Week 5 - FCM + Antigen Receptors

Question 1

What is the orientation of how cells flow in FCM

Answer 1

Centralized flow by electromagnetic forces

Question 2

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

Answer 2

Fluorescence intensity over the Side Scatter

Question 3

Advantages of FCM

Answer 3

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

Question 4

Disadvantages of FCM

Answer 4

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

Question 5

FCM is used for diagnosis of

Mention only the obligatory ones

Answer 5

Hematological Disorders
Immunodeficiency
Monitoring after transplantation
Monitoring of Biological therapies

Question 6

Steps of FCM

Answer 6

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

Question 7

Size measuring with FCM

Answer 7

Forward Scattering Parameter

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

Question 8

Complexity of cell measurement by FCM

Answer 8

Right angle scatter - Side Scatter parameter

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

Question 9

Gating in FCM

Method

Answer 9

Selection of cell population of interest

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

Question 10

Fluorescent Labeling - Direct or Indirect in FCM

Answer 10

DIrect

Question 11

Fluorescence Labeling - Direct or Indirect in Histology

Answer 11

Indirect

Question 12

Graph for Fluorescence labeling

Interpretation

Answer 12

Cell number or Log of Intensity

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

Question 13

Use of FCM (FACS) in Research

Answer 13

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

Question 14

Cell cycle analysis in FCM

Answer 14

PI labeling

Question 15

HLA-DR positive Cell FCM use

Answer 15

Sepsis Follow up over time

Question 16

Cytokines measurement with FCM

Answer 16

Immunolabeled beads for catching Cytokines

Question 17

What are the non specific Immunoreceptors

Answer 17

Pattern recognition Receptors

Opsonic - Fc receptors+Complement receptors

Question 18

What is the difference between TCR and BCR binding?

Answer 18

TCR - MHC Bound Antigens

BCR - Free Antigens

Question 19

Types of Antigen epitope determinant

Answer 19

Linear determinant
Conformation Determinant
Neoantigenic Determinants

Question 20

TCR function in contrast with BCR function

Basic

Answer 20

Never Secreted to the blood

Only serves for T cell recognition antigen

Question 21

What are the ITAM motifs of BCR and TCR

Answer 21

TCR - CD3 and zeta chain

BCR - Ig-beta and Ig-alpha

Question 22

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

Answer 22

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

Question 23

Allotypic and Idiotipic differences within the same class Antibody

Answer 23

Allotype - Structural difference in the non active regions.

Idiot yep - Structural difference in the Active reception part.

Question 24

Papain reaction and Products

Answer 24

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

Question 25

Structural Functions of the Ab

Answer 25

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

Question 26

Importance of C3b and C4b binding on IC

Answer 26

CR1 interaction leads to Macrophages opsinisation in the spleen.

Question 27

Hypervariable parts of VH in Ab

Answer 27

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

Question 28

CDRs binding orientation

Answer 28

CDR1 and CDR2 - Polynorphic Residue of MHC | CDR3 - T cell contact of Residue Peptide

Question 29

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

Answer 29

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

Question 30

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

Answer 30

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

Question 31

DNA Diversity calculation of the VDJ recombination

Answer 31

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

Question 32

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

Answer 32

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

Question 33

Recombinases enzymes (RAG1 and 2) in Pro-Lymphocytes

Answer 33

Clevage of DNA and formation of Hairpin segment

Question 34

Artemis Endonuclease activity in Pro-Lymphocytes

Answer 34

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

Question 35

TdT - Terminal deoxynucleitidyl Transferase | Activity in Pro-Lymphocytes

Answer 35

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

Question 36

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

Answer 36

Alternative splicing of RNA molecule

Question 37

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

Answer 37

Allelic Exclusion- | First recombination capable gene transferred is silencing the other

Question 38

SCID | What could be the cause?

Answer 38

Severe Combined Immunodeficiency | RAG deficiency - No T or B cells diversity

Question 39

Omen Syndrome

Answer 39

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

Question 40

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

Answer 40

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

Question 41

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

Answer 41

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

Question 42

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

Answer 42

S Phase Elevation

Question 43

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

Answer 43

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

Question 44

FACS - What are the Clinical Uses? (3)

Answer 44

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