Future prospects for flow cytometry

The world of flow cytometry is rapidly evolving. During the past few years, there have been significant changes regarding the qualifications for personnel credentialed to perform flow cytometry, assays with revived clinical applications, more elegant fluorochrome development, and the size and sensitivity of instruments. There have also been many developments in reagents, instruments, and applications.

Staff credentials

Clinical flow cytometry laboratories now have an extended pool of expertise available thanks to the development of the Specialist in Cytometry Exam administered by the Board of Certification (BOC) of the American Society of Clinical Pathology (ASCP). The BOC had offered the “qualification in cytometry” (QCYM) designation for many years. In 2015, principal investigators and managers at cytometry core facilities were concerned about the retirement of personnel with extensive expertise. Development of the cytometry qualification exam occurred via collaboration among the International Society for Analytical Cytometry (ISAC), the International Clinical Cytometry Society (ICCS,) and the European Society for Clinical Cell Analysis (ESCCA), and sponsorship by the Wallace H. Coulter Foundation.

Successful qualification and completion of the International Clinical Cytometry Exam (ICCE) led to the credential “Certified Cytometrist” (C.Cy.). Continuing education was required to maintain the certification. In 2016, the original ICCE stakeholders agreed that the BOC would administer the exam. Individuals with ICCE or QCYM credentials became Specialists in Cytometry (SCYM). Highly skilled individuals from cytometry core facilities now have the credential necessary for cytometry positions within the hospital laboratory.

The return of cell cycle analysis

DNA ploidy and cell cycle testing of tumors could return to the clinical laboratory. Krishnan first published this method in 1975¹ using propidium iodide. Today, most solid tumor DNA testing tends to be performed using molecular DNA techniques. The current literature indicates the importance of tumor cell-cycle when selecting or designing clinical therapeutics.^2-5 At GLIIFCA 2016 [Great Lakes International Imaging and Flow Cytometry Association], Hedley presented data showing numerous, different mutated populations within a single pancreatic tumor.^6,7 Because each population may respond differently to various treatment options, accessing the molecular characteristics of each population could enhance clinical treatment and prognosis. The populations were isolated based upon ploidy and S-phase fraction. Therapeutic combinations targeting the different populations within a multivariate tumor could provide better outcomes.

Engineered fluorochromes

Polymer chemistry has developed several fluorochromes. A common example is composed of several light harvesting base polymers that transfer energy via fluorescence resonance energy transfer (FRET) to a reporter dye attached to the polymer.⁸ These 405 nm excitable polymer dyes have six increasingly longer emission wavelengths dependent upon the polymer construction, starting at 421 nm and continuing up to 786 nm.⁹ A new fluorochrome strategy constructs large organic molecules into a “cage” bound by platinum molecules.¹⁰ Specific molecular assembly strategies tune the resonance frequency and fluorescence emission of the cage. Unlike most fluorochromes used today, these structures do not photobleach.

Smaller and more powerful instruments

The development of more sensitive fluorescence detectors has made smaller instrument footprints possible. This change has been occurring through the developmental history of flow cytometry instrumentation. There are clinical flow cytometry models that have the footprint of a standard laser printer. In the clinical laboratory, these smaller instruments usually have fewer lasers and fluorescence detectors.

One current research instrument has three lasers and 13 fluorescence detectors and is the same size as less powerful instruments. One technological advance used in this instrument is the avalanche photodiode detector. These detectors are more sensitive than other photodiodes, principally due to a built in electronic gain. Similar to a typically forward scatter photodiode, only the voltage changes. The avalanche photodiodes can detect emission signal as well as the standard photomultiplier tube but are as little as one-tenth the size of a photomultiplier tube.

REFERENCES

1. Krishnan A. Rapid flow cytometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol. 1975;66(1):188-193.
2. Desjobert C, Mai ME, Hime-Gerard T, et al. Combined analysis of DNA methylation and cell cycle in cancer cells. Epigenetics. 2015;10(1):82-91.
3. Jin J, Lin G, Huang H, et.al. Capsaicin mediates cell cycle arrest and apoptosis in human colon cancer cells via stabilizing and activating p53. Int J Biol Sci. 2014:10(3):285-295.
4. Song X, Zhang Y, Wang X, et al. Casticin induces apoptosis and G0/G1 cell cycle arrest in gallbladder cancer cells. Cancer Cell Int. 2017;17:9. doi: 10.1186/s12935-016-0377-3.
5. Erhardt H, Wachter F, Grunert M, Jeremias I. Cell cycle-arrested tumor cells exhibit increased sensitivity towards TRAIL-induced apoptosis. Cell Death and Disease 2013;4:e661. doi: 10.1038/cddis.2013.179.
6. Hedley D. Studying complex biology in solid tumors. Presentation at the Great Lakes International Imaging and Flow Cytometry Association. September 2016.
7. Chang Q, Chandrashehar M, Ketela T, Fedyshyn Y, Moffat J, Hedley D.
   Cytokinetic effects of Wee1 disruption in pancreatic cancer. Cell Cycle. 2016;15 (4):593-604.
8. Abrams B, Diwu Z, Guryev O, Suni M, Dubrovsky T. New violet-excitable reagents for multiple color flow applications. Cytometry 2013. 83A (8):752-762.
9. Chattopadhyay P, Gaylor, B, Palmer A, et al. Brilliant violet fluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments . Cytometry, 81A: 456–466. doi:10.1002/cyto.a.22043.
10. Yan, X, Cook, T, Wang, P, Huang, F, Stang, P. Highly emissive platinum (II) metallacages. Nature Chem. 2015;7:342-348. doi:10.1038/nchem.2201.

Susan A. McQuiston, JD, MT(ASCP), CCy is a member of the MLO Editorial Advisory Board. She’s currently a faculty member in the Biomedical Laboratory Diagnostics Program at Michigan State University, teaching both the undergraduate and graduate level. She has extensive flow cytometry experience in academic research and private industry.