Banding techniques Flashcards
The best method to process slides for G-banding on the same day that they are dropped is:
Warm the last fix to 37oC during harvest.
Prolong the trypsin treatment time.
Incubate the slide in 60oC for 1-3 hrs before banding (+)
Increase the Giemsa concentration.
You are analyzing a clinical case and discover a small extra marker with satellites. What
staining technique would be most useful in determining the chromosomal origin of this
marker?
QFQ
QFQ
GTG
DAPI/DA
NOR(+)
When using a fluorescein labeled probe during the FISH procedure, the appropriate
counterstain used should be:
5-Bromodeoxyuridine
Ethidium bromide
Propidium iodide(+)
DAPI
When doing sequential staining which of the following is used to remove oil from slides?
Ether
Xylene(+)
Acetic acid
Methanol
What is the best banding technique to distinguish between 47,XY,+18 and 47,XYY?
QFQ(+)
GTG
DAPI/DA
RHG
When the identification of paired chromosomes is the primary goal, which of the following banding techniques should be used?
Q-banding
G-banding(+)
Giemsa staining
C-banding
When the addition of nonhomologous material to the q-arm of chromosome 16 is suspected after G-banding, which of the following banding techniques should be used?
C-banding(+)
Q-banding
R-banding
NOR staining
Which chromosome is most different between C and G banding?
1
9(+)
16
Y
What is not a criteria for G-band identification?
band level
location of bands
location of centromere
location of telomere(+)
What do quinacrine and acridine orange dyes stain?
histones
nonhistones
GC rich DNA
AT rich DNA(+)
Positive heteropyknosis
Chromosome regions which have few genes and contain primarily noncoding regions of DNA remain in a constantly more contracted or supercoiled state. This is because these regions do not actively transcribe mRNA. Positive heteropyknosis implies that these regions will stain darker with DNA stains
negative heteropyknosis
DNA with active transcription of genes is less compacted and is said to have negative heteropyknosis. Regions of negative heteropyknosis will stain lightly with DNA stains
Late Replicating DNA
Dark G and Q bands C bands Light R-bands Positive heteropyknosis Heterochromatin AT rich
Early Replicating DNA
Light G and Q bands Dark R bands Negative heteropyknosis Euchromatin GC rich
Chromosome Banding Code
G(type of banding)
T(general technique)
G(stain used)
GTG = G bands by trypsin using Giemsa QFQ = Q bands by fluorescence using Quinacrine RFA = R bands by fluorescence using Acridine Orange RHG = R bands by heat using Giemsa CBG = C bands by Barium hydroxide using Giemsa
G-banding using trypsin
Slides are treated with a trypsin solution. The treatment time is determined by the following factors:
- age of the slide(Slides become more resistant to trypsin the longer they sit at room temperature (3 days - 4 weeks), the longer they are heat treated (2 hours at 90oC or overnight at 60oC) or the longer they are treated with chemicals that denature or crosslink proteins, such as H2O2.)
- concentration of the trypsin solution (Some investigators use 0.01% crystalline purified trypsin diluted in Hanks without Ca++ or Mg+++ ions. These ions bind to the active site of trypsin and inactivate its enzymatic action. Others use Enzar-T trypsin, a 40x concentrate of porcine pancreatic enzymes (0.1cc in 50 cc of Hank’s = 0.02%)
- pH of the trypsin solution (Trypsin is a pancreatic enzyme which works most effectively at a higher pH (8.5). Trypsin becomes inactive at a pH of 6.8. The best G-banding is achieved at a pH of 7.0 - 7.2 where the pH is just barely active. At a higher pH it becomes too strong and can cause fuzzy, distorted or uneven banding. In addition, the difference between underbanding, optimal, or overbanding becomes a matter of seconds, which is too difficult to control or troubleshoot.)
Q-banding by fluorescence using Quinacrine (QFQ)
Q-banding was the first banding technique to be discovered (Caspersson 1970). Q-banding is induced by staining chromosomes with either Quinacrine dihydrochloride or Quinacrine mustard. Both are fluorescent dyes. Slides are then rinsed and mounted in McIlvaine’s buffer (pH of 5.5 - 6.0). A thin layer of buffer is necessary between the slide and coverslip to 1) swell the chromosomes, 2) increase the contrast between the bands and 3) allow for the travel of fluorescent light to the objective.
QFQ use
Quinacrine preferentially binds to AT rich regions of chromosomes.
Q-banding is very useful in the study of chromosomal variable regions. These regions include the Y chromosome, centromeres of chromosomes 3 and 4 and the satellites of D and G group chromosomes.
Advantage: Extremely quick technique, can finish report the same day you harvest
Disadvantage: Poorer quality of photography, especially true if journal presentation is desired. In addition, the fluorescence is not permanent and fluorescent microscopes are expensive.
Conventional staining
The staining of slides prepared from a cytogenetic harvesting procedure with Giemsa without banding is also useful in the study of human chromosomes. Giemsa is a metachromatic stain. In other words, molecules will stack upon each other. Therefore, darkness of stain is a function of time and concentration. Slides stained for 5 minutes in a 4% Giemsa stain diluted in 0.01 phosphate buffer will give a good solid stain. Banding of chromosomes can distort the morphology of metaphase chromosomes and make it difficult to discern important features such as: primary constrictions (centromeres) secondary constrictions (NORs) fragile sites double minutes satellites (D and G group chromosomes) acquired abnormalities such as dicentrics, rings, fragments, breaks and gaps.
C-banding by Ba(OH2) (CBG)
This technique stains the constitutive heterochromatin which is located around the centromere of all human chromosomes and the constitutive heterochromatin located in the distal long arm of the Y chromosome. Chromosomes 1, 9, 16 and Y generally have the largest C-band regions. These chromosome regions are considered to be genetically inert and contain no functional genes. C-bands are also highly polymorphic.
There are two types of heterochromatin:
Constitutive - contains few to none mendelian genes and is rarely transcribed.
Facultative - euchromatic DNA rendered inactive by cellular processes. (e.g. inactive X in females required for gene dosage compensation).
Technique:
Pretreatment of slides with 0.2N HCL - This acid treatment removes any cytoplasmic debris and some of the histone proteins from the chromosomes.
Extraction of DNA with Ba(OH2) - A saturated Ba(OH2) solution (5% in double deionized water) is a strong alkali that selectively extracts the DNA from the non-C-band regions of DNA.
Incubation in 2xSSC at 60 - 65oC for one hour allows further denaturation and extraction of the DNA in the non-C-band regions.
NOTE: This is a timed reaction. Leaving the slides for too long in the Ba(OH2) solution will result in loss of DNA from the C-band regions as well.
CBG use
C-band as well as Q-band polymorphisms are useful in distinguishing fetal cells from maternal cells, donor cells from host cells, in the identification of marker chromosomes, and in the identification of pericentric and paracentric inversions. Perhaps because these regions are inert, considerable variation (polymorphisms) in size of the C-bands does not seem to affect the phenotype.
Basis of Selective Extraction:
Chromatin is a DNA-histone complex
C-band areas have a tighter interaction with the so called nuclear matrix (that which remains after the removal of all chromatin from the interphase nucleus). The nuclear matrix is comprised of nonhistone proteins of (1) inner nuclear membrane (2) nucleolar matrix and (3) intra-nuclear matrix
Presence of more interaction with nonhistone proteins (support structure for chromatin) prevents or slows extraction of DNA. These regions are highly compact in structure.
NOR stain (Nucleolar Organizer Region)
Silver staining, sometimes called AgNOR staining, stains the NOR's of metaphase chromosomes and the nucleolus of interphase cells. NOR's - nucleolar organizing regions are the chromosomal regions located on chromosomes nos. 13, 14, 15, 21, and 22 in the stalk region. This is the location of the ribosomal cistrons (gene clusters). These ribosomal cistrons code for the 18s and 28s subunits of the ribosomes and are responsible for the formation of the nucleolus in interphase cells. Ribosomes transcribe RNA transcripts into proteins, and the nucleolus is where ribosomal proteins are transcribed. Silver nitrate (AgN03) stains the NOR specific proteins and formic acid or formalin is the catalyst which drives the reaction. Only those NOR's (or cells) which were active in the previous cell cycle will stain positively. These chromosomal regions will contain residual proteins, which will precipitate silver.
Distamycin A - DAPI stain by fluorescence
This is a banding technique that stains only a specific subset of C-bands.
Distamycin A - DAPI stain by fluorescence use
DAPI is a fluorescent dye and Distamycin A is a nonfluorescent antibiotic which binds to AT-rich DNA. Distamycin A quenches all DAPI fluorescence except for the C-band regions of chromosomes 1, 9 16, 15p11 and Yq12. This technique is especially useful in the determination of chromosomal markers originating from the short arm of chromosome 15 and the Y heterochromatin.
Fluorescent in situ hybridization (FISH)
This technique involves hybridization of DNA specific probes on to interphase and metaphase chromosomal DNA.
Satellite probes
Hybridize to specific areas of repetitive DNA (e.g., centromere probes; Y specific probe and short arms of acrocentric chromosomes)
Painting probes
Hybridize to large areas of specific DNA (whole chromosome probes, partial chromosome probes, total genomic)
Unique Sequence probes
Hybridize to small areas containing a specific gene or gene cluster (cosmid probes and telomere specific).
1 Specimen preparation FISH
Painting procedures and mapping of DNA probes on metaphase chromosomes and interphase cells can be performed on conventional methanol/acetic acid fixed cell preparations.
2FISH Denaturation procedures
cause double-stranded DNA to become single-stranded DNA. Single-stranded DNA can hybridize with other single-stranded DNA.
Denaturation of specimen DNA by treatment in 70% Formamide/2x SSC at 70oC (Formamide is an organic solvent that lowers the melting point of DNA which is usually between 90 and 100oC.
Denaturation of the probe.
3FISH Hybridization:
The DNA probe is hybridized onto specimen slides at 37oC for 30 minutes to 16 hours depending on the type of probe used.
4FISH Post Hybridization Wash
This solution is usually 2xSSC or another buffered salt wash and is used to remove free probe or probe bound to non-target DNA. The stringency of the wash solution, or the extent of removal of unwanted probe, minimizes the amount of background, which will be observed under the fluorescent microscope.
NOTE: increasing the temperature or decreasing the salt concentration increases stringency.
5FISH Detection
Direct labeled probes are prelabeled with fluorochromes so this step is unnecessary.
Indirect labeled probes are prelabeled with either the biotin or digoxigenin hapten. These hapten labeled probes can then be detected with avidin or antidigoxigenin antibodies with conjugated fluorochromes. Detection of the probe with antigen-antibody detection systems allows amplification of the signal.
6FISH Probe labeling
Probes are labeled either directly or indirectly with the following fluorochromes:
Fluorescein isothiocyanate (FITC, green) Rhodamine (red) Texas red (deep red)
7FISH Counterstain
A counterstain is used to visualize either the interphase nucleus or the metaphase chromosomes. If the probe is labeled with a red fluorochrome, DAPI is a preferred counterstain (blue). On the other hand, if the probe is labeled with FITC, which produces a yellow-green signal, propidium iodide (PI) is the preferred counterstain (red).
LISH (Light in situ hybridization)
refers to nonfluorescent detection of hybridized probes. These techniques can be either nonisotopic or isotopic.
Nonisotopic techniques allow for the visualization of various substrates through the utilization of enzymatic reactions. These substrates are converted by the enzymatic reaction to products, which form precipitates. A common nonisotopic technique uses an avidin-biotinylated horseradish peroxidase complex followed by H202 - 3,3’ diaminobenzidine-tetrahydrochloride (DAB) reaction. The horseradish peroxidase reduces hydrogen peroxide that turns DAB to a brown or black color that can be observed under the light microscope.
Isotopic- Radioactive techniques utilizing radioactive probes to expose film. A common isotopic technique hybridizes tritiated labeled DNA probes (H3) onto slides, which are then covered with stripping film. After 7-10 days, when the film is developed, silver grains will be seen over those chromosome regions, which contained the hybridized probe.
Coverslips
should be 0.17mm or less in width and have a refractive index similar to that of crown glass (1.515). Coverslips are used when permanent slides are desired. These coverslipped slides will be impervious to scratches and other damage, which can occur to unprotected slides.
Mounting media
Wet mount - these are used in fluorescent microscopy and include both water-based and glycerin-based mounting media used in Q-banding, R-banding, and FISH for example.
Permanent mounting media - These are natural or synthetic resins dissolved in solvents. Again, the refractive index of the mounting medium should match the refractive index of crown glass (1.515) to prevent blurring of the image as seen under the microscope.
Sequential staining
It is possible to perform more than one staining technique on one slide. Sequential staining is necessary under the following circumstances:
harvest yields only one or two slides
harvest yields extremely few metaphases or
special study requires more than one banding procedure be performed (localizing breakpoints of chromatid breaks, FISH on G-banded slides.
The limitation in sequential banding is based on the level of DNA or protein denaturation. Some banding procedures alter the basic structure of chromatin very little, while other banding procedures literally strip chromatin from the chromosomes.
Destaining techniques
Slides can be destained by: Carnoy's fixative Methanol 70% Ethanol These chemicals do not disperse any oil, which may be on the slide from observing the prior banding pattern. Therefore, it is necessary to completely remove all the oil from the slide (Xylene or Hemo-D).
Hydration and Rehydration
Hydration or rehydration of a slide can be accomplished by exposing the slide to various dilutions of ethanol moving from a low percent of water to a higher percent of water
100% 90% 80% 70% EtOH
Dehydration can be accomplished by exposing the slide to these ethanol mixtures in the opposite direction.
Select Slide Storage Methods as Required by Law
By state law slides must be kept for reference for a stated minimum amount of time. In New York slides are required to be stored for six years before disposal. This time varies from state to state. Maintaining an archive of patient slides protects laboratories against litigation.
To prevent destruction of stored slides due to humidity and airborne dust, slides should be stored in an airtight container preferably in a temperature and humidity controlled environment. Slides exposed to the open air can become unreadable after only a short period of time (3 months - 1 year) in areas of the country with a high humidity level.
Troubleshoot
1.Trypsin exposure time
The necessary exposure time in trypsin depends on a variety of variables such as: 1) age of slide, 2) concentration of trypsin, 3) pH of the trypsin, 4) temperature of the trypsin solution, 5) heat treatment of slide.
The best way to have consistently successful GTG banding is to hold as many of these factors constant allowing treatment time to be the only variable.
Undertreated G-banded slides may be retreated in the trypsin solution. The time for the second treatment, however, will be significantly shorter (1 - 2 seconds) if the slide shows some banding. Slides need not be destained before retreatment if 70% ethanol is used following trypsin treatment, but the slides should be free of oil or alcohol. If Hank’s or diluted serum is used to stop the trypsin treatment step, slides should be destained with 100% methanol and dried completely before retreatment in trypsin.
Troubleshoot
2.Slide aging
Slides may be aged by storage at room temperature in an airtight container for a minimum of three days and a maximum of six weeks before G-banding (aging of slides is not as critical in the Q- and AgNOR banding techniques).
Slides may also be aged by high temperatures
overnight at 60oC
2 hours at 75oC
20 minutes at 90oC
Slides may also be aged by chemicals
Staining slides with Giemsa and storing the slides overnight before banding
Treatment of slide with 15% H202 (2 - 7 minutes) prior to banding (important to rinse and dry H202 treated slides thoroughly before placing in the trypsin as this chemical can oxidize your trypsin)
Troubleshoot
3.Environmental factors (e.g., temperature, humidity)
Temperature directly affects the rate of enzymatic activity in the G-banding procedure leading to an indirect relationship in treatment time. For example, cold trypsin solutions (less than room temperature 20oC) will require a longer slide treatment time.
High humidity can cause water to deposit on the surface of the slide. The presence of this small amount of moisture can cause difficulties in achieving even banding. To prevent this, slides should be stored in a dry place or in a desiccator.
What banding technique would give alternating bands, which would distinguish each chromosome?
GTG(+)
CBG
DAPI/DA
NOR
A technologist wants to stain chromosomes using a banding technique that manifests AT-rich regions on chromosomes. What staining procedure should he use?
RHG
CBG
NOR
QFQ(+)
You have just discovered an odd looking number 9 homolog in a patient’s G-banded metaphases. The short arm looks unusually large and the long arm looks smaller between the centromere and the first band. Which of the following banding techniques should be used to investigate?
Q-banding
NOR banding
C-banding(+)
FISH
Which of the following probes would be ideal to detect chromosome aneuploidy in interphase nuclei?
painting probe specific for one chromosome pair
alpha-satellite probe hybridizing uniquely to a specific
centromere
unique sequence probe (e.g., a cosmid) which
hybridizes to one locus on one chromosome pair
either b or c(+)
Which of the following probes would be ideal to detect a small microscopic deletion?
painting probe specific for one chromosome pair
alpha-satellite hybridizing uniquely to a specific
centromere
unique sequence probe (e.g., a cosmid) which
hybridizes to one locus on one chromosome pair.(+)
C-banding stains the constitutive heterochromatin of which chromosomes?
1,9,16,Y(+)
1,9,15,X
1,9,16,X
13,16,X
What is the optimum pH for the trypsin?
- 4-7.6
- 2-7.4(+)
- 0-7.2
- 6-7.8
Chromosome banding properties include:
G-bands are foci of chromatin condensation
R-,G-, and C-bands differ in their time of DNA
replication
R-bands are gene-rich
all of the above(+)
Prior to the development of chromosome banding techniques:
Primary and secondary constrictions could not be
identified
Relative length and centromere position were the
main characteristics used for classification.(+)
All chromosome pairs could be identified.
Satellites on the short arm of acrocentric
chromosomes could not be identified.
Which of the following statement(s) is/are true about C-banding:
Can be used to locate centromere regions of human
chromosomes.
Stains constitutive heterochromatin.
Identifies some normal chromosome variations in
humans.
all of the above(+)
Which of the following does not produce extended chromosomes?
Reduce the Colcemid concentration
Synchronize culture with amethopterin block
Pretreatment with Ethidium Bromide
Decrease the volume of hypotonic(+)
Who developed G-Banding by using trypsin?
Caspersson
Seabright (+)
Arrighi and Hsu
Dutrillaux and Lejeune
Who developed Q-banding?
Caspersson(+)
Arrighi and Hsu
Seabright
Dutrillaux and Lejeune
Who developed C-Banding?
Caspersson
Dutrillaux and Lejeune
Seabright
Arrighi and Hsu(+)
All of the following are true about interphase cytogenetics EXCEPT:
Allows analysis of a large number of cells
Reliable for the detection of monosomy
Provides information about chromosome markers(+)
Useful for evaluating aneuploidy
Quinacrine molecules bind to the DNA by:
Binding to the GC rich regions
Binding to the AT rich regions(+)
Binding to nonhistone proteins
Crosslinking close thymidine nucleotides
Under the electron microscope, what is observed as a consequence of all banding procedures?
Chromatin digestion
Nucleotide crosslinking
Protein melting
Chromosomal collapse(+)
Case #777 still has an uncertain diagnosis after G and C bands were obtained. The director suspects that satellites are involved with the chromosome marker in question. What banding/staining method should be performed to confirm the director’s suspicions?
R-banding
NOR banding(+)
DAPI/DA banding
G-11 banding
Positive heteropyknosis is related to:
Coiling of chromatin filaments(+)
Pale staining
Early replication
Genetic activity
Giemsa is specific for:
Active DNA
RNA
Single-stranded DNA
Double-stranded DNA(+)
Which of the following is not an advantage of Q-banding?
Requires a fluorescent microscope for analysis.(+)
Slides can be scanned for Y chromosomes.
Acrocentric chromosomes demonstrate
polymorphisms.
Q-banding does not alter chromosome morphology.
Which procedure should be performed first if sequential banding is required?
G-banding
C-banding
Q-banding(+)
R-banding
The translocation t(8;14)(q24;q32) is best demonstrated by:
GTG banding(+)
C banding
The NOR stain
The DAPI stain
A marker chromosome is found that appears to have two centromeres by G-banding. Which of the following banding techniques should be used to confirm this observation?
C-banding(+)
NOR-banding
SCE-banding
R-banding
When a staining technique is denoted by the following triplet: GTG, which of the following can be assumed?
The first letter denotes the general technique.
The second letter denotes the type of banding.
The third letter denotes the stain used.(+)
None of the above is correct.
Which of the following statements about G-banding are FALSE?
Methanol-acetic fixation is necessary for chromosome spreading but has no effect on G-banding.
G-banding correlates with the chromomere pattern of pachytene chromosomes of meiosis.
G-dark bands are early replicating DNA regions.
Both a and c(+)
A cytogenetic technologist suspects an inversion of chromosome 9 with G-banding analysis. Which banding technique would help confirm this observation?
A-banding
C-banding(+)
Q-banding
All of the above
A cytogenetic technologist comes across a case with 47 chromosomes. The extra chromosome is roughly the size of a G group chromosome. What banding technique should be used to determine the identity of this chromosome?
C-banding
Q-banding
SCE banding
Either a or b(+)
Which of the following banding solutions DO NOT produce a known banding pattern?
Quinacrine dihydrochloride, McIlvaines’ buffer
Ba(OH)2, 2xSSC
Trypsin, Giemsa
Amethopterin, BrdU(+)
Which of the following banding techniques are used to stain those chromosomal regions that form the nucleolus in interphase cells?
NOR banding(+)
C-banding
Q-banding
DAPI/Distamycin A staining
Of the following factors, which affects G-banding?
Slide aging
Time the slide is treated in trypsin
pH of the trypsin
All of the above(+)
The late synthesizing portions of the chromosomes are:
Genetically active
Euchromatic
Heterochromatic(+)
Recessive
Negative heteropyknosis observed in euchromatin is related to:
Decondensed chromatin(+)
Dark staining
Complementation
Late replication
The dark bands of G-banding:
Are late replicating
Have few genes
Have DNA with a higher A-T base composition
Are all of the above(+)
Which of the following chromosomal polymorphisms can be detected with Q-banding?
Polymorphisms of chromosome Y
Polymorphisms of the centromere of chromosome 3
Polymorphisms of the satellites of acrocentric
chromosomes
All of the above(+)
While G-banding a batch of patient slides processed on the same day, a technician monitoring the slides notices that one patient’s slides show chromosome morphology similar to conventional, solid staining. Which of the following statements is CORRECT?
The slide is under-trypsinized, another slide should be treated for a longer time period.(+)
The trypsin must have deteriorated as all slides dropped on the same day should respond the same to trypsin treatment.
The slide is over-trypsinized, this is why no bands can be observed.
Another slide should be treated for a shorter time period.
Chromosomes with __________contain the ribosomal genes which are involved in protein synthesis.
Primary constrictions
Many bands
Nucleolus organizing regions(+)
Small short arms
After G-band analysis, a cytogenetic director suspects that chromosome number 10 has a terminal deletion. What banding procedure would be most helpful in deciding if a deletion has occurred?
C-banding
NOR-banding
FISH(+)
Q-banding
Giemsa is a metachromatic stain composed of various dyes. Which of the following banding techniques can be used with the Giemsa stain?
R-banding
C-banding
SCE banding
All of the above(+)
Which location can be shown by C-band?
Telomere
Centromere
Constitutive heterochromatin(+)
Facultative heterochromatin
Which one of these abnormalities will produce the brightest Q-band signal?
r(Y)
inv(Y)
i(Y)p
i(Y)q(+)
Which one of these techniques would be MOST helpful in identifying that a marker chromosome is bisatellited?
DAPI/Distamycin
NOR(+)
FISH
SKY
What does the acronym CBG stand for?
C bands by Barium Hydroxide using Giemsa(+)
C banding by Brdu using Giemsa
C Banding by Bleomycin using Giemsa
G bands by Barium Hydroxide using C-banding method
What technique is used to identify a small marker?
DAPI/Distamycin A(+)
FISH
G-Banding
Q-banding
