Immunophenotyping, molecular pathology and tumour genetics
Key Takeaways
IHC, flow cytometry and molecular assays answer complementary questions and require clinical–morphological correlation.
Scant or degraded vitreous material can make a negative lymphoma test inconclusive.
Somatic tumour variants and germline inherited variants have different implications.
HLA-A*02:01 is a specific tebentafusp eligibility requirement, while prognostic testing estimates metastatic risk.
A marker is evidence, not a diagnosis by itself
Immunohistochemistry (IHC) uses antibodies to identify antigens in tissue. Flow cytometry characterizes antigen expression in suspended cells. Molecular tests identify genetic or other nucleic-acid changes. Each addresses a different question, and the final diagnosis integrates morphology, phenotype, molecular findings and the clinical presentation. A single positive marker should not override an incompatible tissue pattern.
Review the suspected lineage before choosing a panel. Cytokeratins support epithelial differentiation, melanocytic markers support a melanocytic phenotype, and lymphoid markers help classify lymphoid populations. These are not perfectly exclusive labels. Controls, staining location, intensity and the distribution among cells affect interpretation. Small samples and prior treatment can limit the result.
Lymphoid assessment
Lymphoma evaluation distinguishes reactive lymphoid tissue from a clonal malignant process and then identifies the subtype. B-cell and T-cell markers, light-chain patterns and proliferation markers may be combined with architecture and molecular studies. Flow cytometry can detect abnormal populations when enough viable cells are available, but a negative result from a scant or degraded sample does not exclude lymphoma.
For suspected vitreoretinal lymphoma, coordinate acquisition and rapid transport with pathology. Cytology, immunophenotyping, molecular tests and cytokine analysis can be complementary. An IL-10 predominance may support the diagnosis in context, but inflammation and sampling limitations prevent treating a ratio as a universal standalone rule. A MYD88 variant can support selected B-cell lymphoma diagnoses but does not replace interpretation of the specimen and systemic context.
Recent corticosteroids may reduce cell yield or alter appearances. When clinically safe, the diagnostic plan should account for this, but do not impose an arbitrary drug-free interval on every urgent patient. Repeat or alternative sampling may be needed after a nondiagnostic result. Evaluate central nervous system involvement through the relevant team rather than assuming an ocular-only disease.
DNA, RNA and cytogenetic methods
Polymerase chain reaction (PCR) amplifies a targeted nucleic-acid sequence. It can assist pathogen detection, clonality or selected variant analysis. A positive pathogen signal may represent contamination or a finding requiring clinical correlation; a negative test can reflect timing, low organism load or an inadequate sample. Test selection should follow the suspected disease and the laboratory's validated assay.
Fluorescence in situ hybridization (FISH) examines specified chromosomal targets. Sequencing can examine individual genes or wider panels, while expression profiling characterizes transcript patterns. These techniques differ in resolution and purpose. A negative targeted test does not exclude a genetic change outside the region assessed. Explain the test's limits when giving a patient or family a result.
| Technique | Main question | Limitation |
|---|---|---|
| IHC | Tissue antigen phenotype | Marker overlap and technical artifacts |
| Flow cytometry | Cellular populations and aberrant phenotype | Needs appropriate cells and transport |
| PCR | Selected pathogen, clonality or variant | Targeted assay and sampling dependence |
| FISH | Specified chromosomal alteration | Does not survey every possible variant |
| Sequencing | Genetic variants in examined regions | Coverage and interpretation limits |
| Expression profile | Prognostic or biological pattern | Assay- and tumour-specific validation |
Uveal melanoma and prognostic testing
Uveal melanoma biology differs from cutaneous melanoma. Chromosomal and molecular features, including chromosome 3 status and alterations involving BAP1, SF3B1 or EIF1AX, can inform prognosis in the appropriate setting. An expression class or molecular marker estimates risk; it is not a certainty that an individual will or will not develop metastasis. Sampling heterogeneity and technical adequacy also matter.
Differentiate somatic tumour testing from germline testing. A BAP1 alteration in a tumour does not automatically prove an inherited syndrome. Young onset, multiple relevant tumours or family history may prompt genetic counselling and a germline pathway. Consent should cover implications for relatives, uncertainty and how results may affect surveillance.
HLA typing has a treatment-selection role for tebentafusp: its European indication requires HLA-A*02:01-positive adults with unresectable or metastatic uveal melanoma. That eligibility marker is different from a general prognostic test. Treatment can cause cytokine release syndrome and requires oncology supervision and an appropriate monitoring setting.
Clinical application
A vitreous sample with few damaged cells and negative flow cytometry is inconclusive if clinical suspicion remains high. A tumour expression result indicating high risk calls for informed surveillance discussion, not a deterministic prediction of survival. A tumour-only genetic variant should not be described as inherited without germline evaluation. State what the test measures, whether the specimen was adequate and what decision the result changes.
Sources: RCPath ophthalmic reporting recommendations, RCPath lymphoma reporting and EMA Kimmtrak.
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