Table 3. Advantages, disadvantages and use of different cytogenetic and molecular testing modalities.

Test	Advantages	Disadvantages	Sensitivity	Best used for
Karyotype	Available in most academic centers; reasonable turnaround time (5–10 days); provides information about all chromosomes present within a cell; easily identifies complex karyotype sarcomas; relatively cheap	Requires fresh tissue; technically demanding assay to set up and perform; dependent on culture and growth of malignant cells; low resolution: ~10 Mb; complex, small or cryptic rearrangements may be missed; provides no information on mutations	5–10%	Identifying large chromosomal rearrangements as well as large gains and losses of genomic material
FISH	Available in most academic centers; can be performed on FFPE samples; rapid turnaround time (3–5 days); alternative FISH modalities, such as multicolor FISH, may provide additional information on specific rearrangements; probes for numerous genes available	Generally, only used to target specific chromosomal alterations; can be technically difficult to interpret signals; relatively low resolution: 200 kb; misses CN-LOH; provides no information on mutations	1–10%	Confirmation/identification of genetic alterations when a specific diagnosis is suspected, particularly rearrangements, deletions and amplifications
Array	aCGH and SNP array available in most cytogenetic laboratories; SNP array can identify CN-LOH; can identify specific areas of gains and losses; relatively fast turnaround time (5–10 days)	Relatively low resolution: 10–100 kb; Cannot detect balanced rearrangements; provides no information on mutations; fresh tissue is preferable, analysis of degraded (such as FFPE) samples is difficult	15–20%	Identifying gains and losses of genomic material, identifying CN-LOH, and identifying unbalanced rearrangements
PCR	Relatively easy assay to set up and perform; equipment widely available; fast turnaround time (3–7 days); works well on FFPE samples; highly sensitive; high resolution: down to 1 nucleotide	Most assays interrogate only specific suspected alterations (although assays such RACE circumvent this); primers must be designed to cover specific areas of interest; RT-PCR assays require RNA which can be difficult to work with and degrade easily	Most assays <1%	Confirmation/identification of rearrangements or mutations when a specific diagnosis is suspected
Sequencing	Older generation sequencing technologies (Sanger, Pyrosequencing) slowly being replaced by NGS; NGS can interrogate tumors for multiple different genetic abnormalities depending on the panel used; high throughput: generates millions of sequencing “reads”; can easily be performed on FFPE samples; high resolution: down to 1 nucleotide	Longest turnaround time (1–4 weeks depending on laboratory workflow); Analysis (NGS) requires complex bioinformatics pipelines to be available; Analysis requires specially trained personnel to interpret sequencing data; NGS equipment and reagents currently very expensive; NGS Primarily only available at larger academic centers and reference laboratories	Sanger ~20%; Pyroseq ~1%; NGS <5%	Analyzing a tumor for various genetic abnormalities including mutations, rearrangements, and copy number alterations

FISH, fluorescence in situ hybridization; aCGH, array comparative genomic hybridization; SNP, single nucleotide polymorphism; RT-PCR, reverence transcriptase-polymerase chain reaction; PCR, polymerase chain reaction; NGS, next generation sequencing; Pyroseq, pyrosequencing; RACE, rapid amplification of cDNA ends; Mb, mega base pair; Kb, kilo base pair; bp, base pair; CN-LOH, copy neutral loss of heterozygosity; FFPE, formalin fixed paraffin embedded.