Cytogenetic Research METHODIC

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KHARKIV NATIONAL MEDICAL UNIVERSITY

FACULTY OF POSTGRADUATE EDUCATION

DEPARTMENT OF MEDICAL GENETICS

METHODICAL RECOMMENDATIONS OF PRACTICAL CLASS

FOR THE STUDENTS OF THE 4TH MEDICAL FACULTY,

OF THE 3RD 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



Theme «Cytogenetic methods of research in clinic» (the third course)

Human cytogenetics has its origin since a discovery of Tjio and Levan in

1956 and then an independent discovery of Ford и Hamerton. Adding water to

suspension of mytotically dividing human cells before their fixing on the glass,

they could separate chromosomes from each other and count their number that was

46. Since that moment a research of qualitative disorders of caryotype, such as

monosomias, trysomias and polyploidia, which are the most common cause of

early abortions and severe inherited diseases, became possible. Next years the

cytogenetic investigations were added by achievements of technological progress

in molecular biology and chemistry and extended knowledge about human

genome. Difficult and minimal changes of chromosomes became accessible to

cytogeneticists. Methodics for estimation of caryotype in cells that are not dividing

became available, receiving of isolated chromosomes for molecular analysis became possible and predicted the detailed investigation of human genome,

mapping of genes and indification of different mutations.

General aim – meeting with cytogenetic methods and their use in medical

practice.

Skills

Specific tasks Aims of final level

1. to know an essence, types, and possibilities of cytogenetic method

1. to choose the patients for providingcytogenetic research

2. to know the region of using

cytogenetic method

2. to interpret the results of cytogeneic

research

Place of providing classes – a classroom at the Department and cytogenetic

laboratory of the Kharkiv Specialized Medical Genetic Center

Duration of the class – 45 minutesPlan of the class

Introduction. 2 min.

1. Essence and types of cytogenetic method 8 min.

2. Methods of coloring chromosomes and the essence of methods 5 min.

3. Readings and the region of using cytogenetic method 10 min.

4. Molecular and cytogenetic methods of diagnostics 10min.

5. Educational control and correction of level of knowledge 10 min.

Equipment of the class – computer system METASYSTEMS, slides, photos,

pictures, and genetic cards.



Tasks for independent preparing and independent correction of final

level of knowledge.

For determination if the final level of knowledge is corresponding to necessary

you should fulfill the following tasks and compare results with answers.

Test tasks.

А. Basic enzyme, providing enzymatic synthesis of gene (DNA):

1. Cytochromoxydase

2. Revertase

3. Endonuclease

4. RNA-polymerase

5. Superoxidase

B. Light stripes on chromosomes in their differential coloring is:1. Heterochromatine

2. Euchromatine

3. Coloring mistake

4. Chiasms

C. Unit of genetic code:

1. Dynucleotide

2. Triplet

3. Pyrymidine base

4. IntronD. For studying of role of genetic and environmental factors a mehod is used:

1. Clinical-genealogical

2. Direct ДNA-probing

3. Microbiological

4. Cytological

5. Twin

E. Preparation that allowed to determinate the correct number of chromosomes

(46) in human caryotype in 1956:

1. Kolchycine

2. Cytoarsein

3. Phytohemaglutinine

4. Fluorescent dyes

F. Preparation of kolchycine stops the cell’s dividing on the stage:

1. Anaphase

2. Prophase

3. Metaphase

4. Thelophase

G. Material for providing polymerase chain reaction can be:

1. chorion cells2. microorganisms



3. biological fluids (sperm, saliva)

4. old blood spots

5. venous blood

6. embryo on the pre-implantation stage

H. Providng indirect method f molecular diagnostics is possible if:

1. original gene is mapped2. mutation is not identified

3. gene is not sequened

4. proband is absent

5. nucleotide sequences planking gene are known and there are DNA-zonds and

oligoprimers for them

Information that is necessary for reinforcement of knowledge level, it’s

possible to find in the following origins:

1. «Медицинская генетика» Под ред. чл-корр. АМНУ, проф. Е.Я.Гречаниной, проф. Р.В. Богатыревой, проф. О.П. Волосовца. Учебник

для студентов высших медицинских заведений ІІІ-ІV уровней

аккредитации. Киев “Медицина” 2007. 534 с. Рекомендовано МОЗ

Украины.

2. Бочков Н.П. Клиническая генетика.- М.: Медицина, 2002

3. Захаров А.Ф., Бенюш В.А., Кулешов Н.П., Барановская л.и. Хромосомы

человека. Атлас. - М.: Медицина, 1982

4. Козлова С.И., Семанова Е., Демикова Н.С., Блинникова О.Е.

Наследственные синдромы и медико-генетическое консультирование.Справочник. - Л.:Медицина,1989.

5. Лазюк Г.И., Лурье И.В., Черствой Е.Д. Наследственные синдромы

множественных пороков развития.-М.:Медицина,19836.

6. Лильин Е.Т., Богомазов Е.А., Гоман- Кадошников П.Б. Генетика для

врачей- М., Медицина, 19903.

7. Наследственные болезни: Справочник/ Под ред. Л.О. Бадаляна.-

Ташкент: Медицина,1980

8. Прокофьева – Бельговская А.А. Основы цитогенетики человека

9. Смирнов В.Г. Цитогенетика. .- М.: «Высш.шк.» 199110.Фогель Ф., Мотульски А. Генетика человека.т.1, «Мир», 1989

Study the following material after adoption of basic data:

1. Виноградова О.А., Савченко В.Г., Домрачева Е.В., Любимова Л.С. и др.

Молекулярно - цитогенетический мониторинг химеризма и минимальной

остаточной болезни у больных хроническим миелолейкозом после

аллогенной и сингенной трансплантации костного мозга // Терапевтический

архив. - 2002. - № 7.

2. Виноградова О.А., Савченко В.Г., Неверова А.Л., Дяченко Л.В. и др.Изучение динамики смешанного химеризма методом флуоресцентной



гибридизации in situ у больных хроническим миелолейкозом после

аллогенной трансплантации костного мозга // Терапевтический архив. - 2001.

- № 7

3. Ворсанова С.В., Юров Ю.Г., Соловьев И.В., Демидова И.А. и др.

Современные методы молекулярной цитогенетики в пре- и постнатальной

диагностике хромосомной патологии // Клиническая лабораторная

диагностика. - 2000. - № 8.

4. Домрачева Е.В., Асеева Е.А. Возможности и перспективы гематологической

цитогенетики // Медицинская генетика. - 2004. - Т. 3, № 4.

5. Цитогенетичні методи дослідження хромосом людини: методичні

рекомендації / Під. ред. Т.Е. Зерової - Любимової, Н.Г. Горовенко. - К., 2003.

6. Юров Ю.Г., Хазацкий И.А., Акиндинов В.А., Довгилов Л.В. и др. Разработка

оригинальной компьютерной программы для молекулярно -цитогенетической диагностики хромосомной патологии // Клиническая

лабораторная диагностика. - 2000. - № 8.

7. Chudoba I., Plesch A., Loerch T. et al. High resolution multicolor-banding: a new

technique for refined FISH analysis of human chromosomes // Cytogenet. Cell

Genet. - 1999. - V. 84.

8. Henegariu O., Neerema N., Bray - Ward P., Ward D. Color - changing

karyotyping: an alternative to M - FISH / SKY // Nature Genetics. - 1999. - V. 23.,

N. 3.

9. Rubtsov N., Karamysheva T., Babochkina T., Zhdanova N. et al. A new simple

version of chromosome microdissection tested by probe generation for 24 - multi -

color FISH, multi - color banding (MCB), ZOO - FISH and in clinical

diagnostics // Medgen. - 2000. - V. 12.

10. Speel E., Hopman A., Komminoth P. Amplification methods to increase the

sensitivity of in situ hybridization: play CARD(S) // The Journal of Histochemistry

and Cytochemistry. - 1999. - V.47, N. 3.

11. Subero M., Ignacio J., Viardot A., Siebert R. et al. Secondary aberration in fillicle

center lymphoma: evaluatio of the diagnostic power of comparative genomic

hybridization (CGH) and standard chromosome analysis (CA) // Medgen - 2000. -

V. 12.



Technological map of providing the class

№ Stage Time,min.

Educational materials Place of providing the

classMeans Equipment

1. Determination of

original level of

preparing on the

questions of

cytogenetic

diagnostics

15 Tasks Classroom

2. Thematic review 55 Genetic cards,caryograms, photos,

and pictures

Electronicmicroscope,

computer

system

Meta-

System

Classroom,cytogenetic

laboratory

3. Results 20 Tasks Classroom

General theoretical questions of the theme.

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

hybridization and readings to using.



Graph of logical structure of the theme « Cytogenetic methods of research in

clinic».

Cytogenetic diagnostics

Region of using C l a s s i f i c a t i o n o f c y t o g e n e t i c m e t h o d s o f

r e s e a r c h

Readings

M u l t i c o l o r e d

c a r y o t y p i n g

Differential paintingLimits

Metaphase

cytogenetics

Molecular genetic

researches

Routine painting

Whole

painti

ng

chromosome

s

Methods of

painting

chromosomes

Caryo

typing

with

changing

color

Comp

arativ

e

geno

michybrid

izatio

n

I n t e r p h a s e c y t o g e n e t i c s

Q p a i n t i n g

C p a i n t i n g

G p a i n t i n g

R p a i n t i n g

Combinatio

n FISH

with other

methodsFluorescent

in situ

hybridization

Multicolore

d banding

chromosom

es



Adition №1.

Methods of painting chromosomes.

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

diagnostics significantly.



Addition №2.

Molecular cytogenetic methods of investigation

• Interphase cytogenetics

1. Multiple banding of chromosomes (МВС)2. Fluorescent in situ hybridization (FISH)

3. Combination FISH with other methods

o Cytology + FISH

o Hystology + FISH

o Immunophenotyping + FISH (FICTION)

• Metaphase cytogenetics

1. Whole coloring chromosomes (Whole painting)

2. Comparative genome hybridization (CGH)

3. Caryotyping with changing color (ССК)

4. Multicolored caryotyping

o Spectral caryotyping (SKY)

o Multicolored FISH (M - FISH, M - BAND)

Interphase cytogenetics allows providing chromosomal analysis on the stage of

interphase, when they are in despringed condition, because hybridization in situ is

independent on the phase of cellar cycle. Such research is impossible using

methods of classical cytogenetics. This new approach to studying caryotype gives

a possibility to make an objective estimation of dimensions of cellar clone,

carrying chromosomal abberation, because the cellar populations become available

for investigation.

Method of fluorescent in situ of hybridization (FISH) was elaborated for detecting

of specific sequences of DNA of cytological preparations. It allows pass to analysis

of DNA sequences of chromosomes.

The base of the FISH method is reaction of hybridization between DNA-probes,

created on special technologies and presented the nucleotide sequence of limited

size, and complementary site of nuclear DNA of investigating cytogenetical

preparation. DNA-probe carries “a mark”- contains nucleotides connected with

fluorochrome directly or with hapten for further visualization by antibodies

carrying fluorochrome. Not connected marked DNA is washed and then detection

of hybridized probe is provided with using fluorescent microscope. Firstly in situ



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



fluorochromes of telomeric DNA-probes.

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-



banding after usual in situ hybridization.

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

with small-size DNA-probes.



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.



3. Repeated (more than two) spontaneous abortions, mortinatality or natality of

children with inborn developmental defects.

4. Disorder of reproductive function of unclear genesis in females and males

(primary amenorrhea, sterility.); anomalies of sexual development in males and

females.

5. Essential mental and developmental retardation in a child.

6. Prenatal diagnostics (late reproductive age, translocation in parents, previous

child with chromosomal disease).

7. Suspicion on syndromes characterized by chromosomal instability (register of

chromosomal aberrations)

8. Leucosis (for differential diagnostics, estimation of treatment efficiency and

prognosis).

9. Estimation of mutagenic influences (radiation, chemical affections).

Addition №4

Karyotyping in sterility is read in following cases:

1. Non-obstructive azoospermia

2. Severe oligozoospermia (<5 mln/ml)

3. Delay of sexual development

4. Primary amenorrhea

5. Secondary amenorrhea (premature menopause)

6. Habitual abortion of early term of pregnancy (two or more spontaneous

abortions at the first trimester of the pregnancy) – both parents are investigated.

Families with gravitated obstetrical anamnesis (habitual abortion, especially of

early terms, mortinatality, multiple inborn developmental defects (MIDD) – chromosomal disorders - 5 - 15% cases.

7. Investigation of donors of sperm and ovums.

Addition №5

Sister chromatid exchane (SCE)

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

xenobiotic.

Cytogenetic Research METHODIC

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