Myeloid Leukemia

Deletion of Derivative Chromosome 9 in CML 111
The following method is based on the FISH method described in Chapter 2,
with particular reference to its use in CML cases.
2. Materials
1. Chromosome suspension produced according to the protocol outlined in Chapter
2. A cell suspension derived from any of the three cultures described would be
suitable for FISH (see Note 1).
2. BCR/ABL dual color dual fusion translocation probe (see Note 2).
3. 9q34 aqua-labeled probe (see Note 2).
4. Reagents outlined in Chapter 2 for use in FISH studies.
3. Methods
In all newly diagnosed cases of CML, FISH should be performed following
the method outlined in Chapter 2, using a BCR/ABL dual-color dual-fusion
translocation probe (see Note 3).
When FISH slides are prepared for analysis, at least 10 metaphase spreads
and/or 50 interphase cells should be scored (see Note 4). All FISH slides should
be scored by two individuals and representative images captured via an image
analysis system or photographed and stored.
A deletion of the der(9) is indicated by the presence of only one fusion signal
per cell, located on the der(22) or Philadelphia chromosome. The second
fusion signal that would normally be found on the der(9) is not present. The
karyotype should be expressed according to the FISH nomenclature outlined in
ISCN (1995) (13) (see Note 5).
To use FISH to look for MRD in a case with a demonstrated deletion of the
der(9), add a 9q34 aqua-labeled probe to the probe/hybridization buffer mixture
(see Subheading 3.3., step 4 in Chapter 2). The combined probe/buffer
mixture consists of: 1 µL BCR/ABL dual fusion probe/1 µL 9q34 probe/1 µL
purified H 2O/7 µL hybridization buffer (see Note 6).
Proceed through the subsequent FISH steps according to the protocol in
Chapter 2.
Cells should be scored, looking specifically for BCR/ABL (red/green) fusion
signals. When assessing MRD, it is preferable to score at least 200 to 300 interphase
cells. Only when a BCR/ABL fusion signal is identified, is it necessary to
score the additional aqua 9q34 probe. If the cell contains a true fusion signal
(instead of an incidental co-localization of ABL and BCR signals), the deletion of
the der(9) that has resulted in only one fusion signal being seen will also result in
the deletion of one aqua 9q34 signal. Thus, a cell containing the t(9;22) will
contain one fusion signal/one red (ABL) signal/one green (BCR) signal/one aqua
(9q34) signal, whereas a false-positive cell will contain one fusion signal/one red
(ABL) signal/one green (BCR) signal/two aqua (9q34) signals (see Note 7).