INTS 4: The Technologies

Question 1

Define morphology

Answer 1

• 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.

Question 2

What is flow cytometry?

Answer 2

• 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.

Question 3

What is cytogenetics?

Answer 3

• 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,

Question 4

What is PCR?

Answer 4

• 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.

Question 5

What is next generation sequencing?

Answer 5

• 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

Question 6

Observe what a blood smear looks like

Answer 6

Question 7

Familarise yourself with the blood smears from normal individuals (1)

Answer 7

Question 8

Familarise yourself with the blood smears from normal individuals (2)

Answer 8

Question 9

Observe this megakaryocyte in the BM

Answer 9

Question 10

Observe the different stages of maturation in the BM

Answer 10

Question 11

What is flow cytometry?

What are the steps?

Answer 11

• a laser-based, biophysical technology employed in cell counting, cell characterisation and sorting, biomarker detection and protein engineering.

Question 12

Explain how foward scatter works (in relation to flow cytometry)

Answer 12

• 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.

Question 13

Explain how side scatter works (in relation to flow cytometry)

Answer 13

• 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.

Question 14

Describe the main steps of flow-cytometry:

1. sample preparation/separation
2. laser light source hitting the cell
3. FSC and SSC detected
4. Signal converted into a readable enumeration of cells and their characteristics (size and number)

Answer 14

Question 15

How does the computer analyse flow cytometry data?

Answer 15

• 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.

Question 16

Describe how flow cytometry works?

Answer 16

Question 17

Observe this flow cytometry image

Answer 17

• In this image, cells have been labelled using two different antibodies labelled in blue (CD3 labelled with one particular fluorochrome or ‘dye’ called fluorescein isothiocynade: FITC) and red (CD4 labelled with another fluorochrome, Allophycocyanin: APC). (You don’t need to remember these, just try to understand the principles.) But it is important to remember that the antibodies are labelled using fluorochromes each one can be excited at different wave-length and so can work together.
• There are distinct cell populations depending on the intensity of the signal: the stronger the signal for the forward scatter, the stronger the positivity and so, more along the horizontal right axis (CD3 FITC).
• If the cells are strongly labelled with the second antibody, the signal will be stronger and higher on the left vertical axis (side scatter; CD4). If cells are negative for both they will be in the left bottom corner.

Question 18

What is immunophenotyping?

Answer 18

• the analysis of heterogeneous populations of cells for the purpose of identifying the presence and proportions of the various populations of interest
• Antibodies are used to identify cells by detecting specific antigens expressed by these cells, which are known as markers.
• Depending of which cluster of differentiation (CD) each cell marks, the different subpopulations can be fully characterised. This is very important when studying abnormal cell proliferation in the bone marrow or peripheral blood.

Question 19

Answer 19

• practise this in the e-module

Question 20

What are the preparation steps in cytogenetics?

Answer 20

• A sample, generally bone marrow or peripheral blood, is collected using an anticoagulant which is necessary to stop the clotting and the cells forming clumps and becoming unsuitable for analysis. Then the sample is put into the culture with stimulants that encourage the cells to divide, which is done overnight. While they are dividing, they are collected and ‘frozen’ so that metaphases can be analysed after they have been dried and appropriately stained. The staining has the ability to ‘mark’ the different bands of the chromosomes. Each band (dark or lighter) tends to highlight how compact a region can be (dark) or lighter structure (light bands) along the chromosome.
• Chromosomes are also ordered by the position of their centromere.
• After appropriate staining of the metaphases, the chromosomes are then ‘ordered’ according to size (from the largest to the smallest chromosomes, in pairs; this is nowadays done by a computer). See the image below where the main steps of Cytogenetic analysis are graphically shown.

Question 21

What is successful cytogenetics dependent on?

What is the solution of this

Answer 21

• cell division
• If there is no cell division (often when the patient has received chemotherapy, which suppresses cell division), fluorescent in situ hybridisation (FISH) can often be used to complement cytogenetics

Question 22

What is Giemsa banding (G-banding) in cytogenetics?

Answer 22

• a technique used in Cytogenetic to produce a visible karyotype by staining condensed chromosomes. It is useful for identifying genetic diseases through the photographic representation of the entire chromosome complement.

Question 23

When is cytogenetics not able to diagnose a genetic abnormality?

Answer 23

• when genetic abnormalities are due to a single base pair mutation in the sequence of a gene
• cytogenetic threshold for detecting conditions is very low
• it is not very discriminatory
• requires a loss of material or transfer of material between chromosomes to be quite a large chunk of DNA

Question 24

What is Florescence in situ hybridisation (FISH)?

Answer 24

• Fluorescent in situ hybridisation (FISH) refers to the use of fluorescently labeled probes to hybridise to cytogenetic cell preparations.
• In fact, it can also be used WHEN cells fail to divide and metaphases are not available; this is called ‘interphase FISH’ or iFISH.

Question 25

Study the image to observe a metaphase cell positive for the BCR-ABL1 rearrangement (associated with chronic myelogenous leukaemia, CML) using FISH.

Answer 25

- The chromosomes can be seen in blue. The chromosome labelled with green and red spots (indicated with a circle in the upper left part of the image) is the one where the rearrangement is present. - The image below is the same karyotype (i.e t(9;22) but done in interphase and so only the signals generated by the labeled probes attached to the specific region of chromatin are seen and not all the separate chromosomes. This can be faster and sometimes as useful as a proper metaphase analysis.

Question 26

Why is cytogenetics important?

Answer 26

- In the last 10-20 years, cytogenetics has become an extremely important test to be carried out especially in Haematological patients because prognosis and overall outcome (survival or not) appear more and more dependent on genetics and cytogenetic abnormalities present in the patients' samples - As illustrated below, the outcome is very poor for some abnormalities ( t(9;22) and 11q23 abnormalities) compared to others (such as t(12;21) and HeL = hyper diploidy cases).

Question 27

How is PCR checked for the correct amplicon production?

Answer 27

- the product of the correct size is tested using gel electrophoresis - can be done in less than 20 mins

Question 28

Describe how Sanger sequencing works

Answer 28

- The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxy-nucleotide triphosphates (dNTPs), and modified di-deoxy-nucleotide triphosphates (ddNTPs), the latter of which terminate DNA strand elongation. - These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. - The ddNTPs may be radioactively or fluorescently labeled for detection in automated sequencing machines. - The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP, and dTTP) and the DNA polymerase. - To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), while the other added nucleotides are ordinary ones. - The dideoxynucleotide concentration should be approximately 100-fold lower than that of the corresponding deoxynucleotide (e.g. 0.005mM ddTTP: 0.5mM dTTP) to allow enough fragments to be produced while still transcribing the complete sequence. - Putting it in a more sensible order, four separate reactions are needed in this process to test all four ddNTPs. - Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. - This is frequently performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes T, C, G, A). - The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.

Question 29

What is Next Generation Sequencing?

Answer 29

- Next-generation sequencing (NGS), massively parallel, or deep sequencing, is related terms that describe a DNA sequencing technology that has revolutionised genomic research. - Using NGS an entire human genome can be sequenced within a single day. - In contrast, the previous Sanger sequencing technology used to decipher the human genome, required over a decade to deliver the final draft. - Although in genome research NGS has mostly superseded conventional Sanger sequencing, it has not yet translated into routine clinical practice. - The aim of this article is to review the potential applications of NGS in paediatrics.

Question 30

When is the Sanger method used over other methods?

Answer 30

- for smaller-scale projects - advantage over short-read sequencing technologies - can produce DNA sequence reads of \>500 nucleotides - for validation of Next-Gen results