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INTS > INTS4 - Technologies > Flashcards

INTS4 - Technologies Flashcards

1
Q

Define morphology.

A

Investigation of cell appearance by observation under a microscope. Requires blood smear with thin layer of cells. Can be used to see nucleus, cytoplasm and other cell structures.

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2
Q

Define flow cytometry.

A

Used to characterise and study different lymphocytes and myelocytes based on expression of different surface antigens. Can also be used to distinguish between different stages of differentiation.

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3
Q

Define cytogenetics.

A

Study of genetic material, to observe normality of chromosomal structure. Can only be used to study large changes to chromosome, not small deletions.

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4
Q

Define FISH.

A

Fluorescence in situ hybridisation. Used alongside cytogenetics for increased accuracy, allowing detection of small changes to DNA.

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5
Q

Discuss general principles of polymerase chain reaction.

A

Process used to amplify specific regions of genome using primers. Requires denaturation, annealing and extension.

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6
Q

How is a blood smear produced.

A

Thin layer of blood is spread onto a glass slide. This is then stained and viewed under a microscope in order to view the morphology of cells.

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7
Q

What blood is used to prepare blood smears.

A

Bone marrow or peripheral blood.

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8
Q

What differences should be observed between peripheral blood and bone marrow aspirate.

A

Peripheral blood shouldn’t contain immature cells. Bone marrow would contain immature cells. Lack of heterogeneity in peripheral blood would be an issue also.

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9
Q

Discuss conclusion from pale red blood cells observed in a blood smear.

A

Lack of haemoglobin in the red blood cell would cause a pale centre.

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10
Q

How are platelets observed under a microscope

A

Observed with purple small circles

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11
Q

What factors can be seen and observed using morphology.

A

Shape, size, amount of cytoplasm, presence of granules.

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12
Q

If cells contain granules, what lineage are they from.

A

Myeloid linage.

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13
Q

If cells don’t contain granules, what cell lineage are they from.

A

Lymphoid lineage.

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14
Q

Discuss overall process of flow cytometry.

A

A suspension of cells is prepared which is placed in a fluid of saline or another fluid. This is then passed through the flow cytometry ensuring that the cells are passing in single line. Light is then passed through the cells, causing the light to become scattered either as forward scatter or side scatter depending on the characteristic of the cell itself. Computer converts light singles into a graph which can be analysed.

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15
Q

Discuss forward scatter in flow cytometry.

A

Forward scatter is dependent on the size of the cell. The larger the cell, the stronger the signal, so the more forward in the diagram the cell is observed.

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16
Q

Discuss side scatter in flow cytometry.

A

Represents the composition of the cell and the different types of fluorescently labelled antibodies used.

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17
Q

For the antibody that is used in flow cytometry, how is the antibody treated,

A

Antibody is raised in mice or rabbits that have been immunised against the specific antigen or protein that is trying to be detected.

18
Q

Define fluorophores.

A

Fluorophores are fluorescent chemicals that re-emit light upon light excitation.

19
Q

How are images obtained from flow cytometry used to distinguish cells.

A

Cells can be positive or negative for specific antigens. Known antigens for specific cells are then compared.

20
Q

Define immunophenotyping.

A

Analysis of heterogenous populations of cells in order to identify presence and proportion of varying populations.

21
Q

When would a flow cytometry test commonly be used

A

Studying of abnormal cell proliferation in bone marrow or peripheral blood.

22
Q

Discuss importance of cytogenetics in haematological patients.

A

Important for maintaining prognosis and survival of haematological patients.

23
Q

How is a sample prepared for cytogenetics.

A

Sample is prepared from bone marrow or peripheral blood by using an anticoagulant. Sample is then stimulated using simulating to promote cell division. Cells are collected and frozen at stages of metaphase then appropriately dried and stained, to observe the various chromosomes.

24
Q

Define a karyogram.

A

Images of the chromosomes stained and organised in pairs in order to be properly observed.

25
Q

Define the autosomes.

A

The 22 pairs of homologous chromosomes.

26
Q

What are the sex chromosomes.

A

One pair of chromosome that determine the sex of an individual.

27
Q

Give the sex chromosomes for different genders.

A

Female - XX

Male - XY

28
Q

Discuss the general process of FISH.

A

Cells fixed in metaphase. Allowed to swell so can be fixed onto a slide. Chromosomes are also fixed. DNA becomes denatured. DNA probes are hybridised with denatured chromosomes. Fluorescent labels are added and the fluorescence is observed.

29
Q

What two technologies are used together to give increased accuracy of test.

A

Cytogenetics plus FISHZ

30
Q

What is a karyotype.

A

Number and visual appearance of chromosomes in a cell nucleus.

31
Q

How are chromosomes ordered when producing a karyogram.

A

Ordered from largest to smallest chromosome.

32
Q

What does FISH allow us to do.

A

Detect physical location of genes on intact chromosomes.

33
Q

How can the amplified products of PCR be checked.

A

Can be checked using agarose gel electrophoresis or real time PCR.

34
Q

What are the characteristics that must be taken into consideration for primer design.

A

Short sequences of DNA. Avoid complementary sequences and repeats. Sequence must be known in order to aid amplification.

35
Q

What reagents are required for PCR.

A

Single strand DNA. Primers. DNA polymerase. dNTPs. ddNTPs.

36
Q

Discuss overview of Sanger sequencing.

A

Method of DNA sequencing based on selective incorporation of chain terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

37
Q

What are ddNTPs.

A

Chain terminating dideoxynucleotides. They lack a 3’ OH group meaning that the chain cannot be elongated.

38
Q

Which concentration should be higher during PCR, ddNTP or dNTP.

A

ddNTP should be 100 fold lower than dNTP

39
Q

How can DNA fragments be observed once separated using gel electrophoresis.

A

Visualised using autoradiography or UV light. Also can be directly read off x ray film or gel image.

40
Q

What is the workflow for next generation sequencing.

A

DNA extraction. DNA shearing. Library preparation. PCR enrichment. Hybrid capture and sequencing. Data analysis is then carried out.

INTS (11 decks)

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