INTS 4: The Technologies Flashcards
1
Q
Define morphology
A
- This is the investigation of the appearance of cells observed under a microscope following their staining.
- This requires the ability to smear and make a very thin layer of cells and then stain the smear to be able to individually visualise each cell.
- In particular, we will learn how to recognise different cells depending on the structure of the nucleus, the shape and size of the cytoplasm, their size relative to other surrounding cells, for instance, the red cells, the presence of granules, whether they are normally present in the blood or should they never be seen in the blood and only in the bone marrow and the structure of the chromatin in the nucleus, condensed or very fine, etc.
2
Q
What is flow cytometry?
A
- Different cells and different stages of their differentiation express different proteins (antigens) on the surface or in the cytoplasm of cells.
- Flow cytometry is used in the lab to characterise and study the different types of lymphocytes or myelocytes based on their expression of different surface antigens. This allows us to identify what stage of the pathway of differentiation they are at and decide if the expression of Ag is normal or aberrant, which is generally associated with leukaemia.
3
Q
What is cytogenetics?
A
- This technology works with fresh cells that are made to divide in vitro and are then blocked during division when all chromosomes can be visualised through staining.
- This allows to study the ‘genetic’ material of a cell and identify if they are normal (as presenting the normal type of chromosomes structurally and numerically) or abnormal, i.e. they present with loss of genetic material (deletions) or material which has migrated from one chromosome to another (translocations)
- This technology has a threshold of resolution which means we cannot see the small region of deletions or translocations between similar staining chromosomal bands and then we need to use FISH to be more accurate and precise,
4
Q
What is PCR?
A
- This is a technology developed around 1985-1990 which helps to amplify a specific region of the genome using primers unique to the sequence flanking the area of interest.
- This is done through three main steps: denaturation, annealing, and extension.
- The 3 steps can be repeated and repeated through many cycles through automated machines that are very efficient and have made this a routine technology in all laboratories.
5
Q
What is next generation sequencing?
A
- Compared to conventional Sanger sequencing using capillary electrophoresis, the short read, massively parallel sequencing technique is a fundamentally different approach that revolutionised sequencing capabilities and launched the second-generation sequencing methods – or next-generation sequencing (NGS) – that provide orders of magnitude more data at much lower recurring cost.
- Next-generation sequencing (NGS), also known as high-throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies. These technologies allow for sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing, and as such revolutionised the study of genomics and molecular biology
6
Q
Observe what a blood smear looks like
A
7
Q
Familarise yourself with the blood smears from normal individuals (1)
A
8
Q
Familarise yourself with the blood smears from normal individuals (2)
A
9
Q
Observe this megakaryocyte in the BM
A
10
Q
Observe the different stages of maturation in the BM
A
11
Q
What is flow cytometry?
What are the steps?
A
- a laser-based, biophysical technology employed in cell counting, cell characterisation and sorting, biomarker detection and protein engineering.
12
Q
Explain how foward scatter works (in relation to flow cytometry)
A
- this depends on the SIZE of the cell.
- The larger the cell, the stronger the light signal capture and more forward (to the right) in the diagram (see image identifying lymphocytes as the smallest, monocytes as the largest cells.
- Granulocytes fit in the middle as the size is concerned almost in between the lymphocytes and the monocytes.
13
Q
Explain how side scatter works (in relation to flow cytometry)
A
- The side scatter reflects the complexity of the cells both as nuclear structure and granularity. Also, side scatter signals reflect the type of fluorescently labeled antibody used. The more granular the stronger the side scatter signal emitted.
- The antibody will have been labeled with a fluorescent tag (Fluorochrome) but will also be able to recognise a specific antigen on the surface or in the cytoplasm of cells (when they are permeabilized and allow the antibody to penetrate and bind to specific proteins).
- Remember that each antibody is normally raised in mice or rabbits that have been immunised against the specific antigen/protein which we want to recognise.
14
Q
Describe the main steps of flow-cytometry:
- sample preparation/separation
- laser light source hitting the cell
- FSC and SSC detected
- Signal converted into a readable enumeration of cells and their characteristics (size and number)
A
15
Q
How does the computer analyse flow cytometry data?
A
- The forwards scatter will collect the size of a cell and the more a cell is located to the right end of the X axis, the larger the cell is;
- so lymphocytes will be located towards the left part of the x-axis and the monocytes will be located to the right side, with the granulocytes up to the right end but higher on the Y axis because they contain a lot of granules.