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Anatomy of a chromosome: centromere, p arm, q arm, telomeric regions

G banding pattern of staining
Image of chromosome 12 shows different banding resolutions

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Deletion: can be interstitial or telomeric
Insertion: rarely involves the centromere
Inversion: paracentric: does not include the centromere, pericentric: includes the
centromere
Translocation: reciprocal, balanced, unbalanced, between both of the same
chromosome t(16;16)
Whole chromosome: trisomy, monosomy, tetrasomy (technically these are
constitutional terms, but they are still used)
Dicentric: two centromeres are present on one chromosome
Rings: loss of telomeric ends, makes the chromosome sticky and the ends join
forming a ring (18 and 19 like to do this)
Isochromosomes: loss of a p or q arm. The remaining arm is duplicate and the
chromosome now has two p arms or 2 q arms on top of each other

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MLPA can do balanced translocations, but you have to know what you are looking for
before testing and also, not many probe sets available for different translocations

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Metaphase at different resolutions

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Left: ALL near haploid
Right: t(9;22) with -7

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Poor morphology typical in ALL even though this is not an ALL.

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Left: dicentric chromosome 8
Right: ring 18

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NOTE: Different labs will culture, harvest, drop and stain differently. However, they
are all based on the same premise and are validated to achieve consistent results
across laboratories.
RPMI = commercially sourced media with nutrients and ability to mimic the environ
of the human body
FBS = Fetal Bovine Serum. Source of protein for the cells. Low antibody level and high
in growth factors.
Hepes = helps keep the pH at a stable rate. Important for replicating human body
environ
Gentamicin = inhibits growth of contaminants.

The stimulated cultures are to produce/enrich as many of the cells of interest as

possible. You will get the occasion where the 24hr unstimulated culture produces
abnormal clones. However, it is more likely to produce abnormal clones in the
stimulated culture. But, in cases of undifferentiated leuks it can go either way. We
have had cases where the unstimulated 24hr culture produces results and the
stimulated doesn’t. It is all about what cells survive in vitro and that can vary from
patient to patient. My point is, talk to your cytogeneticist to determine culture
requirements. If you don’t have a clear cut myeloid, lymphoid lineage then we can

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culture for both to cover all.

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FdU (fluorodeoxyuridine) and Uridine added ~2 hours after culture set up for 24hr
sync (or the afternoon after for 48hr sync) and halts the cell cycle at G1/S. BrdU
(bromodeoxyuridine) added ~16hrs after that
Cells are halted by FdU and Uridine around prophase and prometaphase. Release by
BrdU = maximum metaphase at harvest time = maximum metaphase cells for
analysis.
Sync cultures are harvest ~7-8hours after release to achieve maximum metaphase –
based on S phase approx. 5hrs, G2 phase approx. 3rs in cell cycle
24hr cultures can be harvested the morning after set up. They have no additives and
no stimulation so are not subject to time frames for harvest. It is the BM culture in
it’s natural state.

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Colchicine – prevents the mitotic spindle from forming
24hr cultures – 15mins colcemid
48 sync – 15mins
72o/IL2 – 10mins
96IL4 – 20mins
Lymph nodes – 20mins
Hypotonic time – 15mins

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The dropping action onto the slide further spreads the cell membrane. Hopefully
giving good metaphases on your slide.
Drying time is affected by humidity in the lab. Low humidity = drying will occur too
fast = moist towel. High humidity = drying will occur too slow = hotplate. This ensures
good spread for the metaphases > easier and better analysis.
Drying overnight is important to fix the metaphase and ensure that the trypsin when
banding doesn’t over eat away the light bands. It can be performed faster than
overnight if an urgent result is required, but it is not recommended in most cases due
to a loss of resolution and banding clarity.

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Giemsa = G-banding
FBS= Fetal Bovine Serum
Trypsin and staining times will depend on humidity levels on the day of slide dropping
Trypsin is an enzyme that will partially digest the euchromatin regions (gene rich) and
will not affect the heterochromatin (gene poor and densely wound chromatin
areas). Must be applied in a time frame to avoid over or underbanding. The action of
trypsin is negated by the addition of FBS that has protease inhibitors (a1 antitrypsin).
This stops the action of trypsin.

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NPAAC guidelines determine how many cells must be examined
Some constitutional changes may not affect the patient but are relevant when
analysing for malignancies. If it is in every cell and non-recurrent it will likely be
questioned whether its an incidental finding of a constitutional nature. But if we
know up front, it makes life easier! There are some constitutional changes that can
be similar to malignant changes and unless we know, we cannot determine which
one is which.

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Cryptic rearrangements are ones that appear different on the karyotype than what
would be expected for a standard presentation of the abnormality, but the underlying
changes produce the same result i.e. this is a cryptic 4 way translocation where the
fusion of BCR/ABL occurs but not in the usual way. Not all cryptic translocations can
be identified often due to the complexity of the changes. If a cryptic translocation is
suspected, additional testing will be required.

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Examples of complex karyotypes

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Mitotic index: The number of metaphases seen (or the slides required to reach the
necessary number of metaphases). Low mitotic index can be a result of culture
conditions, poor sample, low WCC. High mitotic index not usually an issue and is
often only seen in PHA (not done on BM only PB). The problem is usually the
opposite. Not enough metaphase for analysis.

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Double minutes identified by FISH as MYC amplification
Also, +6 and iso(17q) > loss of 17p

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Guess the abnormality

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Modal number is often just the number of chromosomes however, it is technically
the number of centromeres
A composite is where two cells may have minor differences but are not different
clones. Often happens in triploid/tetraploid cells where some have floated away.

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Inversions can be pericentric – includes the centromere. Paracentric does not. So,
paracentric will have both breakpoints on either the p or q arms and pericentric will
have a breakpoint on each arm
Intra del – does not involve the telomere only the region described. Terminal deletion
involves everything from the breakpoint to end of telomere

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Resolution for BM in haem malignancies is often <300-300
Karyotyping is quite old, everyone pretty much does it the same way, with some
variation but can be reliably reproduced in any cyto lab.

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Cytogeneticists also tend to refer to a karyotype as cryptic if nothing is detected as
abnormal. i.e. Maybe there is something cryptic going on? In this case, it means that
recurrent changes may be present but below the resolution detection limit of
karyotyping.

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