Chromosome disorders are of conditions, caused by constitutional
numerical or structural abnormalities of chromosomes.
Normally every cell of the human body has 46 chromosomes, organized
in 23 pairs (22 pairs of autosomes, identical in males and females)
and one pair of sex chromosomes – XX in females and XY in males.
The only exceptions are egg–cells and sperm–cells, which have only
haploid set of chromosomes. All normal egg–cells have karyotype 23,X;
the sperm–cells may be 23,X and 23,Y. Fertilization of the egg–cell
by 23,X–sperm will lead to development of female, fertilization by 23,
Y–sperm will produce male organism 46,XY.
Diagnosis of chromosomal disorders requires analysis of chromosomes.
Experienced clinicians (geneticists, dysmorphologists) may diagnose
many chromosomal disorders by clinical examination. But even if clinical
diagnosis is obvious, it has to be confirmed by cytogenetic examination,
because almost all chromosomal disorders may exist in different cytogenetic
variants with very different prognosis for the family. Therefore,
cytogenetic testing is necessary even in patients with a clear clinical
Standard cytogenetic examination requires analysis of chromosomes
on the stage of metaphase (metaphase analysis). At this stage of
cell division all chromosomes became clearly visible structures.
All chromosomes may be recognized by their size, position of a
centromere and characteristic pattern of dark and light bands,
which can be seen after special staining. A cytogeneticist counts
number of chromosomes in each of studied cells and compares its size
and banding pattern with a standard. If the studied cells have 46
chromosomes with normal structure karyotype of the person considered
as normal. If there are some abnormalities it may be evidence of a
Basically (in normal conditions) all cells of the organism have the
same karyotype. Therefore, theoretically all cells may be used for
cytogenetic examination. However, the preferential types of cells
for chromosomal examination are cells of chorionic villi or amniocytes
(in prenatal diagnosis of karyotype) and lymphocytes (for postnatal
Prenatal examination of karyotype is usually performed for several
groups of pregnant women. It was shown that pregnancy by a fetus
with some chromosomal syndromes (trisomy 21 and trisomy 18) is
frequently accompanied by an increase or decrease of several
biochemical components of serum. Almost all trisomies (trisomies 13,
18 and 21) occur more often in fetuses of “older” woman (especially
after 35 years of age). Age and biochemical parameters (taken
together) allow calculation of the risk for Down’s syndrome. If
this risk is higher that arbitrarily chosen level (for example,
higher than 1%) prenatal examination of karyotype is recommended.
Some abnormalities of the fetus, which are noted upon ultrasound
examination may be another indication for prenatal cytogenetic
diagnosis. The examination may be necessary also for the families
where one of the parents is a carrier of a balanced structural
chromosomal rearrangement – translocation, inversion, insertion or
any complex rearrangement.
There are several ways to obtain cells, identical to fetal cells.
The most known test to obtain cells at early stage (~10–11 weeks)
is chorionic villus sampling. Under the control of ultrasound the
special instrument is inserted via uterine cervix or thorough the
abdominal wall. A small piece of placenta with growing chorionic
villi is taken for analysis. Short term cultivation is usually
Amniocentesis is a predominant way to obtain cells for prenatal
diagnosis. Small amount (5–10 ml) of amniotic fluid is taken from
the amniotic cavity via transabdominal amniocentesis. This
procedure is usually performed at 14–17 weeks of pregnancy. Amniotic
fluid has plenty of amniotic cells. After centrifugation almost all
amniotic cells are concentrated at the bottom of the tube. ~1 ml of
suspension from the bottom of the tube is placed on the cover slides
in the small Petri dishes. A special medium is added to facilitate
growth of amniotic cells. After a short-term cultivation (usually
6–7 days) the cells are ready for analysis. A cytogeneticist
counts ~20 cells at least from 2 flasks and karyotypes several cells.
In some centers the cytogeneticist looks on the cells through the
microscope, other centers prefer automatic analysis, when the
cytogeneticist looks on the screen of the special computer designed
for the selection and analysis of metaphases. There is photographic
documentation for every studied person. The results are provided to
the patient and (if the results show a chromosomal disorder) the
family may decide to continue pregnancy or to terminate it.
Technically amniocentesis may be performed also in a more advanced
pregnancy. However amniotic cells obtained after 22 weeks had worse
growth potentials (than amniotic cells at 14–17 weeks). If
karyotype at late pregnancy became really necessary samples of fetal
blood may be obtained by puncture of fetal umbilical cord (under
guidance of ultrasound).
Practically, prenatal cytogenetic diagnosis is a very good method
to reduce numerical abnormalities, mostly trisomies. Its role in
detection of chromosomal disorders, caused by structural abnormalities
is far less, because most women pregnant with fetuses having structural
chromosomal defects are young and do not have biochemical indications
for amniocentesis. The only (but very important) exceptions are families
with structural chromosomal abnormalities in one of the parents. In
these families prenatal diagnosis of the karyotype may be crucial
for decision about fate of the pregnancy. Actually, the last group
of families may benefit from preconceptional diagnosis. This method
(or better these methods) may allow selection of normal egg–cells
for further fertilization in vitro and implantation of the embryo
with already known karyotype. If a balanced rearrangement (usually
translocation) is found in a father, his sperm cells are used for
simultaneous fertilization of several egg–cells with karyotyping
of the very early pre–implantational embryo and implantation
of the embryo having normal karyotype. In that case the family does
not have to decide fate of unborn fetus. However, there are many technical
limitations regarding usage of these methods.
Post–natal cytogenetic diagnosis is based in vast majority of
situations on examination of the lymphocytes of the peripheral blood.
Cells of the peripheral blood are mature cells, they grow and divide
in the bone marrow, spleen and lymphatic nodes. Adding of specific
stimulator phytohemagglutinin (PHA) is necessary to obtain division
of lymphocytes, obtained from peripheral blood. Small amount of blood
(less than 1 ml) mixed with PHA and special medium is cultivated in
thermostat at 37°C during 72 hours. After it the obtained suspension
of dividing cells is treated by Colchicine, which blocks cellular
division. Hypotonic solution is added to provide better spreading
of chromosomes on the slides. Special staining allows visualization
of the chromosomes as structures having an individual pattern of
distribution of dark and light bands. Further steps (analysis itself)
are basically the same as in analysis of amniotic cells for prenatal
However, the standard (visual) cytogenetic analysis does not allow
recognition of small deletions or duplications. Even in ideal technical
conditions level of recognition is about 5-6 millions of base pairs (Mb).
Practically, however, deletions or duplications less than 10 Mb hardly
may be recognizable. Fluorescence in situ hybridization (FISH) is a
method, which may improve quality of cytogenetic diagnosis in patients,
where some structural abnormalities may be suspected. There are probes
to some specific segments of DNA. These probes are tagged by fluorescent
stains. In normal condition the person will have two areas of hybridization
(2 hybridization spots) on the homologous chromosomes. When the patient
has a hybridization spot only on one of the homologous chromosomes
it means that this segment of DNA on the other homologous chromosome
is lost. Vice versa, three spots of hybridization may indicate evidence
of a duplication of this segment of DNA. This method may be used
also for the study of undivided (interphase) cells, obtained, for
example, from a buccal smear (or uncultivated amniotic fluid). Practically,
FISH may be used for exclusion (or confirmation) of trisomies or relatively
frequent deletions, for example del 22q11.2, which causes diGeorge
syndrome or del 7q11.23, which causes Williams syndrome. Limitations
of FISH examination are obvious: a) if you have normal results with
probes “a”, “b” and “c” it means
that a patient does not have deletions or duplications for these regions,
but does not exclude abnormalities for regions “d” and
“e”, which have not been tested; b) FISH does not give
precise coordinates of the deleted segment.
Sometimes, the patient may have mosaicism: the condition, when he/she
has several clones of cells with different chromosomal complement.
Mosaicism is very common for numerical anomalies of sex chromosomes,
but not so common for autosomal trisomies and for structural chromosomal
abnormalities. The methods of cytogenetic examination for diagnosis
of mosaicism are the same but number of studied cells should be increased.
Usually the number of cells with different karyotypes is shown in brackets
after the standard formula. For example, the formula 47,XX,+21 /46,XX 
means that the patient have mosaic trisomy 21 with trisomy in 80% of cells.
There are some rare conditions, where an abnormal karyotype may be
found predominantly (or even exclusively) in fibroblasts, whereas
the lymphocytes show a normal karyotype. This situation is typical
for mosaic tetrasomy 12p (Pallister–Killian syndrome) and frequent
in some “rare” trisomies. Skin biopsy and cultivation
of skin fibroblasts may be necessary for cytogenetic examinations
to confirm (or exclude) these syndromes. FISH examination of interphase
cells using probes for 12p may facilitate diagnosis of Pallister–Killian
The ultimate goal of all these methods is diagnosis of constitutional
(inherited) chromosomal abnormalities. Structure of chromosomes may
be changed in various tumors. The methods for examination of these
acquired chromosome abnormalities are out of our scope.