Module 2 - first half mixed Flashcards
Immunophenotyping
Immunophenotyping
Immunophenotyping means the detection (with or without quantification) of antigens expressed on or in cells
The antigens detected may be expressed on the surface membrane, in the cytoplasm or within the nucleus
They are mainly recognized by means of antibodies, either polyclonal antisera or monoclonal antibodies
What is required for immunophenotyping?
Antibody that is complementary to the Ag
the antibody has bound to the cell must be recognized in some way, e.g. by binding a fluorescent chemical (a fluorochrome) to it or by binding an enzyme such as peroxidase or alkaline phosphatase to it
How are the enzyme-labelled antibodies recognised?
The enzyme-labelled antibodies are recognized by a coloured product when they are supplied with a substrate and the enzyme catalyses a reaction
How are the fluorochrome-labelled antibodies recognised?
Fluorochrome-labelled antibodies bound to cells can be recognized with a fluorescence microscope of by a light detection system in a flow cytometer
When using fluorochrome labelled-antibodies, detection of antigens within cells requires the cell to be ‘permeabilised’ in some way.
How does the flow cytometer work?
Stream of cells in single file
You shine a laser at them at a perpendicular angel and you get a FSC that comes straight out the other end (in the same direction as laser). The rest is refracted as SSC along a series of dichroic mirrors.
What is the role of a dichroic mirror?
Dichroic mirrors reflect light of some wave lengths and transmit light of other wavelengths. This separates the light into different fluorescent signals which are transmitted along the series of mirrors.
Why do we need immunophenotyping?
1 differentiation AML from ALL and determining the subtype of AML/ ALL
2 distinguish acute leukaemia from a lymphoproliferative disorder/ other tumour
3 revealing the abnormal phenotype which is useful in monitoring minimal residual disease after remission is achieved
4 identify antigens for therapeutic targets
- you rarely need immunophenotyping to diagnose CML because usually blood film/ count/ cytochemistry will give you the diagnosis accurately
Which markers are myeloid?
CD13, CD33, CD117, CD235, anti-MPO, CD41, CD42,
Which markers are not lineage resrictied?
CD45 (common leucocyte Ag), CD34 (HSCs, immature cells), TdT (ALL and AML blasts can express) HLA-DR CD56
T lineage markers
Primary: CD2, cCD3,
Supp: CD1a, CD4, CD6, , CD7, CD8
B lineage markers
Primary CD10, CD19, CD20, cCD22, CD79a
Supplementary panel: SmIg, CD138
CD15
?
MRD what is it and what do we use to monitor it?
(MRD) is residual leukaemia that cannot be detected by morphological analysis
Conventional cytogenetic analysis is too insensitive to permit detection
FISH is somewhat better
Best is molecular analysis (e.g. PCR) or multiparameter flow cytometry
How is flow cytometry used to monitor resdual disease?
Multiparameter flow cytometric immunophenotyping for this purpose needs lots of antibodies and simultaneous measurement of 4 or 5 characteristics of the leukaemic cells
A leukaemic-associated immunophenotype specific for the patient is identified at diagnosis and then sought when the patient appears to be in remission
Which lymphoid malignancies are “early” in ontogeny?
Early in ontogeny Precursor cells
B cell Acute lymphoblastic leukaemia/lymphoblastic lymphoma (B-ALL)
T cell Acute Lymphoblastic Leukaemia/lymphoblastic/lymphoma
(T-ALL)
Which lymphoid malignancies are “late” in ontogeny?
B or T/NK cell Neoplasms
CLL
Myeloma
Follicular, DLBCL, MZL
Why are lymphocytes prone to malignancy?
- Rapid cell proliferation in immune response (Proliferating cells risk DNA replication error)
- DNA molecules are cut and rejoined plus undergo deliberate point mutations: generates immunoglobulin and T cell receptor diversity (Potential for rejoining errors and new point mutations)
- They are dependent on apoptosis (90% of normal cells die!) Ensures antibody specificity and excludes auto-immune disease. Apoptosis is “switched off” in germinal centre.
CGH for Hodkgin’s Lymphoma
Comparative genomic hybridization (CGH) was applied with PCR for cytogenetic analyses of reed Sternberg cells. These analyses indicated higher rates of numerical aberrations of individual chromosomes than had previously been found by banding analysis. Gains and losses in more than 50% of:
classical HL tumors were identified on 2p, 7q, and 16q (Joos, 2000),
NLPHL, chromosomal arms 1q, 3p, 5q, and Xq were affected (Franke 2001)
EBV pathogenesis in HL
- In EBV-positive cases there is expression of EBNA1, LMP1, LMP2a and LMP2b
- LMP1 is oncogenic; it mimics the action of activated CD40, a co-receptor on B cells, and activates NFκB, favouring proliferation and survival
- LMP2 is also oncogenic, down-regulating genes needed for B-cell maturation and upregulating genes for proliferation and resistance to apoptosis
Prognostic factors of HL
Age, stage, LDH (high is worse), ESR, Lymphocyte count (<0.6 is worse), anaemia is worse
How do you give autologous vs allogenic transplants?
Autologous
•Give GCSF 5-16mg/kg every day for 4days; 3 hrs in total
•4 days later, Collect HSCs (CD34+ve) from PB & freeze
•Thaw HSCs & reinfuse back to host after high dose chemo
Allogeneic:
• Give cancer sufferer high dose chemo +/- radio
• Transplant BM cells from a different donor, who is ideally matched for HLA-type aka sibling/identical twin
o Increasing the specificity requirement of matching decreases the chance that a donor will be available
•Mortality rate is upto 50% and it costs roughly £150k per transplant done
How do you get donor stem cells
•Donor stem cells can be obtained from BM/ PB/UC
BM - procedure is done under GA and involves taking a lot of BM samples (can only take 5ml at a time and you need about a litre)
PB - like autologous, needs GCSF prior to HSC extraction
UC - can only get a little sample - not useful for adults because you need roughly 2mill CD34+ cells/kg body weight
Factors that have a negative impact on BMT outcome?
>40 Female -> Male transplant Advanced disease >1 year from diagnosis Unmatched donor
Treatment of GvHD
Corticosteroids Cyclosporin A FK506 Mycophenylate mofetil Monoclonal antibodies Photopheresis Total lymphoid irradiation
Prophylaxis of GvHD
Methotrexate Corticosteroids Cyclosporin A CsA plus MTX FK506
T-cell depletion
Post-transplant cyclophosphamide
Commonly deleted gene in CLL?
ATM (Ataxia telangectasia mutated) gene is on 11q
Abnormalities of the ATM gene are among the most commonly occurring somatic mutations in cancer and generally have been associated with inferior prognosis.
Major role in repairing ds breaks of DNA
Associated with 17q deletions and/or TP53 gene mutations.
Stressing cells and checking how they respond is used to assess ATM function.
ATM’s function
Following DNA DSBs, the MRN complex senses and initiates DNA repair and recruits ATM. As ATM is brought to the site of DNA damage, it dissociates from homo-dimers into active monomers and is catalytically activated by auto-phosphorylation.
Once @, ATM serves as a transducer and phosphorylates and activates other protein kinases. E.g. phosphorylation of checkpoint kinase 2 (CHK2), which in turn modulates its own substrates, > in cell-cycle arrest.
ATM also @ p53, by direct ATM-mediated phosphorylation through CHK2
ATM also has effects on chromatin relaxation, which aids in repair, through phosphorylation of KRAB-associated protein 1 (KAP1) and others.
Finally, ATM can result in prosurvival signalling that is balance with the apoptotic signals, including phosphorylation of AKT, and activation of NF-κB (not shown).
The different types of molecular techniques
STEP ONE PCR. THEN YOU CAN DO THE REST
•Agarose gel analysis/ Qiaxel segregate fragments within the product according to size/molecular weight - qualitative information only
oQiaxel is preferred because it avoids the need for the bromide (carcinogen) and is much quicker
•Gene scanning is more sensitive/ specific/qualitative
oIt can determine sequences and hence is useful in segregating fragments that vary by only a few bases, which would otherwise have a similar weight and be missed on agarose gel/Qiaxel
•Heteroduplex gel analysis is useful when one allele is mutated and the other is not
oIt uses slow re-annealing which can generate mismatches in such cases
oHowever this is laborious and you need a lot of sample material
•Real time PCR uses 2 primers and 1 probe
oThe probe is degraded by TAQ-polymerase after about 15 cycles, and then produces light
oConcentration of your molecular target at the beginning of the reaction effects the number of cycles needed to produce light (cycle threshold. Hence you can use RQ-PCR to tell how much of your target was in a particular sample. (All samples reach the same plateau, but after a different number of cycles)
•Sanger Sequencing is similar to heteroduplex but it will tell you what nucleotides are mutated, hence it is quantitivate, whereas heteroduplex can only tell you if a mutation is present or not (qualitative)
Describe step 1 of all the molecular techniques
•You have to extract DNA/RNa from cellular (usually WCC) suspensions
•Then you do PCR - you can use upto 70 cycles if you are looking for a rare molecular target
•Primer design is important
o 18-30bp long
o Avoid repeats & complimentary sequences to prevent self-annealing
o Limit C/G because they require more heat to denature (4degs as opposed to 2degs with A/T)
Clinical applications of some molecular techniques in haematology
- Establish clonality in T/B cell proliferations
- Demonstrate chimerism/ monitor treatment post BM transplant
- PCR detection of chromosomal abnormalities/ fusion genes
What is class switching Why is this bit prone to malignancy
Immunoglobulin class switching, also known as isotype switching, isotypic commutation or class-switch recombination (CSR), is a biological mechanism that changes a B cell's production of immunoglobulin from one type to another, such as from the isotype IgM to the isotype IgG. During this process, the constant-region portion of the antibody heavy chain is changed, but the variable region of the heavy chain stays the same (the terms "variable" and "constant" refer to changes or lack thereof between antibodies that target different epitopes). Since the variable region does not change, class switching does not affect antigen specificity. Instead, the antibody retains affinity for the same antigens, but can interact with different effector molecules. Double-stranded breaks are generated in DNA at conserved nucleotide motifs, called switch (S) regions, which are upstream from gene segments that encode the constant regions of antibody heavy chains; these occur adjacent to all heavy chain constant region genes with the exception of the δ-chain.
