Week 5 - FCM + Antigen Receptors Flashcards
What is the orientation of how cells flow in FCM
Centralized flow by electromagnetic forces
In Routine hematology test what is is measured in the two axes of he graph
Fluorescence intensity over the Side Scatter
Advantages of FCM
Large Amount of cells examined and sorting possible
Quick 2 min examination, Diagnosis in an hour (critical in leukemia)
Objective, Automated and Multi-parametric
Disadvantages of FCM
Expensive for special equipment
High training needed
Cell suspension is needed for examination
FCM is used for diagnosis of
Mention only the obligatory ones
Hematological Disorders
Immunodeficiency
Monitoring after transplantation
Monitoring of Biological therapies
Steps of FCM
Sample preparation - conjugation with Ab
Instrument setup
Data description
Data analysis
Size measuring with FCM
Forward Scattering Parameter
Absence of Laser signal scattered by the cell (Speed of flow is known)
Complexity of cell measurement by FCM
Right angle scatter - Side Scatter parameter
Detection of the scattered light that bounces back from the cell in 90 degrees
Gating in FCM
Method
Selection of cell population of interest
Circling the cell population on the graph - Possible by labeling with luminescent Ab binding the CD
Fluorescent Labeling - Direct or Indirect in FCM
DIrect
Fluorescence Labeling - Direct or Indirect in Histology
Indirect
Graph for Fluorescence labeling
Interpretation
Cell number or Log of Intensity
When the peak is more on the right the more Ab binding we have
Use of FCM (FACS) in Research
Sorting by Electrical force pulling of detected cells of interest flowing down
Cell cycle analysis in FCM
PI labeling
HLA-DR positive Cell FCM use
Sepsis Follow up over time
Cytokines measurement with FCM
Immunolabeled beads for catching Cytokines
What are the non specific Immunoreceptors
Pattern recognition Receptors
Opsonic - Fc receptors+Complement receptors
What is the difference between TCR and BCR binding?
TCR - MHC Bound Antigens
BCR - Free Antigens
Types of Antigen epitope determinant
Linear determinant
Conformation Determinant
Neoantigenic Determinants
TCR function in contrast with BCR function
Basic
Never Secreted to the blood
Only serves for T cell recognition antigen
What are the ITAM motifs of BCR and TCR
TCR - CD3 and zeta chain
BCR - Ig-beta and Ig-alpha
Classification of Antibodies
According to -
(Light chains are always kappa or Lambda)
Heavy chains Type (Isotypic Differences)
Glycosilation
Mono- , Di- , Pentameric Forms
Allotypic and Idiotipic differences within the same class Antibody
Allotype - Structural difference in the non active regions.
Idiot yep - Structural difference in the Active reception part.
Papain reaction and Products
Lysis of Ab to Fab and Fc
Fab- Antigen Binding Fragment
Fc- Crystallizable Fragment
Structural Functions of the Ab
Idiotypic antigen Binding - Variant domains
C3b and C4b binding - First constant domain
Fc Binding - Constant Stem part
C1q binding - Second constant domain
Importance of C3b and C4b binding on IC
CR1 interaction leads to Macrophages opsinisation in the spleen.
Hypervariable parts of VH in Ab
Hypervariable CDR - Ionic interaction, Disulfides..
Complementarity Determining Region.
CDR1, CDR2, CDR3
CDRs binding orientation
CDR1 and CDR2 - Polynorphic Residue of MHC
CDR3 - T cell contact of Residue Peptide
How many Antigens do we meet in Life ?
How many genes are there in the Human DNA?
What is the problem arising?
10 Million kinds
35,000 Genes
Hypothetically Insufficient readily produced Antibodies
What is the composition of the gene for the Heavy chain?
VDJ segments (Combined togethein the Differentiation of the CLP) C segments- Mio , Delta, Gamma, Alpha, Epsilon
DNA Diversity calculation of the VDJ recombination
51~ V
27 D
6 J
51 x 27 x 6 equals 8262
DNA Diversity out of Kappa and Lambda arranged independently - Which Segments?
Kappa - 40(V) x 5 (J) equals 200
Lambda - 30 (V) x 4 (J) equals 120
(Numbers don’t matter)
Recombinases enzymes (RAG1 and 2) in Pro-Lymphocytes
Clevage of DNA and formation of Hairpin segment
Artemis Endonuclease activity in Pro-Lymphocytes
Cleavage of the Hairpin structure from RAG marked by the DNA in Palindromic Sequences, No filling of gap yet..
TdT - Terminal deoxynucleitidyl Transferase
Activity in Pro-Lymphocytes
Adds nucleotides and Forms ligation between the separated strands filling the gap between the palindromic
How does Recombination of C segments of the Ab gene occur?
Alternative splicing of RNA molecule
How is it that BCR show only Paternal or Maternal Ig never both?
Allelic Exclusion-
First recombination capable gene transferred is silencing the other
SCID
What could be the cause?
Severe Combined Immunodeficiency
RAG deficiency - No T or B cells diversity
Omen Syndrome
RAG or Artemis partial activity
Rash and Erythoderma
caused by IgE overproduction with IL4 IL5 eosinophil driving and leakage forming
Mechanisms responsible for the full diversity of antigen receptors:
V(D)J rearrangement steps -
1) Germline diversity
2) Combinatorial diversity
3) Junctional diversity
Mechanisms responsible for the full diversity of antigen receptors:
Post-V(D)J rearrangement -
4) Receptor editing
5) Somatic hypermutation
6) Receptor revision
What would be the sign for Malignancy in FACS detection of PI labeled Cell cycle intermediates?
S Phase Elevation
What are the different graphs that we can obtain from FACS? (3)
1) Granularity Vs Size
2) Fluorescent Intensity(Log) Vs Cell Count (or another Intensity)
3) PI labeling for Cell cycle measurement
FACS - What are the Clinical Uses? (3)
1) Hematological -Leukemia/Lymphoma/Bone Marrow Transplantation
2) MDR/MRD Diseases
3) Immunodeficiencies (AIDS)
