KHARKIV NATIONAL MEDICAL UNIVERSITY FACULTY OF POSTGRADUATE EDUCATION DEPARTMENT OF MEDICAL GENETICS METHODICAL RECOMMENDATIONS OF PRACTICAL CLASS FOR THE STUDENTS OF THE 4 TH MEDICAL FACULTY, OF THE 3 RD YEAR OF EDUCATION (ELECTIVE COURSE) ON THE THEME: «CYTOGENETIC METHODS OF RESEARCH IN CLINIC» Established at the Department’s meeting «___»______2007 Kharkiv 2007 Head of the Department, MD, professor_________________E.Ya. Grechanina
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Introduction. Determination of cytogenetic method of diagnostics.
Significance of cytogenetic method in clinical practice: diagnostics of chromosomal diseases; diagnostics of range of mendeling diseases connected with
chromosomal instability; diagnostics of oncological diseases; mutagenic influence
of chemical matters etc. on stability of genome.
Types of cytogenetic methods. Methods of collecting samples for providing
cytogenetic research. Methodical bases for receiving preparations of chromosomes,
and investigating tissues.
Methods of coloring chromosomes (routine and differential).
Readings for using cytogenetic method with the aim of diagnostics of
chromosomal diseases.Promethaphase analysis of chromosomes. Autoradiographic research.
Method of molecular cytogenetic diagnostics. Essence of the method of fluorescent
The central task in providing cytogenetic analysis is determination of caryotype.
Caryotyping is the start point of whole research, determining a possibility of using
all other methodical approaches. Investigation of chromosomal set can be provided
using different methodics of routine coloring when all the chromosomes have thesame even color (Fig. 1). Such method allows determining the qualitative disorders
of caryotype and detecting some marker chromosomes without confirmation of
their origin.
Fig. 1. Dycentric chromosome found with using method of routine painting
As such approach is informative a little, the main method of analysis of caryotype
is technique of G – differential coloring chromosomes (G - bending), that Torbjorn
Caspersson was elaborated in the end of 1960-s. The base of the method is the
acquisition by chromosomes the pattern from light and dark stripes in processing
them by trypsine and then painting by Hymza dye. The character of draught is
specific for each pair of chromosomes. Dark stripes (G - stripes, G - bends) are inthe places of localization of intersticial heterochromatine. Such sites don’t contain
the same DNA practically, and they are full of А-Т - pairs. In prometaphase when
the chromosomes are on the early stage of condensation it’s possible to count about
2000 stripes, in other cases – 400-800 stripes (Fig. 2). This method of painting
allows identifying the chromosomes, detecting the deletions, inversions, insertions,
translocations, fragile sites and more completed rebuilding. Pre-processing of
cytogenetic preparations precedes to painting by the G - method. In connection
with methodic it can be an incubation in buffers without Ca2+ и Mg2+, in t° <= 37°С
or t° >= 60°С; incubation in solutions of proteolytic enzymes (trypsine, pepsineand others); incubation with deproteinized matters (urea, 2-merkaptoetanol);
incubation in standard salt solution (SSC) under action of alkaline and high
temperature. There are different methodics of G – differential coloring
chromosomes, but as a rule GTG (pre-processing by trypsine, coloring by Hymza
dye) is used. Reaction of trypsyne provides transition of regular spiral structure to
irregular segmentation that creates obstacles to perception of Hymza de in G –
negative sites and increases it in G – positive segments of chromosomes.
Fig. 2. Segment of metaphase plate (G – differential painting chromosomes of
leukocytes of peripheral blood)
R - method allows getting diametrical draught of chromosomes, that’s converse the
same in G – painting (light R – stripes are corresponding to dark G - stripes and
vice versa). Preparations, received in R – coloring, are analyzed by phase –
contrast microscopy. In getting of R – draught of chromosomes for pre-processing
the balanced salt solution of Arl is used in t° = 78 - 96°С, and also in specific
indexes of рН and time of exposition, which are various in different methodics.
Preparing of preparations is more completed than in G – differential coloring,
because the pre-processing by Ва(ОН)2 in 60°С or NаОН and formaldehyde are
often used.
If the painting on R – method provides in lower indexes of рН solutions and with
longer exposition, telomeric regions will be colored only (Т - method).Q –
differential coloring chromosomes is received with using of fluorochromes,exciting by blue-violet light, imitate red, yellow and orange lighting. Among the
dyes usually acrihin is used, but preparations are more qualitative in painting by
acrihin - iprit or acrihin - propyl; a bit worse result – in using of derivative
bibenzymidazolum Hoechst 33258. Much fluorescenting chromosomal sites are
corresponding to dark G - discs; difference is that the secondary pulling of
chromosomes 1 and 16 are colored by G – method only, but intensity of painting
segments of chromosomes 3, 4, 13 - 15, 21 - 22 and Y is weaker in using Q -
method.
If we put a cytogenetic preparation in alkaline and then stay it in SSC solution in t°= 60 - 65°С for a long time, the structural chromatine of pericentromeric regions
will be colored only. This approach that received the name of С - method is used
for detecting pericentric inversions.
Appearance of new technologies of molecular cytogenetics that are based mainly
on in situ hybridization of nucleic acids, increased possibilities of chromosomal
hybridization with 32P-marked probe was described in 1969. Improving methods
of marking DNA-probes, providing hybridization and detection of marked DNA on
cytological preparations allow passing to localization of unique sequences.
Elaboration of non-radioactive systems of marking and detecting DNA-probes has
done the method of in situ hybridization safe, simple for providing and processing
results. Development of technologies of digital record of microscopic pictures andtheir computer elaboration resulted in creation of new approaches to visualization
and identification of chromosomal material. At present time there are some basic
approaches using by modern molecular cytogenetics:
1. Identification of material of long chromosomal regions and whole
chromosomes.
2. Detection of specific DNA sequence in interesting region.
3. Analysis of disorder of balance of single chromosomal regions.
For identification of material of chromosomal regions and whole chromosomeschromosome-specific and region-specific probes are used (painting probes),
“coloring” the whole chromosomes or chromosomal regions in using FISH
methodics. Parallel development of methods of in situ hybridization, creation and
detection of DNA-probes and improving epyfluorescent microscopy, digital record
and computer processing of micro pictures resulted in appearance of new
technologies allowing using some dozens DNA-probes in one experiment.
DNA-probes are central and leading link in FISH providing because determination
of chromosomal anomaly is possible if corresponding DNA-probe is available.
DNA-probes for centromeric chromosomal sites (CEP - centromeric probe) work
for identification of centromeric regions of chromosomes and detection of
quantitative disorders of caryotype both in interphase and metaphase. They contain
short nucleotide sequences that are complementary to multiple chromosome-
specific α – satellite DNA - repeats, localized at centromeric chromosomal site.
Pan – centromeric probes allow detecting the centromeric regions of all
chromosomes and they are used for detection of aneuploidia, polyploidia, dycentric
chromosomes and other complex aberrations. It’s possible to use individual
centromeric probes to some chromosomes if investigating pathology is connected
with quantitative disorders of the chromosomes (monosomias, trysomias). To
exclude cells with haploid and polyploid chromosomal set in analysis the controlcentromeric DNA-probe for any chromosome without diagnostic meaning is used.
DNA-probes to telomeric sites of chromosomes (TEL - telomeric probe) are
intended for detection of deletions and rebuildings affected the ending sites of
chromosomal arms. Such probes are specific for р- or q- arms of chromosomes and
complementary to the site that is 300 т.п.н. from the end of chromosome. As a
rule, DNA-probes for short and long chromosomal arms are connected with
different fluorochromes that allow detecting both telomeric chromosomal sites on
one cytogenetic preparation. To specify differential diagnostics of deletions andmonosomias it’s proposed to use the DNA-probe to centromeric site of
investigating chromosome connected with fluorochrome that is differ from
Locus-specific DNA-probes (LSI - locus - specific identifiers) hybridize with
unique sequences of DNA. They are intended for detecting significant
chromosomal aberrations in different pathological conditions.
On the base of interphase FISH – analysis the integration of molecular cytogenetics with other methods occurs. The material can be not only cytogenetic
preparations but also standard colored blood and medullar smears, or traces of
lymphonodes and hystological material stored in the form of paraffin blocks.
Combination FISH with morphological methods of investigation allows comparing
cytogenetic and morphological peculiarities of cells directly. Unfortunately, such
method isn’t enough standardized for wide using in clinical diagnostic researches.
In 1992 Weber-Matthiesen et al. had proposed using the combination of methods
of immunophenotyping and for investigation of turnoral cells called FICTION
(Fluorescence Immunophenotyping and Interfase Cytogenetics as a Tool for Investigation of Neoplasms). For such analysis uncoloured blood and medullar
smears or preparations of other tissues are used. On the first stage the preparations
incubate with specific monoclonal antibodies and then conjugate with
fluorochromes for further visualization of antigen-monoclonal body complex. Then
they provide hybridization in situ with DNA-probes. Different colored
fluorochromes are used for detection of monoclonal antibodies and DNA-probes.
Investigation of preparation by fluorescent microscope with necessary set of filters
allows providing analysis of immunophenotype and hybridization signals in the
interphaze nuclei.
Digital record of micro images has opened a possibility to transfer both
combination of fluorochromes and correlation of their intensity into pseudo colors.
The method was productive for elaboration of the method of multicolored
chromosomal banding (MultiColor Banding - МСВ). It provides not the whole
chromosomal analysis but the detail analysis of a single chromosome. Region-
specific DNA – tests are marked by different fluorochromes of combination of
fluorochromes. The level of signal of each DNA-probes varies on intensity
achieving maximum in the centre and falling practically to zero on its limits.
Recovering of profiles of intensity of the signals of DNA-tests provides variationsof correlations of intensity of fluorescence of different fluorochromes along the
chromosome. Relations of intensities can be transferred to pseudo colors and each
point of imaging. Thus, each chromosomal region has its own pseudo color. Such
variant of multicolored FISH has high effectiveness in analysis both
interchromosomal and intrachromosomal rebuilding in turnoral diseases. However,
for its successful providing it’s necessary to determine a chromosome previously
for investigation. Thus, the method of molecular cytogenetics is suitable for
analysis of disorders of caryotype associated with specific chromosomes. At
present time there are sets of DNA-probes for providing MCB of all humanchromosomes (Fig. 3). Due to saving of aerial chromosomal organization on all
stages of cellar cycle, MCB allows providing analysis of chromosomal
reorganization both in interphase nuclei and metaphase nuclei.
Fig. 3. Multicolored banding of human chromosome 5 (in situ hybridization
of region-specific of DNA-libraries on chromosome 5; profiles of signals of
region-specific of DNA-libraries on chromosome 5; multicolored banding of chromosome 5; ideogram of chromosome 5; fluorochromes, used for МСВ).
Metaphase cytogenetics that historically appeared earlier than interphase, allows
determining a wide spectrum of chromosomal disorders, but it’s necessary that the
investigating cells are at the metaphase of meiosis.
Method of comparative genome hybridization (Comparative Genome Hybridization
- CGH) was created for detection of quantitative disorders in genome. It is based
on the providing reaction of hybridization in situ with using whole genome like
DNA-probe. Extracted and normal donor DNA is marked by different colour fluorochromes converting them into DNA-probes. Equivalent numbers of these
probes are mixed and used in hybridization with control cytogenetic preparation.
After providing FISH metaphases are analyzed by fluorescent microscope
determinating with the help of specialized program of computer analysis of image
the intensity of fluorescence of two fluorochromes along each chromosome. In
absence of quantitative changes in caryotype of investigating sample a correlation
of intensity of lighting of two fluorochromes is watched. In the case of
amplification of genes the intensity of the signal of corresponding fluorochrome is
increasing, but in the case of partial loosing genetic material it is decreasing. Thus,CGH allows detecting a genome dysbalance. But this method can’t be used for
finding balanced translocations and inversions, and trysomias and deletions can be
determined in the case if the size of imbalanced site is not less than 10 mln. pairs
of nucleotides.
Multicolor caryotyping has found a wide using for determination of origin of
different marker chromosomes in turnoral haematological diseases. Such analysis
is provided in using DNA-probes of whole painting to arms or whole
chromosomes (probes WCP - whole chromosomal painting). Such probes arehybridized with multiple short DNA sequences situated along the chromosome.
Thus, in studying by fluorescent microscope a chromosome looks like whole
colored in specific color. Technique of whole chromosomal painting became a
reason for new type of caryotype research – multicolour caryotyping. DNA-probes
using for the analysis consist of mix of probes of whole painting for whole human
chromosomal set marked with the help of combination of some fluorochromes,
correlation of which is chosen for each chromosomal pair. Using corresponding
methods of registration and computer programs of analysis of image estimating theintensity of lighting of all fluorochromes for each point of image allows providing
caryotyping in which each chromosomal pair has its unique “pseudo color”.
Simultaneous using n number of fluorochromes allows providing simultaneous
analysis of 2n-1 DNA-probes.
Combination using of direct- and hapten-marked DNA-probes allows decreasing
number of fluorochromes necessary for visualization in metaphase of material of
all human chromosomes to three. But the version of multicolour FISH is more
complicated.
There are different variants of multicolour FISH for decision of different tasks in
cytogenetic diagnostics.
At present time there are two basic types of providing pseudo multicolor images.
In the simplest variant the sets of specific combinations of exciting and closing
filters and apparatus for black and white digital registration of signal are used.
Modern technique is able to provide registration both signal intensity and its
spectral characteristics. Such technical equipment is the base of spectral
caryotyping (SKY).
M-FISH (multitarget, multifluor, multicolor or multiplex FISH) is general name of
traditional multicolour FISH with using sets of fluorochrome-specific filters.
Principe of M-FISH is separated digital registration of signal of all information and
using fluorochromes in change of filter sets. Processing of whole recorded
information connected with separating signal and background and also quantitative
estimation of signal is conducted with the help of special program providing that
transfers the information about level of signals of fluorochromes in each point of
image to pseudo colors. One of the main limits of the number of using DNA- probes is the number of available fluorochromes with uncovering spectra of
exciting and emission and corresponding filter sets.
24-color FISH (variant of M-FISH) has the widest spreading. It is intended for
simultaneous identification of material of all human chromosomes. It has high
efficiency in detection of chromosomal translocations because each chromosomal
pair is colored into its pseudo color, but it doesn’t allow detecting deletions and
inversions. Such problem can be partially solved by simultaneous painting
chromosomes DAPI that gives a possibility of providing analysis of differentialdraught of chromosomes. Unfortunately, the quality of DAPI-banding of
chromosomes after M-FISH is worse than GTG-differential painting and DAPI-
Method of multicolor chromosomal banding (R x FISH) is based on the intertype in
situ hybridization. It allows detecting a part of interchromosomal rebuilding
providing analysis of whole human genome in one experiment. DNA-probes, using
in R xFISH, are marked by combination of three fluorochromes that provides 7 pseudocolors. They paint specifically single regions of chromosomes, creating
their coloured draught. Such peculiarity of DNA-tests for R xFISH depends on type
of their getting. These are chromospecific DNA – libraries of two types of
hylobates: Hylobates concolor and Hylobates syndactylus (Fig. 4). As a result after
intensive chromosomal rebuilding in formation of modern types of hylobates the
material of their chromosomes was mixed a lot compared with organization of
human chromosomes. In hybridization the DNA sites of different chromosomes of
hylobates connected with different fluorochromes connect with complementary
DNA sites on the same human chromosome creating its colored draught ininvestigation with using fluorescent microscopy (Fig. 5). Unfortunately, providing
R xFISH has enough serious limits of chromosomal rebuilding. Those which had
place inside of one colored R x-band can’t be detected by this method if they don’t
lead to significant and visible changes of its size. The probes paint some
chromosomal regions of different chromosomes into one pseudo color. Also there
is a lack of R xFISH such as a big size of many colored bands, but each of human
chromosomes 15, 18, 19, 21, 22, x and Y presents one colored band. Using large
number of fluorochromes and new DNA-tests in future can significantly increase
abilities of the method. But in increasing number of using fluorochromes R xFISH
will be more informative than routine 24-colored FISH.
Fig. 4. Colored draught of human chromosome 1 in R x FISH.
Fig. 5. Colored draught of human chromosome in R x FISH. а). Metaphase plate. b).
Chromosomal distribution
The main principles of analysis of microscopic images in spectral karyotyping
(spectral karyotyping - SKY) don’t differ from using in M-FISH. The differences
are connected with the type of registration of image requiring correct describing
spectral characteristic of object. In one measure there are all spectral curves for all
points of image. For spectral karyotyping of all human chromosomes 5
fluorochromes are used: one is in green spectrum, two are in red spectrum and two
are in infrared spectrum. Exciting and emission of all fluorochromes using in
marking DNA-probes is provided in one filter set that allows avoiding their
sequent changing, intermediate focusing and problems connected with it, such asaerial moving of image, determination of cut-off values and segmentation masks.
In one act of exposition for each point of image the whole spectral characteristic of
light is recorded. On the base of analysis of spectral curves an availability and
absence of specific fluorochromes in the point is determinated. The next step is a
procedure of classification conducting with the help of special program providing
that allows determining chromosomal belonging of analyzing material. The great
advantage of SKY is that painting DAPI is registered parallel with spectral image.
The possibility of parallel analysis of spectral image and qualitative differential
chromosomal painting simplify significantly an interpretation of results of the SKYand allow determining correctly the points of chromosomal gaps. Classical
methods of cytogenetic analysis allow detecting about 15% chromosomal
rebuildings identifying with the help of SKY. This technology is highly effective
for analysis of FISH results in interphase nuclei (spectral FISH). Next advantage of
the SKY is possibility of using fluorochromes with covering spectra of exciting
and emission that extends the list of fluorochromes that are suitable for using and
increases the number of using fluorochromes. For providing SKY a block of filter
for spectral FISH and a block of filters for DAPI are necessary.
The lack of the SKY is long time for record of microscopic images. Also it can be
waiting that SKY is a bit less effective than М-FISH in providing investigations
Color - changing karyotyping is proposed by range of authors like alternative
method to M-FISH and SKY. This method is based on analysis of difference of
signal length between samples of DNA hybridized with DNA-probes connected
with fluorochromes and DNA-probes carrying antibodies. This method requires 3
filters only and doesn’t require a special ward and program providing. For identification of chromosomes hybridization is provided once only and two images
are received. The base of the method is difference of the length of fluorescent
signal because the time of exposition of DNA-tests connected with antibodies is
less on 80 - 90% than in tests connected with fluorochromes. After reaction of
hybridization the smears are exhibited with corresponding primary antibodies and
then visualization is provided receiving the first photo. Then the preparations are
exhibited with secondary antibodies and avidine connected with the same
fluorochromes. The smears can be contrpainted using DAPI. Then the photos of
preparations are made with using three filters. These images are exhibited withusing special program providing and the second photo with visible chromosomes
connected with specific antibodies is made. Thus, some chromosomes will
fluorescent on the first or the second photo only, but other ones will change their
color on different photos.
From written above the conclusion can be done that the fullest analysis of
karyotype (determination of disorders of chromosomal set, diagnostics of
translocations, deletions, duplications, inversions and insertions) can be provided
by different methods of differential painting (G- and R-banding), and also R xFISH
and SKY in combination with DAPI, that allow to get finally the diametrical
chromosomal draught. Methodic FISH with using locus - specific DNA – probes is
suitable perfectly for detection of specific chromosomal aberrations that are
important for the diagnostics and prognosis. For qualitative changes of genetic
information the CGH method can be used, however it doesn’t allow to determinate
localization of the sites with amplified and deleted genes. As a result, the type of
methodic depends on the tasks of investigators.
Addition №3
Readings for providing cytogenetic investigations
Readings for cytogenetic investigations are enough wide, especially in obstetrical-
gynecological practice. Below there is a list of conditions in which it’s necessary
to provide a cytogenetic investigation of a patient (proband) or, if it’s necessary,
of his relatives with diagnostic aims.
1. Suspicion on chromosomal disease on clinical symptomatic (for confirmation
of diagnosis).
2. Multiple inborn developmental defects in a child (not genic syndrome); + thechild’s parents have multiple developmental defects.
Genotoxic action of chemical matters in vivo is often accompanied by
destabilization of genome and its rebuildings. If the contact with mutagens leads
to formation of the DNA lacerations, in the process of their reparation the
exchanges of homological sites of sister chromatids in the interphase nuclei arewatched. Their frequency is the measure of mutagenic interaction for cells. There
were elaborated the methods, which allow finding SCE in somatic cells of animals
and plants. Cells (lymphocytes of peripheral blood usually) incubates in vitro in
accompanying 5-bromdezoxiuridine that’s included into DNA instead of timidine
for two cellar cycles. One of the chromatids includes the analogue of nucleoside
into both DNA chains and another one includes it into one DNA chain only.
Fluorescent dye has different interaction with such homological chromatids thatcan be detected on intensity of painting with the help of microscope – one
homological chromatid looks lighter than another one. In SCE light and dark sites
in chromatids of individual chromosomes are sequenced and the number of such
sites is the measure of SCE frequency. The higher SCE the more intensive was
predicative mutagenic interaction on the somatic cell. Determination of SCE can
be used for retrospective estimation of mutagenic interaction because SCE are
prolonged for some time after initiating action of mutagen in its absence, for
example, for removal of origin of ionizing radiation of whole detoxication of