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174 Part IV: Molecular and Cellular Hematology Chapter 13: Cytogenetics and Genetic Abnormalities 175

successfully. An involved lymph node or tumor mass specimen may be the most frequently used commercially available FISH probes. Several
examined for the analysis of lymphoma cells. types of probes can be used to detect chromosomal abnormalities by
For specimen collection, 1 to 5 mL of marrow are aspirated asep- FISH. Hybridization of centromere-specific probes has been used to
tically into a syringe coated with preservative-free sodium heparin and detect monosomy, trisomy, and other aneuploidies in both leukemias
transferred to a sterile 15-mL centrifuge tube containing 5 mL of culture and solid tumors, as well as the sex chromosome complement in the
medium (RPMI 1640, 100 units sodium heparin). The use of Vacutainer transplant setting (Fig. 13–1).
tubes containing heparin as an anticoagulant should be avoided, as the Translocations and deletions can also be identified in interphase
heparin contains preservatives that suppress cell growth. If a marrow or metaphase cells by using genomic probes that are derived from the
aspirate cannot be obtained, a marrow biopsy may be taken and placed breakpoints of recurring translocations or within the deleted segment
into the collection tube. Approximately 75 percent of marrow biopsies (see Fig. 13–1). In some cases, FISH analysis provides more sensitiv-
can be minced to generate suspension of cells that will yield adequate ity, in that cytogenetic abnormalities have been identified by FISH in
numbers of metaphase cells for complete analysis. For blood specimens, samples that appeared to be normal by conventional cytogenetic anal-
10 mL are drawn aseptically by venipuncture into a syringe coated with yses. Advantages of FISH include (1) the rapid nature of the method
preservative-free heparin. To avoid loss of cell viability, it is critical that and the ability to analyze large numbers of cells; (2) its high sensitivity
the specimen be transported at room temperature to the cytogenetics and specificity; and (3) the ability to obtain cytogenetic data from sam-
laboratory without delay. Overnight shipment of specimens frequently ples with a low mitotic index or from terminally differentiated cells. A
results in loss of cell viability, and most laboratories experience a high further increase in sensitivity in cases with a low percentage of malig-
proportion (25–50 percent) of inadequate analyses using such speci- nant cells can be achieved by performing FISH analysis on samples that
mens. For optimally handled specimens, approximately 95 percent of have been enriched previously for specific subpopulations of cells. For
all cases should be adequate for cytogenetic analysis. Those cases that example, the use of FISH in combination with plasma cell enrichment
are inadequate generally represent samples from patients with hypocel- techniques is routinely applied in the clinical setting to maximize the
5,6
lular marrows. detection rate of specific chromosome rearrangements in myeloma.
The major disadvantage of FISH testing is the inability to interrogate
CHROMOSOME NOMENCLATURE more than a few abnormalities. FISH is most powerful when the analy-
sis is targeted toward those abnormalities that are known to be associ-
Chromosomal abnormalities are described according to the Interna- ated with a particular tumor or disease. In a clinical setting, cytogenetic
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tional System for Human Cytogenetic Nomenclature (Table 13–1). To analysis could be performed at the time of diagnosis to identify the
describe the chromosomal complement, the total chromosome num- chromosomal abnormalities in an individual patient’s malignant cells.
ber is listed first, followed by the sex chromosomes, and numerical and Thereafter, FISH with the appropriate probes could be used to detect
structural abnormalities in ascending order. The observation of at least residual disease or early relapse, and to assess the efficacy of therapeutic
two cells with the same structural rearrangement, for example, translo- regimens. For example, the use of FISH to detect the t(9;22) in CML
cations, deletions or inversions, or gain of the same chromosome, or patients following therapy with an oral tyrosine kinase inhibitor, or sex
three cells each showing loss of the same chromosome, is considered chromosome determination after a sex-mismatched transplant, is wide-
evidence for the presence of an abnormal clone. However, one cell with spread. Material from patients newly presenting are often analyzed most
a normal karyotype is considered evidence for the presence of a normal efficiently by conventional cytogenetic analysis, combined with quanti-
cell line. Patients whose cells show no alteration or nonclonal (single tative reverse transcriptase polymerase chain reaction (qRT-PCR) anal-
cell) abnormalities are considered to be normal. An exception to this is ysis if a specific chromosome rearrangement is suspected, for example,
a single cell characterized by a recurring structural abnormality. In such a BCR-ABL1 fusion. Molecular qRT-PCR monitoring of the blood and
instances, it is likely that this represents the karyotype of the mutated marrow of CML patients is now part of the recommended testing for
subclone in that particular patient. patient followup. 7

METHODS THAT COMPLEMENT MICROARRAY ANALYSIS
KARYOTYPE ANALYSIS Several microarray-based technologies play an important role in the
diagnosis and experimental analysis of hematologic malignancies,
FLUORESCENCE IN SITU HYBRIDIZATION including high-density copy number/single nucleotide polymor-
Cytogenetic analysis of human tumors is often technically difficult phism (SNP) array testing (also known as chromosomal microarray
because of the presence of multiple abnormalities and requires highly analysis [CMA]), microarray-based gene expression profiling, and
skilled personnel. These factors have led investigators to seek alterna- high-throughput SNP genotyping. CMA allows genome-wide detec-
tive methods for identifying chromosomal abnormalities, such as fluo- tion of copy number abnormalities (deletions and duplications) at a
rescence in situ hybridization (FISH). The FISH technique is based much higher resolution than karyotyping; it also enables detection of
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on the same principle as Southern blot analysis, namely, the ability of loss of heterozygosity (LOH) that occurs without concurrent changes
single-stranded DNA to anneal to complementary DNA. FISH can be in the gene copy number, that is, uniparental disomy (UPD) (Chap. 10),
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performed on marrow or blood films, or fixed and sectioned tissue, as and can be attributed to somatic mitotic recombination (also referred
it does not require dividing cells. The target DNA is the nuclear DNA to as copy-neutral LOH) (Fig. 13–2). CMA is clinically available as
of interphase cells, or the DNA of metaphase chromosomes that are an adjunct test to karyotyping and FISH, and it facilitates detection
affixed to a glass microscope slide. Commercial probes are now avail- of genomic abnormalities in a substantial proportion of patients with
able for the most common abnormalities, and are directly labeled with myelodysplastic syndromes (MDSs) and leukemia with a normal kar-
fluorochrome, which simplifies the technique by eliminating the probe yotype; it can also be used as a cost-effective alternative to large panels
preparation and detection steps. With the development of dual- and of FISH probes, and as a very useful tool to characterize chromosomal
triple-pass filters, most laboratories now have the capacity to hybridize abnormalities of uncertain significance. Microarray-based gene expres-
and detect two to three probes simultaneously. Table 13–1 summarizes sion profiling has been applied to study a variety of hematopoietic

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