Topic 1 Flashcards
- What are the limitations of histology?
Invasive and often require hospitalisation, therefore, this will result in increased anxiety for the patient and more costly. Moreover, due to its invasiveness, it is not suitable for monitoring disease.
- What are the advantages and limitations of cytology?
Advantages:
• Most procedures are outpatient procedures
• Can determine which patient requires surgery
• Easy and safe procedures – less invasive
• Less anxiety on the patient
• Can be used to monitor disease
Limitations:
• Cannot observe architecture
• May be obstructed by necrosis, fibrosis or haemorrhagic cells
• Cells may not be exfoliative
• Sample may not be representative or cellular enough for diagnosis
a) Density gradient centrifugation
This technique is used for non-gynae haematogenous samples to remove RBCs which may obstruct vision. Histopaque is added to a test tube. This is followed by addition of patient sample slowly on top. The solution is centrifuged and this becomes separated into layers. The middle layer contains epithelial, inflammatory and tumour cells which we are interested in and this is transferred into a new test tube from which a smear is made.
b) Cyto-centrifugation
Samples which are hypocellular such as CSF require cytocentrifugation to concentrate the cells in one area, increasing the sensitivity of detection. The cytocentrifuge consists of three parts – a funnel, absorbent pad and centrifuge. The sample is placed in the funnel and the centrifuge is turned on. The cells are dense and are forced onto the slide while the liquid is absorbed by the pad.
c) Cell block technique
A Cell block technique is used to obtain a paraffin-embedded block so that immunocytochemistry, genetic or even histochemical testing can be performed and enhance diagnosis. The sample is washed with saline and centrifuged, after centrifugation the supernatant is discarded. This washing procedure is performed 3 times to ensure removal of formalin as it interferes with the thrombin. A few drops of plasma are then added to the deposit and this is vortexed. An equal amount of thrombin is added to the mixture and a clot forms. The coagulum is placed in a container with 10% NBF and sent to histology to obtain a paraffin-embedded block.
Four phases of cell cycle
- G1 Phase: the cell spends a majority of its time in this stage, it produces ribonucleic acid (RNA) including mRNA, rRNA and tRNA required for transcription and translation to synthesize proteins from DNA. Proteins are required for repairing cell structures, and carrying out cell function and may also be needed for mitosis and continuation of the cell cycle.
- S-Phase: DNA replication occurs, the cell replicates all of the DNA resulting in 46 pairs of chromosome so that each daughter cells has 23 pairs of chromosomes each.
- G2 Phase: The cell increased its energy in preparation of mitosis and also synthesis tubulins. Tubulin is an important component of microtubules which are used to separate sister chromatids during anaphase.
- Mitosis: the nucleus divides after 4 sequential steps that take place, prophase when sister chromatids become wider and shorter, metaphase when chromosomes line at the centre of the cell, anaphase when sister chromatids are pulled apart by microtubules and telophase which results in splitting the nucleus into two. After this cytokinesis takes place which is cytoplasmic division to bring about two daughter cells.
- State the three key components of the cell cycle and briefly indicate their role in the control of its highly complex machinery. (9 marks)
- Cyclin-Dependent Kinases (CDKs) – these are enzymes encoded for by the cell-division cycle gene cdc-gene). . The concentration of these in the cell is constant but are found in their inactive form and become activated by cyclins.
- Cyclins – these are present depending on the phase that the cell cycle is in, they bind to CDKs and activate them. The CDK-cyclin complex is designated a maturation promotion factor (MPF) and allows the cell to enters the next phase.
- CDK Inhibitors – these bind to CDK and inhibit their action, therefore, they slow down or arrest the cell cycle depending on the extent of DNA or other structural damage that was detected.
- What are the key components which control the cell cycle mechanism, and clearly indicate how they interact with each other to ensure that the cell progresses correctly from one phase to the next? (15 marks)
• Cyclin-Dependent Kinases (CDKs) – these are enzymes encoded for by the cell-division cycle gene cdc-gene). They are capable of phosphorylating proteins to allow the cell to enter the next phase. The concentration of these in the cell is constant but found in their inactive form and become activated by cyclins.
• Cyclins – these are present depending on the phase that the cell cycle is in, they bind to CDKs and activate them. The CDK-cyclin complex is designated a maturation promotion factor (MPF) and allows the cell to enters the next phase.
• CDK Inhibitors – these bind to CDK and inhibit their action, therefore, they slow down or arrest the cell cycle depending on the extent of DNA or other structural damage that was detected.
When the cell is required to proliferate and/or differentiated, growth factor produced from protooncogenes such as c-erbB which is an epidermal growth factor are produced and these bind to receptors on the cell surface. After a cascade of events occurs transcription factors are activated. These go on to transcribe and translate cyclin genes which produce cyclins. The cyclins produced depend on which phase in the cell cycle the cell is in. Cyclin-dependent kinases (CDK) are produced from cell-division cycle (cdc) genes continuously, but they are found in the inactive form. The cyclin produced binds to the CDK and activates it. In the cell there are E2F transcription factors which are required for proliferation, although these are continuously expressed they are inhibited by retinoblastoma (Rb) proteins. However, CDK once inactivated by cyclins goes on to phosphorylate this E2F-Rb complex which leads to dissociation. Therefore, E2F is transcribes DNA sequences that produces proteins required for cell cycle continuation. The cyclin produced then disintegrates, and CDK is inactivated again to allow production of a new type of cyclin required for the next phase.
If there is damage in the cell this is detected by p53, a potent tumour suppressor protein. If the DNA damage is not severe it initiates DNA repairing enzymes but if there is damage beyond repair it induces apoptosis. However, it also acts as a transcription factor for genes that produce CDK-inhibitors such as p15, p16 and p27. These CDK-inhibitors bind to CDK and inactivate it, therefore the cyclin-CDK complex cannot phosphorylate the retinoblastoma protein and cell cycle is halted, until DNA repair mechanisms repair the damage.
- Define FNA and evaluate the importance of this sampling technique
Fine needle aspiration is an aspirated cell sampled obtained from a solid tumour using a high-gauge needle. This technique has replaced frozen sections because it is more accurate, less invasive and has a faster turn around time. Therefore, it provides less anxiety to the patient, decrease the need for hospitalisation and is more cost effective. As a result this technique is important at determining which patients may require further investigations or treatment, monitor disease or treatment and also used as a way for pre-operation planning together with the patient.
- List six main advantages of FNA. (6 marks)
- Complication and pain is minimised when compared to biopsy, and since it is non-invasive it is an outpatient procedure
- It has a fast turn around time, therefore it will relief the anxiety of the patient and can initiate further tests or treatments faster.
- Highly sensitive and specific – if the correct sampling procedure is performed and cytological sample is processed accordingly.
- Repeatable – can be used to monitor treatment
- Cost effective when compared to a biopsy and uses less equipment
- Allow for pre-operative diagnosis and the patient will know what is going happen before going into surgery, so it allows for active planning.
- During the FNA sampling procedure, highlight the crucial steps that one has to follow to ensure a safe, adequate, representative sample. (6marks)
Lump identified by feeling it or by radiological imaging (ultrasound)
If possible immobilise the lesion and push narrow gauge needle through the lump and ensure that the needle is in the centre of the lesion.
Suction is applied by negative pressure and the lump is gently probed sideways while Mainting position in the lesion to ensure a representative sample is obtained.
The pressure is removed while still in the lesion to prevent tumour seeding into pareas without the lesion.
The needle is detached from the plunger and aspirated on slide in cases of ROSE or placed in fixative e.g. alcohol for PAP stain or RPMI for flow cytometry.
- Distinguish between palpable and non-palpable lesions, and mention the commonest body sites for FNA sampling. (6marks
Palpable lesions are those that can be felt as they are superficial such as those in the thyroid gland. These kind of lesions can be sampled without radiological imaging while non-palpable masses are those that cannot be seen or felt as they are embedded deep inside the body such as those in the lungs, and require imagining such as CT scans to be detected.
The most common sites: thyroid, breast, lymph node, lung, liver and spleen.
- List two disadvantages of the FNA technique. (2marks)
Increased chance of obtaining an inadequate sample due to necrosis, fibrosis and haemolytic sample.
Provides less information than biopsy because the architecture cannot be determined.
FNA has now become an established method of investigation in clinical practice.
- Define FNA, evaluate the importance of this sampling technique, outline the vital steps involved in the sampling procedure and list the factors that determine its efficiency. (20 marks)
Fine needle aspiration is when a narrow needle is inserted into a solid tumour and an aspirated sample is taken.
This is an important technique because there are decreased complication and cost when compared to histological section. It also has a fast turnaround time (TAT) and is highly sensitive and specific if sampled correctly. FNA can be repeated, so can be used to monitor therapy given to a patient and even disease progression if needed. One of the most important advantages for this technique is that its fast TAT allows for pre-operative diagnosis so that the patient is aware of what is going to happen prior to surgery, allowing for active planning. This was not allowed with other methods as they had a slow TAT.
method for sampling FNA
- Lump identified by feeling it or by radiological imaging (ultrasound)
- Immobilise and push 22-25 gauge needle through the lump and ensure that the needle is in the middle of the lump.
- Suction is applied by negative pressure and the lump is gently probed sideways to ensure a representative sample is obtained.
- The pressure is removed while still in the tumour to prevent tumour seeding and aspiration from areas without the lesion.
- The needle is detached from the plunger and aspirated on slide in cases of ROSE or placed in fixative e.g. alcohol for PAP stain or RPMI for flow cytometry.
Efficiency of FNA depends on the quality of the sample, this needs to be representative and can be improved by rapid onsite evaluation where the sample is aspirated onto a slide and screened to ensure sample is representative. FNA can be performed in conjunction with ultrasound to ensure that the needle is inserted into the middle of the lesion. Smear preparation, that is ensuring the correct fixative is used for the stain and the staining procedure needs to be done appropriately and microscopic interpretation should be accurate and efficient to ensure diagnosis is done appropriately.
