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GBA Immunology > Week 5 - FCM + Antigen Receptors > Flashcards

Week 5 - FCM + Antigen Receptors Flashcards

1
Q

What is the orientation of how cells flow in FCM

A

Centralized flow by electromagnetic forces

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2
Q

In Routine hematology test what is is measured in the two axes of he graph

A

Fluorescence intensity over the Side Scatter

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3
Q

Advantages of FCM

A

Large Amount of cells examined and sorting possible
Quick 2 min examination, Diagnosis in an hour (critical in leukemia)
Objective, Automated and Multi-parametric

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4
Q

Disadvantages of FCM

A

Expensive for special equipment
High training needed
Cell suspension is needed for examination

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5
Q

FCM is used for diagnosis of

Mention only the obligatory ones

A

Hematological Disorders
Immunodeficiency
Monitoring after transplantation
Monitoring of Biological therapies

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6
Q

Steps of FCM

A

Sample preparation - conjugation with Ab
Instrument setup
Data description
Data analysis

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7
Q

Size measuring with FCM

A

Forward Scattering Parameter

Absence of Laser signal scattered by the cell (Speed of flow is known)

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8
Q

Complexity of cell measurement by FCM

A

Right angle scatter - Side Scatter parameter

Detection of the scattered light that bounces back from the cell in 90 degrees

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9
Q

Gating in FCM

Method

A

Selection of cell population of interest

Circling the cell population on the graph - Possible by labeling with luminescent Ab binding the CD

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10
Q

Fluorescent Labeling - Direct or Indirect in FCM

A

DIrect

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11
Q

Fluorescence Labeling - Direct or Indirect in Histology

A

Indirect

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12
Q

Graph for Fluorescence labeling

Interpretation

A

Cell number or Log of Intensity

When the peak is more on the right the more Ab binding we have

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13
Q

Use of FCM (FACS) in Research

A

Sorting by Electrical force pulling of detected cells of interest flowing down

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14
Q

Cell cycle analysis in FCM

A

PI labeling

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15
Q

HLA-DR positive Cell FCM use

A

Sepsis Follow up over time

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16
Q

Cytokines measurement with FCM

A

Immunolabeled beads for catching Cytokines

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17
Q

What are the non specific Immunoreceptors

A

Pattern recognition Receptors

Opsonic - Fc receptors+Complement receptors

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18
Q

What is the difference between TCR and BCR binding?

A

TCR - MHC Bound Antigens

BCR - Free Antigens

19
Q

Types of Antigen epitope determinant

A

Linear determinant
Conformation Determinant
Neoantigenic Determinants

20
Q

TCR function in contrast with BCR function

Basic

A

Never Secreted to the blood

Only serves for T cell recognition antigen

21
Q

What are the ITAM motifs of BCR and TCR

A

TCR - CD3 and zeta chain

BCR - Ig-beta and Ig-alpha

22
Q

Classification of Antibodies
According to -
(Light chains are always kappa or Lambda)

A

Heavy chains Type (Isotypic Differences)
Glycosilation
Mono- , Di- , Pentameric Forms

23
Q

Allotypic and Idiotipic differences within the same class Antibody

A

Allotype - Structural difference in the non active regions.

Idiot yep - Structural difference in the Active reception part.

24
Q

Papain reaction and Products

A

Lysis of Ab to Fab and Fc
Fab- Antigen Binding Fragment
Fc- Crystallizable Fragment

25
Q

Structural Functions of the Ab

A

Idiotypic antigen Binding - Variant domains
C3b and C4b binding - First constant domain
Fc Binding - Constant Stem part
C1q binding - Second constant domain

26
Q

Importance of C3b and C4b binding on IC

A

CR1 interaction leads to Macrophages opsinisation in the spleen.

27
Q

Hypervariable parts of VH in Ab

A

Hypervariable CDR - Ionic interaction, Disulfides..
Complementarity Determining Region.
CDR1, CDR2, CDR3

28
Q

CDRs binding orientation

A

CDR1 and CDR2 - Polynorphic Residue of MHC

CDR3 - T cell contact of Residue Peptide

29
Q

How many Antigens do we meet in Life ?
How many genes are there in the Human DNA?
What is the problem arising?

A

10 Million kinds
35,000 Genes
Hypothetically Insufficient readily produced Antibodies

30
Q

What is the composition of the gene for the Heavy chain?

A 
VDJ segments (Combined togethein the Differentiation of the CLP)
C segments- Mio , Delta, Gamma, Alpha, Epsilon
31
Q

DNA Diversity calculation of the VDJ recombination

A

51~ V
27 D
6 J
51 x 27 x 6 equals 8262

32
Q

DNA Diversity out of Kappa and Lambda arranged independently - Which Segments?

A

Kappa - 40(V) x 5 (J) equals 200
Lambda - 30 (V) x 4 (J) equals 120
(Numbers don’t matter)

33
Q

Recombinases enzymes (RAG1 and 2) in Pro-Lymphocytes

A

Clevage of DNA and formation of Hairpin segment

34
Q

Artemis Endonuclease activity in Pro-Lymphocytes

A

Cleavage of the Hairpin structure from RAG marked by the DNA in Palindromic Sequences, No filling of gap yet..

35
Q

TdT - Terminal deoxynucleitidyl Transferase

Activity in Pro-Lymphocytes

A

Adds nucleotides and Forms ligation between the separated strands filling the gap between the palindromic

36
Q

How does Recombination of C segments of the Ab gene occur?

A

Alternative splicing of RNA molecule

37
Q

How is it that BCR show only Paternal or Maternal Ig never both?

A

Allelic Exclusion-

First recombination capable gene transferred is silencing the other

38
Q

SCID

What could be the cause?

A

Severe Combined Immunodeficiency

RAG deficiency - No T or B cells diversity

39
Q

Omen Syndrome

A

RAG or Artemis partial activity
Rash and Erythoderma
caused by IgE overproduction with IL4 IL5 eosinophil driving and leakage forming

40
Q

Mechanisms responsible for the full diversity of antigen receptors:
V(D)J rearrangement steps -

A

1) Germline diversity
2) Combinatorial diversity
3) Junctional diversity

41
Q

Mechanisms responsible for the full diversity of antigen receptors:
Post-V(D)J rearrangement -

A

4) Receptor editing
5) Somatic hypermutation
6) Receptor revision

42
Q

What would be the sign for Malignancy in FACS detection of PI labeled Cell cycle intermediates?

A

S Phase Elevation

43
Q

What are the different graphs that we can obtain from FACS? (3)

A

1) Granularity Vs Size
2) Fluorescent Intensity(Log) Vs Cell Count (or another Intensity)
3) PI labeling for Cell cycle measurement

44
Q

FACS - What are the Clinical Uses? (3)

A

1) Hematological -Leukemia/Lymphoma/Bone Marrow Transplantation
2) MDR/MRD Diseases
3) Immunodeficiencies (AIDS)

GBA Immunology (15 decks)

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