For over 20 years, clinical flow cytometry has been a mainstay in the diagnosis and prognosis of hematological neoplasms. Precise immunophenotyping of a patient by flow cytometry at diagnosis and throughout subsequent stages of chemotherapy conveys invaluable information to clinicians on the specific type, status, and resolution of the disease. The World Health Organization (WHO) sets the minimum diagnostic criteria for hematological neoplasms in an effort to ensure that patients embark on the proper treatment plan. However, while these criteria are used worldwide, there is a lack of standardization in diagnostic protocols and in the interpretation of diagnostic information. Despite proposals for the use of standardized panels and markers in flow cytometric immunophenotyping, many levels of subjectivity continue to exist, resulting in suboptimal diagnostic and prognostic outcomes.


Let it Flow graphic_small
Designed by Catherine Schrankel.

Flow cytometry antibody panel design and analysis of the acquired data give pathologists several opportunities to introduce bias. Subjectivity of panel design can first be introduced when a pathologist consults the literature to determine the number of markers required to make an accurate diagnosis. With more than 100 interchangeable diagnostic markers that can be used to accurately characterize hematopoietic malignancies, pathologists can choose what they believe is the best combination. Furthermore, the decision of which markers to use could be driven by criteria other than disease biology. When choosing “backbone” markers, or the minimal markers required to compare lineages between panels, decisions may be based on fluorochrome conjugate availability and compatibility, rather than on the pure diagnostic value of each marker. Variability in the type of flow cytometer and lasers available can also lead to different panel designs between institutions, as the unique set of fluorescence channels in each machine directs the combinations of fluorochromes available for diagnostics. Finally, after panel design and acquisition, how the data is analyzed and interpreted can also be entirely subjective. In light of these issues, many data scientists have called for the design of algorithms for analyzing n-dimensional data to ensure that diagnosticians can accurately distinguish disease phenotypes.

Attempts to standardize panels and protocols for diagnostic flow cytometry have been carried out for multiple hematopoietic diseases, including lymphomas, leukemias and myeloproliferative disorders. These attempts have focused almost entirely on biological marker choice and usually fail to address the most appropriate fluorophore combination of said biological markers for immunophenotypic diagnosis. One exception to this pattern is the EuroFlow consortium, an independent group of 20 European diagnostic research groups, which over a period of six years identified key markers and the combinations of their respective antibody clones and fluorochrome conjugates for use in multicolour panels. They also tackled issues encompassing instrument settings and data analysis. While this is arguably the most comprehensive attempt to standardize the flow cytometric diagnostic process to date, their proposed panels and settings have not yet been readily adopted.

Another way to address the inherent subjectivity in flow cytometry diagnostics may be to look towards alternative multi-parameter analysis platforms, such as mass cytometry (known as CyTOF). CyTOF is a single cell analysis technique that combines the cellular analysis principles of flow cytometry and the precision and quantitative power of mass spectrometry. It relies on antibodies coupled to rare earth metals of distinct molecular weight to interrogate dozens of markers in one staining tube without worrying about fluorochrome-conjugated antibody compatibility and spectral overlap. While this could eliminate some of the bias associated with flow cytometry, CyTOF still has unique technical issues of its own, such as the non-specific loss of 30-40% of cells prior to vaporization, which could affect diagnostic processes aimed at enumeration of malignant cells. Once the barrier to entry is lower for this burgeoning new technology, mass cytometry may be the answer to the issue of standardization on an international level.

Investigators seeking better definitions of disease and improvements in diagnosing these diseases drive the field of hematological diagnostics forward. While this drive is good for discovery and technical advancement, subjectivity and variability in panel design should not be present in diagnostic laboratories. Standardization would narrow down the reagents and instrument settings appropriate for use, streamline costs, and lend further confidence to diagnoses, allowing for prognoses that are more accurate. This will ultimately lead to better care for patients suffering from hematopoietic neoplasms and other diseases in which pathologists employ flow cytometry as a diagnostic tool.

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Mark Gower

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