Molecular Diagnostics and Cytogenetics
Technology map
PCR amplifies a target; real-time PCR quantifies it (and, with allele-specific probes, detects single-nucleotide variants). Sanger sequencing reads one amplicon; next-generation sequencing reads millions in parallel and is the tool for panels and whole exome. Southern blot detects DNA, Northern detects RNA, Western detects protein. FISH uses fluorescent probes on interphase or metaphase cells and detects specific loci; karyotyping shows the whole genome at roughly 5–10 Mb resolution; chromosomal microarray detects copy-number changes at kilobase resolution but not balanced rearrangements.
Nomenclature and classic translocations
t(9;22)(q34;q11.2) is the Philadelphia chromosome, BCR::ABL1, chronic myeloid leukemia (and some ALL) - targeted by tyrosine kinase inhibitors. t(15;17) PML::RARA defines acute promyelocytic leukemia and responds to ATRA/arsenic. t(8;21) RUNX1::RUNX1T1 and inv(16) are core-binding factor AML. t(14;18) IGH::BCL2 defines follicular lymphoma. t(11;14) CCND1::IGH is mantle cell lymphoma.
Quality and variant classes
Distinguish missense, nonsense, frameshift, and splice-site variants. Know the difference between analytical sensitivity (limit of detection) and clinical sensitivity, and know that contamination, PCR inhibitors (heme, heparin), and poor DNA quality cause false negatives. Always include positive and negative controls plus a no-template control.
Traps
Do not use karyotype to find a 200 kb deletion, and do not use microarray to find a balanced translocation. FISH is targeted - a normal FISH panel does not exclude a genome-wide abnormality.
Worked example: a young patient with pancytopenia, DIC, and promyelocytes on marrow → order PML::RARA testing. Prompt ATRA therapy is started before confirmation because the disease is a coagulopathic emergency.