Ancillary Testing: IHC, Flow Cytometry, Cytogenetics and Molecular
Core concepts
Understand the indications, limits, and interpretation of ancillary tests in pediatric pathology: immunohistochemistry for lineage (CD45, desmin, myogenin, WT1, synaptophysin), flow cytometry for leukemia/lymphoma immunophenotyping and minimal residual disease, cytogenetics (karyotype, FISH) and molecular testing (PCR, sequencing, microarray). Know when each is appropriate-FISH for rapid targeted loci, karyotype for structural/ploidy, microarray for copy-number and congenital anomalies.
Grasp preanalytic variables: tissue handling, fixation, decalcification, and their effects on IHC and molecular yield. Understand test performance concepts-sensitivity, specificity, positive/negative predictive value, and the effect of prevalence.
Traps
Questions conflate diagnostic vs prognostic vs predictive markers. Another trap: assuming FISH excludes all abnormalities-it targets specific loci. Do not forget that flow cytometry requires viable cells, so necrotic or heavily fixed tissue may fail. Microarray detects copy number, not balanced translocations.
Worked example / rule of thumb
A suspected Ewing sarcoma with equivocal morphology: IHC (CD99, NKX2.2) plus molecular confirmation of EWSR1 rearrangement. Rule of thumb: "FISH = targeted; karyotype = genome-wide structural; microarray = copy number; sequencing = sequence."