

Lower

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22
Q

Lymphoproliferations: Why do NGS-based techniques allow for higher resolution than PCR-based fragment analysis?

A

Allows for analysis of all sequences

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23
Q

Lymphoproliferations: Which two NGS assays can be used to investigate lymphoproliferations?

A
  • Amplicon PCR amplification
  • DNA-based capture assay
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24
Q

Lymphoproliferations: Describe the amplicon PCR amplification workflow (2)

A
  • Multiplex expansion of IGH-and IGK regions
  • Sequencing expanded products
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25
Q

Amplicon PCR amplification: how can a result suggest clonality?

A

One peak clearly stands out from the curve

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26
Q

Amplicon PCR amplification: If there are a lot of different sequences represented in a curve (even though there is a clear peak that stands out), what does this suggest?

A

No clonal proliferation

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27
Q

Lymphoproliferations: Describe the DNA-based capture assay workflow

A

Probes pulldown ROI from total genomic information

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28
Q

DNA-based capture assay: Probes can target what? (4)

A
  • Rearrangements
  • Translocations
  • Copy no. alterations
  • Mutations (indells)
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29
Q

DNA-based capture assay: What is meant with probes being used to target rearrangements?

A

Simultaneous reporting of clonality at all Ig/TR loci

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30
Q

DNA-based capture assay: Probes can/can’t be specific for all different V-, D-, and J-genes

A

Can

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31
Q

DNA-based capture assay: What is the advantage of using probes to target translocations?

A

Detecting previously unknown translocation partners

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32
Q

DNA-based capture assay: What does using a probe to target copy number alterations allow for?

A

Clonality detection in difficult B-cells cases exhibiting somatic hypermutation

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33
Q

DNA-based capture assay: Adding mutations as an addition method does not help/help in detecting clonality

A

Help

34
Q

What is meant with the heterogeneity in lymphoproliferations?

A

Specific subgroups have different prognosis/require different treatment

35
Q

Lymphoproliferations: which techniques are available for prognosis? (2)

A
  • Molecular immunodiagnostics
  • Cytogenetics
36
Q

Lymphoproliferations: What is analyzed using molecular immunodiagnostics?

A

Somatic mutation analysis of rearranged Ig-genes

37
Q

Lymphoproliferations: Which sequencing techniques are used in molecular immunodiagnostics? (2)

A
  • Sanger
  • NGS-based immune repertoire sequencing
38
Q

Lymphoproliferations: What is analyzed using cytogenetics? (2)

A
  • Chromosome abberations/translocation/deletions
  • Mutations/SNVs
39
Q

Which physiological mutations are studied in CLL? Which technique?

A

IGHV-genes -> Sanger

40
Q

Prognosis: Why are mutations in IGHV-genes studied in CLL?

A

They give information about whether the origin cell has been through SMH or not -> gives big difference in prognosis

41
Q

What is the percentage identical IGHV in U-CLL?

A

> 98%

42
Q

Lymphoproliferations: Patients with U-CLL have a better/worse prognosis than patients with M-CLL

A

Worse

43
Q

Lymphoproliferations: Describe the process of sequencing IGHV mutations (3)

A
  • Clonal rearrangement is amplified
  • V-gene almost fully sequenced
  • Blast sequence in IMGT database
44
Q

Lymphoproliferations: How is the clonal rearrangement amplified in IGHV mutation sequencing?

A

Using IGH FR1 or leader multiplex PCR

45
Q

Lymphoproliferations: What is determined in the blast of IGHV mutation sequences? (2)

A
  • V-, D- and J-gene usage
  • Total no. mutations in sequence compared to reference sequence
46
Q

Lymphoproliferations: when do you consider a IGHV sequence non-mutated?

A

If more or the same as 98% is similar to the reference sequence

47
Q

Lymphoproliferations: What factor can also impact CLL prognosis (sequencing)?

A

Amino acid sequence of the junctional region

48
Q

Lymphoproliferations: what is CLL monitoring aimed at?

A

Evaluation of therapy effect

49
Q

Lymphoproliferations: What is the main monitoring analysis?

A

Minimal residual disease analysis

50
Q

Minimal residual disease analysis: Which patient data is used for this analysis?

A

Patient-specific Ig/TCR-rearrangements

51
Q

Minimal residual disease analysis: What do patient-specific Ig/TCR-rearrangements allow for?

A

High-resolution monitoring

52
Q

Which techniques are used to monitor CLL? (2)

A
  • Real-time quantitative PCR
  • NGS-based MRD monitoring
53
Q

What is the common principle in both real-time quantitive PCR and NGS-based MRD monitoring?

A

Clone sequencing upon diagnosis

54
Q

CLL monitoring: What are the steps of the PCR-based process? (3)

A
  • Identification of targets at diagnosis
  • Testing patient-specific RQ-PCR
  • Analysis of MRD during follow up using RQ-PCR
55
Q

CLL monitoring: In over 95% of CLL patients, at least two … markers are found through sequencing

A

Ig/TR markers

56
Q

CLL monitoring: What does identification of targets at diagnosis allow for…?

A

Design of a patient-specific primer

57
Q

What region is also called the DNA fingerprint of a leukemic cell?

A

Junctional region

58
Q

PCR-based process CLL monitoring: On what are sensitivity tests performed?

A

Dilutions

59
Q

CLL monitoring: Primers are designed and tested in silico for…(3)

A
  • Hairpin formation (internal looping)
  • Duplicate/dimer formation
  • Aspecific binding
60
Q

What is MRD?

A

Minimal residual disease -> remaining leukaemic cells after cancer treatment

61
Q

MRD analysis: how is the standard curve obtained?

A

Serial dilution of diagnostic sample

62
Q

MRD analysis: what does the yellow line show?

A

CT-value measured in follow-up samples

63
Q

Monitoring allows for detection of slowing in the decrease of leukaemic cells. What can be done if such a decrease is spotted?

A

Therapy can be adapted to mitigate this higher risk of relapse

64
Q

CLL monitoring: What are validation normalization reagents used for in NGS-based monitoring techniques?

A

To trace back the number of reads of a leukaemic sequence to the actual no. of cells

65
Q

CLL monitoring: How can validation normalization be performed?

A

Central in tube quality/quantification control

66
Q

CLL monitoring: NGS-based MRD detection allows/doesn’t allow for deeper sampling than PCR-based techniques

A

Allows

67
Q

What is the most polymorphic gene locus of the human genome?

A

HLA

68
Q

Genetic variation in MHC II is mainly in the … chains?

A

B-chains

69
Q

HLA: Which class II genes show some variation in the a-chain?

A

DP and DQ

70
Q

HLA: Which class II gene shows only variation in the B-chain?

A

DR

71
Q

True or false: polymorphisms contribute to amino acid substitutions in the peptide binding cleft of the HLA molecule

A

True

72
Q

What does diversity of HLA within the population lead to?

A

Diversity of adaptive immune response

73
Q

What are reasons to perform HLA-typing? (3)

A
  • Transplant rejection
  • Predisposition for certain (auto-) immune diseases
  • Adverse reactions to medication
74
Q

Which HLA-genes need to be matched for a kidney transplant? (3)

A
  • HLA-A
  • HLA-B
  • HLA-DRB1
75
Q

True or false: For hematopoietic stem cell transplantation, matching of all HLA genes is required

A

True

76
Q

Do you need to perform HLA matching for all solid organs (except kidneys)?

A

No

77
Q

Why is a HLA matching test not performed during solid organ transplant (except kidney)?

A

No time in the transplantation setting for solid organs

78
Q

HLA can typed through.. (2)

A
  • Serological techniques -> using antibodies
  • Molecular techniques -> mostly PCR/sequencing
79
Q

What is a problem in serological HLA typing?

A

No. of HLA alleles is far higher than the amount of serological HLA specificities

80
Q

On what principle is HLA typing mostly based?

A

Lysis

81
Q

Cells that need to be HLA-typed are incubated with… (2)

A
  • Serum containing known anti-HLA antibodies, OR
  • Specific monoclonal anti-HLA antibodies
82
Q
A

I&I SC2 (13 decks)

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