CYTOGENETIC TECHNIQUES Flashcards
1
Q
- Study of the physical size and structure of chromosomes
- A specialized laboratory discipline that examine the structure and behavior of chromosomes at the cellular level
A
Cytogenetics
2
Q
cells are arrested in what phase of mitosis?
A
end prophase or in early
metaphase
3
Q
cells are arrested in end prophase or in early metaphase of mitosis because of
A
- Compact and densely staining of chromosomes
- Characteristic size and shape of chromosomes
4
Q
- Checks the number of chromosomes in the normal diploid cell
- Check the size distribution
A
Karyotype
5
Q
Comparing chromosomes are based on:
A
- Length
- Location of centromeres
- Location and sizes of G-bands
6
Q
Two Cytogenetic Techniques
A
- Classical or Standard Cytogenetic
- Fluorescence in situ hybridization (FISH)
7
Q
Allows visualization of loss or gain of material
A
Classical or Standard Cytogenetic
8
Q
- Allows much smaller changes to be seen
- Far more advanced
A
Fluorescence in situ hybridization (FISH)
9
Q
Blood additives
A
EDTA, ACD
10
Q
what is EDTA?
A
EthyleneDiamineTetraAcetic Acid
11
Q
- Prevents the coagulation of blood
- Allows the qualitative and quantitative determination of HIV, Hepatitis C and Cytomegalovirus (CMV)
A
EDTA
12
Q
- Prevents the coagulation of blood
- Preserve the form and function of cellular components
A
Acid Citrate Dextrose
13
Q
top color of EDTA?
A
purple top
14
Q
top color of ACD?
A
yellow top
15
Q
- Uncommon
- Used for:
- Bladder cancer screening
- Monitor therapy of bladder cancer
A
Urine
16
Q
type of urine used
A
midstream
17
Q
how much urine is needed?
A
first 10ml
18
Q
- Differential and quantitative analysis as a sensitive specific biomarker for the detection of colorectal cancer
A
Feces
19
Q
- Rapid identification of infectious agents
- Comes from brain and spinal cord
A
Cerebrospinal Fluid
20
Q
how many tubes are used in CSF? what tube is used?
A
4 tubes are collected; 2nd tube is used
21
Q
why is the 2nd tube used for CSF collection?
A
it is the purest CSF specimen
22
Q
normal CSF protein
A
50-80 mg/dL
0.18-0.45 g/L
23
Q
normal CSF glucose
A
15-45 mg/dL
2.5-3.5 mmol/L
24
Q
normal CSF RBC count
A
0 cells/mm3
25
normal CSF WBC count
0-3 cells/mm3
26
normal CSF xanthochromia (yellowish color)
none
27
normal CSF appearance
normal (water)
28
bacterial CSF appearance
turbid
29
viral CSF appearance
clear
30
fungal CSF appearance
fibrin web (clotting)
31
bacterial CSF protein
more than 1
32
viral CSF protein
less than 1
33
fungal CSF protein
0.1-0.5
34
bacterial CSF glucose
less than 2.2 mmol/L
35
viral CSF glucose
normal (2.5-3.5)
36
fungal CSF glucose
1.6-2.5
37
bacterial CSF WBC
more than 500
38
viral CSF WBC
less than 1000
39
fungal CSF WBC
100-500
40
bacterial CSF increased in what type of WBC?
neutrophils
41
viral and fungal CSF increased in what type of WBC?
monocyte
42
* Assess the presence of infectious microorganisms
* Fluid found in joints
synovial fluid
43
process of collecting fluids from the joints
arthrocentesis
44
tube used in arthrocentesis?
Sterile plain tube
45
clarity, color, and viscosity of normal SVF (synovial fluid)
transparent, clear, high
46
clarity, color, and viscosity of non-inflammatory SVF (synovial fluid)
transparent, yellow, high
47
clarity, color, and viscosity of inflammatory SVF (synovial fluid)
translucent, yellow, low
48
clarity, color, and viscosity of septic SVF (synovial fluid)
opaque, dirty/yellow, variable
49
clarity, color, and viscosity of hemorrhagic SVF (synovial fluid)
bloody, red, variable
50
wbc count and PMNs% of normal SF
less than 200, less than 25%
51
wbc count and PMNs% of non-inflammatory SF
200-2000, less than 25%
52
wbc count and PMNs% of inflammatory SF
200-10000 (up to 100000), more than 50%
53
wbc count and PMNs% of septic SF
more than 80,000, more than 75%
54
wbc count and PMNs% of hemorrhagic SF
200-2000, 50-75%
55
* Prenatal diagnosis of congenital disorder
* Assess fetal maturity
* To look for Rh isoimmunization or intrauterine infection (blood type infections)
amniotic fluid
56
when to collect amniotic fluid?
15-20 weeks AOG (4th-5th month)
57
how many days does amniotic fluid culture take?
9-12 days
58
* For earlier diagnosis than amniotic fluid
* part of the placenta
chorionic villus
59
when to collect chorionic villus?
10-12 weeks (2nd-3rd month)
60
how many days does chorionic villus culture take?
3 weeks
61
* For infectious agents identification
* Identification of cancer cells
Pleural, pericardial, ascitic fluid
62
collection of fluid from the lungs, heart, and abdomen
Paracentesis
63
* Less invasive
* Excellent source of genomic DNA
* For patients who had blood transfusion
and bone marrow transplantation
Buccal cells
64
method of collecting buccal cells
* Rinsing with mouthwash
* Use of swab
65
* Formalin-fixed, paraffinembedded tissue
* Frozen specimen in an Optimal Cutting Temperature Compound (OCT)
solid tissue
66
* Forensic analysis (Genomic DNA Identification)
* Trace metal and drug analysis
Hair and nails
67
specimens transported in room temp
* blood
* bone marrow
* amniotic fluid
* chorionic villi
68
specimens transported in ice
solid tissue
69
Techniques in Classical Cytogenetics
1. Cell culture
2. Sufficient metaphase plates are produced
3. Cells are subjected to hypotonic swelling
4. Fixation
5. Staining
6. Photomicroscopy
7. Case report
70
multiplication of cells using mitogenic stimulating agents
cell culture
71
chemical agent which stimulates mitosis?
phytohemagglutinin (PHA)
72
arresting the cells at end prophase using mitotic inhibitors
Sufficient metaphase plates are produced
73
mitotic inhibitor used
colcemid
74
To further disperse the chromosome within the cell and to lyse any red cells present (to harvest the chromosomes)
hypotonic swelling
75
buffer solution used in hypotonic swelling
0.075M KCl
76
how long are cells stored in hypotonic swelling?
10-20 mins, 37 deg Celsius
77
Alters the cell membranes and chromosomes by removing lipids and water molecules and denature proteins
Fixation
78
modified Carnoy's fixative
3:1
absolute methanol : glacial acetic acid
79
* Artificial aging of the cells at 65C for 30-60 mins improves the quality of the staining
* Dehydrate the chromosome
Staining
80
dehydration of chromosomes are treated with?
proteolytic enzyme
solution
81
what dye is used in staining?
metachromatic dye
82
Lightly stained
Euchromatin
83
Dark stained
Heterochromatin
84
Use computer imaging systems with software to capture and karyotype 2 or more representative metaphase spreads per specimen
Photomicroscopy
85
* Description of the chromosomal finding in International System of Cytogenic Nomenclature (ISCN)
* Recommendations are made
– When abnormalities are detected
– Genetic counseling
– Referral to specialist
– Additional genetic testing
Case report
86
* Uses fluorescent dye to mark abnormalities
* The principal molecular technique currently used in clinical cytogenetic laboratory
* Called Molecular Cytogenetics
* Involves applying DNA probes to a chromosome spread
Fluorescent In Situ Hybridization
87
Process of FISH
1. Chromosomes are placed on a microscope slide and then denatured
2. Complimentary DNA probes are constructed
3. When the DNA is denatured, the probe can then hybridize (form hydrogen bonds) to the complementary single-stranded DNA
4. The labeled probes can be used to identify different chromosomes or targeted chromosome regions
88
separates the ouble helix into single strands using heat, chemicals and pH change
Denaturing
89
* For the sequence of interest and labeled with a fluorescent dye.
* Ultraviolet microscope
(fluorescence microscope)
Complimentary DNA probes are constructed
90
the things that light up under the microscope
fluorophores
91
* Detected using a fluorescence microscope
* The probe will be visible as one or more fluorescent signals in the microscope
When the DNA is denatured, the probe can then hybridize (form hydrogen bonds) to the complementary single-stranded DNA
92
To help recognize:
– Trisomies
– Translocations
– Deletions
The labeled probes can be used to identify different chromosomes or targeted chromosome regions
93
Uses of FISH
* To form a diagnosis
* Evaluate a prognosis
* Evaluate remission of a disease
– Cancer
94
FISH vs Classical Cytogenetics
* Detect diseased cells more easily
* Does not require living cells
* Can be quantified automatically
* A computer counts the fluorescent dots present
