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LQB490 > Introduction to cytogenetics > Flashcards

Introduction to cytogenetics Flashcards

1
Q

What is clinical cytogenetics?

A

The branch of genetics that studies the relationship between chromosomal aberrations and pathologic conditions; the study of chromosome morphology, structure, pathology, function, and behaviour.

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2
Q

What is molecular pathology?

A

A discipline that deals with the origins and mechanisms of diseases at the most fundamental level - macromolecules such as DNA and protein - in order to provide dx and discover possible avenues for rx.

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3
Q

What is the difference between clinical cytogenetics and molecular pathology?

A

Low versus high resolution genetic analysis.

Cytogenetics analyses chromosomes at 1000X magnification and can detect changes (deletions/duplications) between 5 - 10 megabases (million bp) in size.

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4
Q

What is the minimum size of genetic changes that can be detected under a microscope?

A

Changes that are 5 - 10 megabases (million base pairs) in length at 1000X magnification.

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5
Q

How many base pairs are in the human genome?

A

3 billion.

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6
Q

What is the minimum size of genetic changes that can be detected using molecular techniques?

A

A single base pair.

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7
Q

What are the three main benefits of organising DNA into chromosomes?

A
  • Packing large amounts of material into small spaces
  • Less chance of breakage due to entanglement with other chromosomes
  • Easier to separate during cell division
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8
Q

How much does a DNA strand decrease in size by being packaged into a chromosome?

A

7000-fold decrease

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9
Q

Which phase of the cell cycle is used for cytogenetics?

A

Metaphase (or prometaphase [border]).

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10
Q

Which phases of the cell cycle make up interphase?

A

G1 (growth), S (synthesis [DNA replication]), and G2 (growth [preparation for mitosis]).

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11
Q

What are the four main phases of mitosis?

A

Prophase, metaphase, anaphase, and telophase.

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12
Q

What is the difference between mitosis and cytokinesis?

A

Mitosis is nuclear division, while cytokinesis is cellular division.

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13
Q

What is chromatin?

A

Chromosomes of an interphase nuclei; decondensed chromosome

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14
Q

What is the term for chromosomes of an interphase nuclei?

A

Chromatin

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15
Q

What is the term for a decondensed chromosome?

A

Chromatin

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16
Q

A non-dividing cell (G1) will have one chromosome consisting of one ____.

A

Chromatid

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17
Q

A non-dividing cell (G1) will have one ____ consisting of one chromatid.

A

Chromosome

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18
Q

What is replicated during S phase in a non-dividing cell?

A

The chromosome (consisting of one chromatid) so that the chromosome then consists of two sister chromatids.

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19
Q

What is the difference between a chromosome of a non-dividing cell before and after S phase?

A

Before S phase the chromosome consists of one chromatid; after S phase the chromosome consists of two sister chromatids.

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20
Q

The two sister chromatids of a non-dividing cell after S phase are bound at what structure?

A

The centromere

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21
Q

What happens to the two sister chromatids of a chromosome following metaphase?

A

The sister chromatids separate and become daughter chromosomes (and are each referred to as chromosomes in their own right).

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22
Q

Why is the classic ‘x’ form of chromosomes not visible when examining a metaphase cell under the microscope?

A

The sister chromatids are so closely bound that the gap cannot be seen; what looks like one chromosome under a microscope contains two chromatids.

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23
Q

How many chromosomes are in a dividing cell?

A

46

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24
Q

How many chromatids are in a dividing cell?

A

92

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25
Q

How many chromatids are in two paired chromosomes?

A

Four

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26
Q

How many chromosomes are in a gamete cell?

A

23

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27
Q

How many chromosomes are in a somatic cell?

A

46

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28
Q

Are diploid cells somatic or gamete cells?

A

Somatic

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29
Q

What is the meaning of ‘diploid’?

A

Two copies of each chromosome

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30
Q

How many pairs of autosomes are in a somatic cell?

A

22

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31
Q

How many autosomes are in a somatic cell?

A

44

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32
Q

How many pairs of chromosomes are in a somatic cell?

A

23

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33
Q

How many sex chromosomes are in a somatic cell?

A

2

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34
Q

How many pairs of sex chromosomes are in a somatic cell?

A

1

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35
Q

Which type of chromosomes are labelled 1 to 22?

A

Autosomes

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36
Q

Which type of chromosomes are labelled 23?

A

Sex chromosomes

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37
Q

How many autosomes are in a gamete cell?

A

22

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38
Q

How many sex chromosomes are in a gamete cell?

A

1

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39
Q

Are haploid cells somatic or gamete cells?

A

Gamete

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40
Q

What is the meaning of ‘haploid’?

A

One copy of each chromosome

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41
Q

Name the structure at both ends of a chromosome.

A

Telomere

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42
Q

What is a telomere?

A

The structure found at both ends of a chromosome.

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43
Q

What is the name for the short arm of a chromosome?

A

P arm

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44
Q

What is the name for the long arm of a chromosome?

A

Q arm

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45
Q

What are telomeres?

A

Regions of DNA at the ends of chromosomes that consist of TTAGGG repeats and are required for replication and stability.

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46
Q

Telomeres consist of ____ repeats.

A

TTAGGG

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47
Q

TTAGGG repeats are a feature of what chromosome structure?

A

Telomeres

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48
Q

How do telomeres protect chromosomes?

A

TTAGGG repeats are lost during each cell division which has a protective role in preventing end-to-end fusion and therefore promoting cell survival.

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49
Q

What is end-to-end fusion?

A

An open chromosome attaching to the end of another open chromosome, resulting in translocation or abnormality.

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50
Q

What is the centromere?

A

The constricted region in a chromosome where sister chromatids are held together, and the region where attachment to the mitotic spindle occurs.

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51
Q

What is the term for the constricted region where attachment to the mitotic spindle occurs?

A

Centromere

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52
Q

Which region is responsible for the accurate segregation of the replicated chromosomes to daughter cells during mitosis/meiosis?

A

Centromere

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53
Q

What is a metacentric chromosome?

A

A chromosome with a centromere in the middle (p and q arms are ~ the same length).

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54
Q

What is the term for a chromosome with a centromere in the middle (p and q arms are ~ the same length)?

A

Metacentric

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55
Q

What is a submetacentric chromosome?

A

A chromosome with a centromere positioned off the middle but not fully at one end (p arm is longer than the q arm).

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56
Q

What is the term for a chromosome with a centromere positioned off the middle but not fully at one end (p arm is longer than the q arm)?

A

Submetacentric

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57
Q

What is an acrocentric chromosome?

A

A chromosome with a centromere positioned at one end of the chromosome (leaves a satellite on stalks that is the p arm).

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58
Q

What is the term for a chromosome with a centromere positioned at one end of the chromosome (leavies a satellite on stalkds that is the p arm)?

A

Acrocentric

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59
Q

What is a karyogram?

A

A diagram/photograph of a cell with chromosomes ordered into pairs and in numerical order based on banding patterns.

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60
Q

What is the term for a diagram/photograph of a cell with chromosomes ordered into pairs and in numerical order based on banding patterns?

A

Karyogram

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61
Q

What is a karyotype?

A

A written description of a detected chromosome complement (e.g. 46,XY).

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62
Q

What is the term for a written description of a detected chromosome complement (e.g. 46,XY)?

A

Karyotype

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63
Q

List the four pathogenic mechanisms by which chromosome abnormalities result in a pathogenic cytogenetic abnormality (affect normal function).

A
  1. Dosage effect
  2. Direct damaging effect
  3. Position effect
  4. Imprinting
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64
Q

Describe the ‘dosage effect’ pathogenic mechanism of cytogenetic abnormality.

A

Lack or excess of chromosomal material (which directly corresponds to the amount of protein and gene expression).

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65
Q

What is the term for a pathogenic mechanism of cytogenetic abnormality caused by lack or excess of chromosomal material?

A

Dosage effect

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66
Q

Describe the ‘direct damaging effect’ pathogenic mechanism of cytogenetic abnormality.

A

Disruption of a gene that causes dysfunctional/non-functional genes.

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67
Q

What is the term for a pathogenic mechanism of cytogenetic abnormality caused by disruption of a gene that causes dysfunctional/non-functional genes?

A

Direct damaging effect

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68
Q

Describe the ‘position effect’ pathogenic mechanism of cytogenetic abnormality.

A

A gene in a new position functions inappropriately.

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69
Q

What is the term for a pathogenic mechanism of cytogenetic abnormality caused by a gene in a new position functioning inappropriately?

A

Position effect

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70
Q

Describe the ‘imprinting effect’ pathogenic mechanism of cytogenetic abnormality.

A

Unequal parental origin of a chromosome causing some of the chromosomes/DNA to be silenced.

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71
Q

What is the term for a pathogenic mechanism of cytogenetic abnormality caused by unequal parental origin of a chromosome causing some of the chromosomes/DNA to be silenced?

A

Imprinting effect

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72
Q

What is a constitutional abnormality?

A

A chromosome abnormality that is present from contraception and usually affects all cells of the body.

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73
Q

What is the term for a chromosome abnormality that is present from contraception and usually affects all cells in the body?

A

Constitutional abnormality

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74
Q

Infertility, problems of early growth and development, and family history complications are usually due to what kind of abnormality?

A

Constitutional abnormality

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75
Q

Constitutional abnormalities commonly cause what three issues?

A

Infertility, problems of early growth and development, and family history complications.

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76
Q

What kind of specimen should be collected when investigating constitutional abnormality?

A

Peripheral blood (from a whole blood sample).

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77
Q

What kind of specimen should be collected when investigating stillbirth/neonatal death (miscarriage)?

A

Products of contraception (POC - chorionic villi [contains foetal DNA and maintains its health well after foetal demise], amniotic sac, umbilical cord, foetal parts).

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78
Q

When are products of contraceptions usually taken?

A

In the first trimester which is when most large cytogenetic imbalances will cause pregnancy loss.

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79
Q

What kind of specimen should be collected when investigating the chromosome complement of a living foetus?

A

Amniotic fluid or chorionic villi.

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80
Q

What procedure is performed to obtain amniotic fluid for cytogenetic investigation of a living foetus?

A

Amniocentesis

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81
Q

What is amniocentesis?

A

A procedure in which a large needle is used to transabdominally aspirate amniotic fluid for cytogenetic analysis.

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82
Q

What are the limitations of amniocentesis for cytogenetic investigation of a living foetus?

A
  • Can only be performed from 14 - 18 weeks gestation (~somewhat advanced)
  • The cells in the fluid have been sloughed off by the foetus and are usually already dead so grow poorly in culture and contain poor DNA
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83
Q

What is chorionic villus sampling (CVS)?

A

A produce in which a large needle is used transabdominally or transvaginally to remove chorionic villus for cytogenetic analysis.

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84
Q

What are the advantages of chorionic villus sampling (CVS)?

A
  • Can be collected from 10 - 12 weeks gestation

- The cells are healthy so grow well in culture and yields better DNA

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85
Q

What is the risk that amniocentesis or chorionic villus sampling (CVS) will cause miscarriage?

A

0.5 - 1%

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86
Q

Are amniocentesis and chorionic villus sampling (CVS) a normal component of prenatal care?

A

No.

Amniocentesis and CVS are invasive sampling techniques that have a 0.5 - 1% chance of inducing miscarriage so are not performed unless indicated.

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87
Q

Amniocentesis and chorionic villus sampling (CVS) are both procedures guided by ____.

A

Ultrasound

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88
Q

What three kinds of specimen should be collected when investigating neoplasias (predominantly leukaemias)?

A

Peripheral blood, bone marrow aspirate and trephine, and solid tumours (e.g. tumours, lymph node, skin).

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89
Q

How long does mitosis typically take?

A

One hour

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90
Q

In which stage of mitosis do replicated chromosomes condense to form identifiable chromosomes?

A

Prophase

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91
Q

At which stage of mitosis does the nuclear envelope break down?

A

Prophase

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92
Q

At which stage of mitosis do spindle fibres form from the centrioles at each end of the poles?

A

Metaphase

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93
Q

What occurs in prophase?

A

Replicated chromosomes condense to form identifiable chromosomes and the nuclear envelope breaks down.

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94
Q

At which stage of mitosis do the chromosomes line up at the metaphase plate?

A

Metaphase

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95
Q

What occurs in metaphase?

A

Spindle fibres form from the centrioles at each end of the poles and the chromosomes line up at the metaphase plate.

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96
Q

At which stage of mitosis do the spindle fibres pull the sister chromatids to opposite poles and begins to form two daughter cells?

A

Anaphase

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97
Q

What occurs in anaphase?

A

The spindle fibres pull the sister chromatids to opposite poles and begins to form two daughter cells.

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98
Q

At which stage of mitosis is the nuclear envelope reformed?

A

Telophase

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99
Q

What occurs in telophase?

A

The nuclear envelope is reformed.

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100
Q

What is the purpose of cell culture and synchronisation?

A

To maximise chromosome length and the number of cells simultaneously in metaphase.

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101
Q

What are the two broad methods for culturing cells?

A

Cell suspensions and monolayer cultures.

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102
Q

Which broad type of culture is used for blood and bone marrows?

A

Cell suspensions

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103
Q

Which broad type of culture is used for amniotic fluids, CVS, tissues (not including bloods or bone marrows), and some tumours?

A

Monolayer cultures

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104
Q

What types of tissue are cell suspensions used to culture?

A

Bloods and bone marrows

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105
Q

What types of tissue are monolayer cultures used to culture?

A

Amniotic fluids, CVS, tissues (not including bloods or bone marrows), and some tumours.

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106
Q

What determines how successfully a culture has maximised the number of cells in metaphase?

A

Mitotic Index (MI)

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107
Q

What is the Mitotic Index (MI)?

A

The ratio of interphase to metaphase cells in a culture.

MI determines how successfully a culture has maximised the number of cells in metaphase.

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108
Q

What is the term for the ratio of interphase to metaphase cells in a culture?

A

Mitotic Index (MI)

109
Q

A high Mitotic Index (MI) means that there are a lot of cells in ____ in that culture.

A

Metaphase

110
Q

A ____ Mitotic Index (MI) means that there are a lot of cells in metaphase in that culture.

A

High

111
Q

A low Mitotic Index (MI) means that there are a lot of cells in ____ in that culture.

A

Interphase

112
Q

A ____ Mitotic Index (MI) means that there are a lot of cells in interphase in that culture.

A

Low

113
Q

List the seven factors required for cell culture.

A
  • Nutrients
  • Hydration
  • Warmth
  • Respiration
  • Waste removal
  • Protection from pathogens
  • Protection from physical harm
114
Q

List the three properties of base media.

A
  • Nutrients, vitamins, trace minerals
  • Microenvironment
  • Buffers pH changes
115
Q

List the three components of complete media.

A
  • Base media
  • Growth serum
  • Antibiotics
116
Q

What is commonly used as growth serum in complete media?

A

Foetal calf serum (contains growth factors which promote cell division).

117
Q

Which antibiotics are commonly used in complete media?

A

Penicillin and streptomycin.

118
Q

Base media, growth serum, and antibiotics are the three components of what substance?

A

Complete media

119
Q

What is marrowmax?

A

A commercial complete media commonly used for bone marrow cell culture.

120
Q

Name a commercial complete media that is used for bone marrow cell culture.

A

Marrowmax

121
Q

What is amniomax?

A

A commercial complete media commonly used for amniotic fluid cell culture.

122
Q

Name a commercial complete media that is used for amniotic fluid cell culture.

A

Amniomax

123
Q

What is PB max?

A

A commercial complete media commonly used for peripheral blood cell culture.

124
Q

Name a commercial complete media commonly used for peripheral blood cell culture.

A

PB max.

125
Q

What are the incubation conditions for normal peripheral blood cultures?

A

37’C
5% CO2
~72 hours

126
Q

Why are peripheral blood cultures incubated at 5% CO2?

A

The CO2 reacts with the buffers in the media to keep the pH under control.

127
Q

What cell type is the goal of culturing peripheral blood?

A

T cells

128
Q

What is a mitogen?

A

A substance that stimulates cells to enter cell division.

129
Q

What is the most common mitogen used to stimulate T cell division for constitutional blood work?

A

Phytohaemagglutinin (PHA)

130
Q

What is phytohaemagglutinin (PHA)?

A

The most common mitogen used to stimulate T cell division for consitutional blood work.

131
Q

Which mitogen can be added to oncology cultures to stimulate T and B cell proliferation?

A

Pokeweed mitogen

132
Q

What is pokeweed mitogen?

A

A mitogen that can be added to oncology cultures to stimulate T and B cell proliferation.

133
Q

Name the two DNA complements that work together and target B cells, stimulating B cell division and preventing apoptosis.

A

Interleukin-2 (IL-2) and DPS30

134
Q

What are interleukin-2 (IL-2) and DSP30?

A

DNA complements that work together and target B cells, stimulating B cell division and preventing apoptosis.

135
Q

A cell culture fixed at random would yield a very ____ MI.

A

Low

136
Q

A cell culture fixed at random would show a slide full of cells in which phase of mitosis?

A

Interphase

137
Q

What is synchronisation?

A

The process of getting or keeping most cells in a culture into metaphase.

138
Q

Name the two techniques of synchronisation.

A

Block and release.

139
Q

What is blocking (synchronisation)?

A

Addition of a reagent which blocks the cell cycle from proceeding past a specific stage, causing dividing cells to accumulate at the same mitotic point.

140
Q

Name the technique in which a reagent is used to prevent the cell cycle from proceeding past a specific stage, causing dividign cells to accumulate at the same mitotic point.

A

Blocking, a synchronisation technique

141
Q

Name a blocking reagent commonly used in cytogenetics.

A

Thymidine

142
Q

How does thymidine work as a blocking reagent?

A

Thymidine blocks DNA synthesis, causing the cell cycle to slow to a near halt at the G1/S boundary.

143
Q

What is releasing (synchronisation)?

A

Addition of a reagent which releases cell from blocking and allows them to resume division in a similar phase to other cells in the culture.

144
Q

What is done to continue synchronisation when the majority of cells have reached the blocked stage of the cycle?

A

Addition of a releasing agent to resume the cell cycle with most cells in a similar phase.

145
Q

What reagent is used to release a thymidine block?

A

2-dioxycytidine

146
Q

What is 2-deoxycytidine used for?

A

Releasing a thymidine block.

147
Q

What happens after 2-deoxycytidine is added?

A

The cells resume the cell cycle in a synchronised fashion.

148
Q

What happens to cells during harvesting?

A

Cell swelling to make them brittle, fixing of cells in their current state, and removing unwanted cells (RBCs).

149
Q

Why are RBCs removed during harvesting?

A

RBCs have no nucleus and no DNA so do not contain chromosomes to analyse.

150
Q

What is the aim of harvesting cells for cytogenetics?

A

To fix cells in metaphase and prepare them for spreading.

151
Q

What are the three stages of harvesting?

A
  • mitotic arrest
  • hypotonic treatment
  • fix cells
152
Q

What is the purpose of mitotic arrest in cell harvesting?

A

To optimise chromosomes in the desired condensed state.

153
Q

Optimising chromosomes in the desired condensed state is the purpose of which stage of harvesting?

A

Mitotic arrest, the first stage of harvesting.

154
Q

What is the purpose of hypotonic treatment in cell harvesting?

A

Swelling cells to disperse chromosomes and weakening the cell membrane to facilitate spreading.

155
Q

Swelling cells to disperse chromosomes and weakening the cell membrane to facilitate spreading is the purpose of which stage of harvesting?

A

Hypotonic treatment, the second stage of harvesting.

156
Q

What is the purpose of fixing cells in cell harvesting?

A

Preserve cells in their current state, hardening membranes and chromatin.

157
Q

Preserving cells in their current state, hardening membranes and chromatin is the purpose of which state of harvesting?

A

Fixing cells, the third stage of harvesting.

158
Q

What is used to optimise mitotic index (MI) and arrest cells in metaphase?

A

A spindle inhibitor (examples: colchicine and colcemid).

159
Q

Colchecine and colcemid are examples of what reagent?

A

Spindle inhibitor.

160
Q

What is the purpose of a spindle inhibitor?

A

To block cells in the optimal state by inhibiting polymerisation of the mitotic spindle.

161
Q

How do spindle inhibitors block cells in the optimal state?

A

Inhibiting polymerisation of the mitotic spindle.

162
Q

Addition of a spindle inhibitor such as colchicine is part of which stage of harvesting?

A

Arresting cells in metaphase, the first stage of harvesting.

163
Q

Additional of a spindle inhibitor such as colchicine is part of which stage of sample processing?

A

Harvesting.

164
Q

What substance added in harvesting increases the number of metaphase cells at harvest?

A

Colchicine, a spindle inhibitor.

165
Q

Why are cells synchronised by blocking and releasing rather than simply adding colchicine (spindle inhibitor)?

A

Long exposure to colchicine shortens chromosomes and kills cells.

166
Q

What two factors have an inverse relationship when deciding how long to expose cells to colchicine?

A

Number of metaphases : chromosome length.

Increased time = increased metaphases but decreased chromosome length.

Decreased time = decreased metaphases but increased chromosome length.

167
Q

Would chromosomes exposed to colchicine for 15 minutes be long or short?

A

Long

168
Q

Would chromosomes exposed to colchicine for 1 hour be long or short?

A

Short

169
Q

How does adding a hypotonic solution remove RBCs?

A

Influx of water through their semi-permeable membrane via osmosis causes them to lyse.

170
Q

What type of solution causes RBCs to lyse?

A

Hypotonic

171
Q

What effect does adding hypotonic solution have on lymphocytes during harvesting?

A

Swells lymphocytes which spaces out chromosomes and makes cytoplasm fragile (enables chromosome spreading).

172
Q

What type of solution is added during harvesting to swell lymphocytes, spacing out chromosomes and making the cytoplsam fragile for chromosome spreading?

A

Hypotonic solution.

173
Q

How long should cells be left in hypotonic solution during harvesting?

A

~10 minutes.

174
Q

Which hypotonic solution is commonly used for harvesting?

A

0.075M KCl (potassium chloride).

175
Q

Is 0.075M KCl a hypertonic or hypotonic solution?

A

Hypotonic

176
Q

At what stage of harvesting is 0.075M KCl added?

A

Hypotonic treatment, the second stage of harvesting.

177
Q

What temperature is hypotonic treatment conducted at?

A

37’C

178
Q

Why would hypotonic treatment be conducted at 37’C?

A

Warming the solution to 37’C softens the cell membrane and speeds cell swelling.

179
Q

What is done to soften the cell membrane and speed cell swelling during hypotonic treatment stage of harvesting?

A

Warming the solution to 37’C

180
Q

What is the optimal exposure time to hypotonic treatment during harvesting?

A

15 minutes.

181
Q

Exposing cells to hypotonic treatment for 5 minutes would result in what kind of cell spreading?

A

Under-spreading.

182
Q

Exposing cells to hypotonic treatment for 1 hour would result in what kind of cell spreading?

A

Over-spreading.

183
Q

Exposing cells to hypotonic treatment for 15 minutes would result in what kind of cell spreading?

A

Optimal spreading.

184
Q

What substance is universally used as a cytogenetic fixative?

A

Carnoy’s fixative (3:1 methanol : acetic acid).

185
Q

A 3:1 solution of methanol to acetic acid is commonly known by what name?

A

Carnoy’s fixative.

186
Q

What is Carnoy’s fixative?

A

A 3:1 solution of methanol to acetic acid that is universally used as a cytogenetic fixative.

187
Q

Why should Carnoy’s fixative be added to cells slowly?

A

The solution can be harsh on cells.

188
Q

What is done after adding Carnoy’s fixative for cell fixation during harvesting?

A

The culture is centrifuged and supernatant removed - this is repeated until the cell pellet is white and the supernatant is clear.

189
Q

How many times should the culture be centrifuged and supernatant removed after adding Carnoy’s fixative during harvesting?

A

Until the cell pellet is white and the supernatant is clear, usually ~3 fixes (repeats).

190
Q

What is the aim of chromosome spreading?

A

To produce well-spread metaphases with minimal chromosome overlap whilst avoiding chromosome loss.

191
Q

What is the primary factor that affects chromosome spreading?

A

Drying time

192
Q

List six variables that can effect chromosome spreading.

A
  • Drying time
  • Cell concentration
  • Drop height
  • Wicking effects (drying slide edges)
  • Localised humidity
  • Additional fix
193
Q

What effect does high humidity have on chromosome spreading?

A

The slide dries slower which can cause overspreading.

194
Q

What effect does high temperature have on chromosome spreading?

A

The slide dries faster which can cause underspreading.

195
Q

What happens to Carnoy’s fixative over time?

A

Methanol evaporates, leaving a higher percentage of acetic acid in the solution.

196
Q

What effect does an older preparation of fixative have on chromosome spreading?

A

Methanol evaporates and leaves more acetic acid in the solution which can cause overspreading.

197
Q

What effect does fanning the slide have on chromosome spreading?

A

The slide dries faster which can cause underspreading.

198
Q

What effect does a high cell concentration have on chromosome spreading?

A

Less room in solution can result in underspreading.

199
Q

What effect does drop height have on chromosome spreading?

A

The higher the drop the faster it expands when it hits the slide.

200
Q

What effect does wicking (drawing liquid away by drying the edges of the slide) have on chromosome spreading?

A

The more the slide is dried, the more underspreading will occur.

201
Q

What effect does adding additional fixative to the slide have on chromosome spreading?

A

Additional fixative slows the drying, causing more spreading.

202
Q

What is the purpose of banding and staining?

A

To digest and stand chromosomes to create distinct banding patterns that allow identification of individual chromosomes and detection of structural and numerical arrangements.

203
Q

What is a metacentric chromosome?

A

A chromosome with a centomere in the middle and an even p:q ratio.

204
Q

What is a submetacentric chromosome?

A

A chromosome with a centromere between the middle and an end and an uneven p:q ratio.

205
Q

What is an acrocentric chromosome?

A

A chromosome with a centromere at one end with a large q arm and a very small p arm (satellites and stalks).

206
Q

What is the first step of banding and staining?

A

Ageing

207
Q

What is the purpose of ageing when banding and staining?

A

Ageing gives sharper contrast to banded chromosomes.

208
Q

How are chromosomes aged when banding and staining?

A

Being heated at 90 - 95’C for 1 hour.

209
Q

List three different banding techniques.

A
  • High resolution banding (Giemsa [G] banding)
  • Constituative heterochromatin (C) banding
  • Nucleolar organising region (NOR) banding
210
Q

What is the most common banding used in cytogenetics?

A

Giemsa (G) banding

Also accepted: GTG banding (G-bands by trypsin using giemsa).

211
Q

What is constituitive heterochromatin (C) banding?

A

Banding that stains gene poor regions, typically the centromere.

212
Q

When would constituitive heterochromatin (C) banding be used?

A

Investigating variants or abnormal dicentric (two centromeres) chromosomes.

213
Q

What banding technique should be used when investigating variants or abnormal dicentric abnormalities?

A

Constituitive heterochromatin (C) banding.

214
Q

You examine a slide and see chromosomes with darkly stained centromeres. What kind of banding has been used?

A

Constituitive heterochromatin (C) banding.

215
Q

What is nucleolar organising region (NOR) banding?

A

Banding that stains chromosome satellites (acrocentric chromosomes) with active NOR genes.

216
Q

When would nucleolar organising region (NOR) banding be used?

A

Investigating rearrangements involving satellites.

217
Q

What banding technique would be used when investigating rearrangements involving satellites?

A

Nucleolar organising region (NOR) banding.

218
Q

You examine a slide and see that the satellite regions of some acrocentric chromosomes are darkly stained. What kind of banding has been used?

A

Nucleolar organising region (NOR) banding.

219
Q

What is GTG banding?

A

G-banding by trypsin using giemsa - the most common type of giemsa banding in which chromosomes are treated with trypsin prior to giemsa staining.

220
Q

What does trypsin do when used in GTG banding?

A

Trypsin partially digests some of the chromosomal histones, relaxing the chromosome structure so making DNA stains more accessible.

221
Q

Which reagent used in GTG staining partially digests chromosomal histones, relaxing chromosome structure so making DNA stains more accessible?

A

Trypsin

222
Q

GTG banding darkly stains gene poor ____-rich regions.

A

AT

223
Q

GTG banding darkly stains gene ____ AT-rich regions.

A

Poor

224
Q

Does GTG banding stain gene poor AT-rich regions light or dark?

A

Dark

225
Q

GTG banding lightly stains gene rich ____-rich regions.

A

GC

226
Q

GTG banding lightly stains gene ____ GC-rich regions.

A

Rich

227
Q

Does GTG banding stain gene rich GC-rich regions light or dark?

A

Light

228
Q

When examining GTG banded chromosomes you realise a large part of a band is missing. Which is more likely to have a significant impact - a missing dark section or a missing light section?

A

A missing light section as these are gene-rich regions.

229
Q

How would the bands appear on an under-trypsinised slide?

A

Faint and poorly defined.

230
Q

How would the chromosomes and bands appear on an over-trypsinised slide?

A

Chromosomes would look wide and ‘motheaten’ with fuzzy banding.

231
Q

What is a G positive band?

A

A dark band in a chromosome.

232
Q

What is a G negative band?

A

A pale band in a chromosome.

233
Q

What is resolution in banding and staining?

A

The number of G positive and negative bands.

234
Q

List four factors that can impact resolution.

A
  • Cell type
  • Colchicine time
  • Ageing
  • Trypsin time
235
Q

How is resolution measured?

A

Resolution in measured in bands per haploid set (bphs) which directly correlates with quality assurance (QA) level.

236
Q

What is the target resolution/QA level?

A

QA 6 (550 bands)

237
Q

What is the aim of chromosome analysis?

A

To find and examine quality metaphase chromosomes from slides via microscopy.

238
Q

What does chromosome analysis via microscopy entail?

A

Detection of numerical and structural abnormalities, and recording details of the cells examined including karyotyping and counting cells.

239
Q

What is the minimum number of cells per sample required for karyotyping?

A

5 cells per sample.

240
Q

What is the minimum number of cells per sample required for chromosome counting?

A

10 cells per sample.

241
Q

In the minimum 5 cells required for karyotyping, how many times much each chromosome must have all regions unobscured?

A

At least twice.

242
Q

What type of abnormality is found by counting chromosomes in karyotype analysis?

A

Numerical abnormalities

243
Q

What type of abnormality is found by cross-examining banding patterns between chromosome pairs (homologues) in karyotype analysis?

A

Structural abnormalities

244
Q

How many karyotypes are analysed per patient before reporting?

A

Five (minimum).

245
Q

What is an ideogram?

A

A diagrammatic representation of a chromosome.

246
Q

What two things are ideograms particularly useful for?

A
  • Comparison of sex chromosomes in males

- Breakpoint determination (finding where breaks occurred in a chromosome)

247
Q

What is the format of a written karyptype (ISCN)?

A

Number of chromosomes, sex chromosomes, abnormality (if any).

248
Q

What is the karyotype of a normal/healthy male?

A

46,XY

249
Q

What is the karyotype of a normal/healthy female?

A

46,XX

250
Q

What is the karyotype of a female with an additional (aneuploid) chromosome 21 (trisomy 21/Down syndrome)?

A

47,XX,+21

251
Q

What is the karyotype of a male with a deletion between 13q21.1 and 13q31.1?

A

46,XY,del(13)(q21.1q31.1)

252
Q

What is the karyotype of a female with a translocation between 6q27 and 15q22?

A

46,XX,t(6;15)(q27;q22)

253
Q

What is fluorescence in situ hybridisation (FISH)?

A

A technique to detect and locate specific DNA sequences on a chromosome by hybridising complementary fluorescently labelled DNA probes.

254
Q

What is a probe (FISH)?

A

DNA sequences correspondign to a particular region of a chromosome and fluoresces when excited.

255
Q

What is hybridisation (FISH)?

A

The process of combining two complementary single-stranded DNA molecules and allowing them to reform a single double-stranded molecule through base pairing.

256
Q

List the seven steps of fluorescence in situ hybridisation (FISH).

A
  1. Fix sample to slide
  2. Apply FISH probe to slide
  3. Co-denaturation
  4. Hybridisation
  5. Stringency wash
  6. Apply DAPI and antifade
  7. Excite probe with filtered UV light and examine under a microscope.
257
Q

How is co-denaturation achieved (FISH)?

A

Heating the slide to 74’C for 2 - 5 minutes.

258
Q

What is co-denaturation (FISH)?

A

Third step of FISH - separation of target and probe dsDNA into ssDNA.

259
Q

What is hybridisation (FISH)?

A

Fourth step of FISH - reannealing sample DNA and with incorporated probe DNA.

260
Q

How is hybridisation achieved (FISH)?

A

Incubating the slide at 37’C for 2 - 48 hours.

261
Q

What is a stringency wash (FISH)?

A

Fifth step of FISH - washing in SSC buffer to remove unbound probe.

262
Q

What will happen if the SSC buffer used for a stringency wash is too hot or the salt too weak?

A

The wash will remove bound and unbound probe, stripping the slide.

263
Q

What will happen if the SSC buffer used for a stringency wash is too cool or the salt too strong?

A

The wash will not remove unbound probe from the slide.

264
Q

What is done after a stringency wash (FISH)?

A

Application of DAPI and antifade.

265
Q

What does DAPI do (FISH)?

A

DAPI lightly stains chromosomes.

266
Q

What does antifade do (FISH)?

A

Antifade protects probes (fluorophores) from light.

267
Q

Can interphase cells be used for FISH?

A

Yes, interphase cells show probe location in relation to other probes and allows enumeration of signals.

268
Q

What is the benefit of using metaphase cells over interphase cells for FISH?

A

Metaphase cells show probe location in relation to other probes, allow enumeration of signals, AND show probe position on chromosomes.

LQB490 (2 decks)

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