Limitations

Although flow cytometry provides valuable immunophenotyping information, it does have its limitations. Awareness of such limitations is important in interpreting flow reports and making a diagnosis. Flow cytometry should be used in conjunction with other diagnostic methods (e.g. morphology, immunohistochemistry, chromosomal karyotyping and molecular methods) for accurate diagnosis and risk stratification. Healthcare resources also sometimes limit the amount of information that can be gained from flow cytometry – resource-constrained approaches may be used to confirm a malignancy, rather than aid in high-resolution diagnostic classification.

Pre-Analytical Issues

Panel Selection

Flow cytometry can only provide immunophenotyping information if the correct antibody panel is applied. For example, in patients with diffuse large B cell lymphoma, a panel designed for characterisation of acute myeloid leukaemia would be entirely useless, as it would not contain CD20, CD19, surface light chains or other B cell markers. Abnormal populations may be “missed” if the incorrect panel is applied.

It is hence important for the ordering clinician to be cognizant of this and either select the correct panel, or provide enough clinical information that the haematologist / pathologist in the flow lab can ensure that the appropriate panels are applied.

Sample Requirements

Flow cytometry requires fresh samples, processed as soon as possible following collection. Incorrect sample storage or storage for long periods can result in natural apoptosis of cells, reducing the specificity of flow cytometric analysis. Flow cannot be run on formalin-fixed tissue.

Apoptotic cells and cellular debris also causes non-specific antibody adsorption, which can lead to false positives.

Flow also lacks morphological correlation (in contrast with immunohistochemistry, where morphology can be observed simultaneously). Sampling errors such as aparticulate, haemodiluted bone marrow aspirates can hence influence the sensitivity of flow cytometry and result in reports that are not representative of the actual bone marrow microenvironment.

Technical Issues

Like all laboratory investigations, technical issues may reduce the quality of analysis. Flow cytometry equipment is complex, and requires trained operators. Potential areas for error include:

• “Wrong blood in tube” errors – e.g. mislabeled samples.
• Operator issues:
  • Antibodies not added or incorrect quantities added.
  • Incorrect antibodies added / selected.
  • Cells not washed adequately.
  • Inadequate cytoplasmic permeabilisation.
  • Inadequate RBC lysis.
• Reagent issues
  • Expired reagents.
  • Contaminated reagents.
  • Wrong reagents selected.
• Antibody and fluorochrome issues.
  • Spectral overlap (see Fluorescence section).
  • Drug-treatment leading to reduced antigen expression (e.g. rituximab treatment leading to lack of CD20 on B cells).
  • Antibody binding blocking antigen-antibody binding on adjacent antigen sites (steric hindrance).
  • Autofluorescence.
  • Non-specific antibody / fluorochrome binding.

Analytical Issues

Data Interpretation

• No morphological correlation – especially in cases with aberrant phenotypes.
• Not excluding debris / doublets.
• Not recognising autofluorescence / non-specific binding.
• Incorrect gating strategy.
• Incorrect interpretation of immunophenotype.