The global burden of infectious disease, even before Covid-19 was staggering, with HIV/AIDS accounting for more than 4.5 million deaths per year. As we are all now fully aware, new microbial pathogens are emerging at an alarming rate. From 1940 to 2004, more than 335 new infectious diseases were described. Their emergence is believed to be driven by a combination of socio-economic, ecological, and environmental factors, fueled by poorly allocated global resources. As areas of high population densities, the adoption of poorly planned agricultural practices and antibiotic drug resistance continue to be established, the problem is only going to worsen.  We, therefore, need access to robust and sensitive tools for infectious disease diagnosis, and more comprehensive solutions to characterize the process of infection and disease epidemiology. 

Flow Cytometry Applications

In affluent regions, molecular techniques and flow cytometry are displacing many manual methods for infectious disease diagnosis and monitoring; but potential flow cytometry applications in infectious disease management are vast and largely untapped. This is somewhat due to the large expense of instrumentation, and the complex nature of flow cytometry.  The strategy to distill flow cytometry down to core component functions has driven amazing innovation around single cell analysis. Heavy and delicate laser systems have been replaced with light emitting diodes, and acoustic focusing is providing a valid path to eliminating the large volumes of sheath fluid required for traditional flow cytometry. Novel solutions are being developed by pioneers in the field such as PointCare Technologies who have developed flow cytometers that detect gold labeled CD4+ antibody binding, using simplified optical and detector systems that are both more robust and durable than traditional hospital flow cytometers. And now with open reconfigurable digital data acquisition systems becoming mainstream, data handling and processing is less arduous and can be automated. All in all, recent technological advances have resulting in the development of many streamlined flow cytometry systems for field medicine applications to tackle the global challenges that infectious diseases pose to public health. 

Success Stories- Wide-Spread Adoption of Flow Cytometry Solutions

One well publicized example of where flow cytometry has been widely adopted is with HIV diagnosis and monitoring. The CD45+ pan-leukocyte antibody has been readily applied to flow cytometry applications to provide robust absolute total white blood cell counts and white blood cell differentials. Similarly, CD4+/CD8+ ratios have been determined using flow cytometry to examine babies born to HIV-seropositive mothers. These types of screens are now replacing expensive HIV-1 DNA and RT-PCR analysis. Simplified flow is also compatible with HIV viral load-associated lymphocytes activation tests as well as antigen specific cellular immune response assays that rapidly diagnose active Tuberculosis in both HIV-negative and HIV-TB coinfected individuals.

But these applications are just the tip of a growing ice burg of clinical possibilities, and as the technology advances, the use of flow cytometry has now diversified well beyond lymphocyte profiling, into many areas of research and biological manufacturing.   

Attributes of Flow Cytometry for Infectious Disease Research

There are several key attributes of flow cytometry that make compelling arguments for its translation into infectious disease research and testing.

Sensitivity

By definition, flow cytometry is single cell analysis, meaning each cell within a sample is analyzed independently. Analysis like this at the cellular level provides access to discrete observations that are just not possible with bulk sample analysis. Flow cytometers can now detect single-fluorescent molecules on or in a cell, providing previously unattainable analytical insights. 

Throughput 

Although we traditionally think of flow cytometry as analyzing cells, the same approach can be applied to micro-bead analysis for multiplexed capture assays. Micro-bead assays utilize a suspension of microparticles, of different sizes and colors that are coated with different antigens or capture antibodies. Much like a sandwich, ELISA is used to capture and detect analytes in samples, distinct micro-beads can be designed to selectively capture and measure different analytes from a complex sample. This concept can be used to measure levels of various types of molecules, from cytokines and chemokines to targeted immunoglobulins, viral and bacterial antigens, and with so many bead size and color combinations, it is possible to design 100 plex assays.  Coupling this platform with molecular techniques has extended its application to include such techniques as clonal screening and single-nucleotide polymorphism analysis, providing high throughput, high complexity analysis, and maximize data from clinical samples. 

Statistics

Carefully designed flow cytometry panels can generate Big Data sets- with every cell/particle providing 18+ parameters of data, statistical analysis of this data can provide profound and valuable clinical insights. Flow cytometry therefore not only provides a qualitative clinical read-out, but also a quantitative read-out of many relevant biomarkers, and biomarker combinations. 

Reagent Availability

In recent years there has been a push towards the development of novel fluorophores and probe designs that can be applied to the measurement of different cellular components from DNA and RNA, to proteins, glycan structures, and membrane lipids, as well as provide signals of cellular function, metabolism and gene expression. This advancement of chemistry allows us to maximize the application of 2 or 3 laser flow cytometry systems and achieve high-content flow cytometry.   

Versatility

Flow Cytometry is incredibly versatile in that it can be optimized to analyze almost all types of cells, and subcellular particles such as micro-vesicles and exosomes. Such versatility enables it to be applied to many aspects of infectious disease research such as pathogen detection, characterization, enumeration, and functionality such as drug susceptibility and resistance, without the need for pathogen isolation and culturing. 

Applications of Flow Cytometry for Infectious Disease Research and Diagnosis. 

Clinical Microbiology

Traditional microbiology relies on the isolation and culturing of pathogens in the lab, this is both time-consuming and challenging and sometimes unsuccessful. However, there are many examples of methods employing flow cytometry to examine clinical samples for the presence of pathogens (Srikuman, et. al. 1992; McClelland et. al 1994; Huges et. al. 1996) and for even more diverse applications such as the testing of victims of a bioterror attack or exposure. This isn’t without challenges though- flow cytometry requires single cell suspensions and microbes are often sticky and form aggregates, therefore sample processing using surfactants and enzyme processes to preserve pathogen integrity but minimize aggregation may need to be employed.

Microbial Finger Printing 

Flow Cytometry fingerprinting is emerging as a powerful means of studying bacterial populations and discriminating different growth phases within a microbial culture. The ability to accurately enumerate and simultaneously assess different physiological parameters without subjective gating is achieved using a combination of dyes (SYBR green and PI) that when coupled with scatter signals generate a unique signature profile or fingerprint. This enables efficient screening of samples for rapid and comprehensive microbial characterization for research and biological manufacturing applications. However, this has yet to be translated to clinical applications.

Automation of Microbial-Flow Cytometry 

Automation of microbiology using flow cytometry will significantly promote its adoption in clinical settings for critical applications such as screening of urinary tract infections (Mejuto et. al. 2017), antimicrobial susceptibility testing, and treatment monitoring. In addition, to direct microbe detection, rapidly serology testing for immunoglobulin responses to infection are possible, and the ability of flow cytometry to be multiplexed enables the simultaneous screening for multiple microbial infection on a single sample. 

The Study of Pathogen-Host Cell Interactions 

Many aspects of the pathogen-host cell interaction can be evaluated using flow cytometry, and this single-cell approach provides much finer resolution and representative analysis of the host-pathogen interaction than traditional bulk analysis. Measurable parameters include the expression of cell surface receptors that are critical for pathogen invasion, as well as the immune response to the infection, and the triggering of apoptosis. Imaging flow cytometry has been particularly valuable in this area since it enables researchers to distinguish cellular location of the pathogen throughout the infection, as well as track the infection through therapeutic response. By enabling the examination of pathogen morphology, subcellular localization, and the co-localization of pathogens with cytoplasm organelles, we now have unprecedented capabilities to follow the life cycle of pathogens and detail the tipping points of the host-pathogen interaction, such as cell differentiation during biofilm formation and the mechanisms triggering sporulation.  This research has been made possible through the implementation of novel reagent advancement such as red-shifted fluorescent protein development as well as enhanced data analytical software (Haridas, et. al. 2017). 

Pathogen Detection. 

The direct detection of pathogens in blood is also an evolving area of technology development. The Los Alamos National Laboratory pioneered the application of flow cytometry for high sensitivity, DNA fragment sizing to provide a rapid DNA fingerprint of pathogens in clinical samples (Ferris et al 2004, Larson et al 2000). This process was shown to take minutes rather than days for the Gold Standard Pulsed Field Gel Electrophoresis method. The coupling of flow cytometry and mass spectroscopy analysis now provides unprecedented pathogen detection and identification. 

Another sample preparation technique that has accelerated pathogen detection and identification is the adoption of gel microdrop encapsulation, which also enables us to sort out microbes from clinical samples for genomic evaluation (Ryan et. al.1995).

Other application of these techniques includes food and water safety. According to CDC estimates, 1 in 6 people get food poisoning each year. The majority of these outbreaks are caused by 31 pathogens (viruses, bacteria, and parasites) with the top offenders being Norovirus and Salmonella. Traditional methods of food surveillance employ enumeration of bacteria through plate counting. This process takes several days and misses many microbes in an unculturable state (known as viable but not culturable, VBNC) that still may cause sickness. Modern food production requires more rapid screening techniques to keep up with the pace of food distribution. The flow cytometry-based solutions such as the RAPID-B platform are portable and simple to implement. Using flow cytometry analysis of a complex food matrix environment analysis can be completed within a 6-hour window and detect one single bacterial cell. Such real time results afford great improvements in product control and safety and provide the regulatory authorities with dramatically higher throughput and accuracy (Buzatu et. al. 2014). 

Flow Virometry

 At between ~10 to 300 nm in size, viruses are generally too small for directed measurement by traditional flow cytometry, since the signal from these particles can easily get lost into the background noise. However, the field of virometry is another area of rapid technology growth both in the resolution of the instrumentation and reagent development. These advancements enable single-molecule detection and researchers have been able to successfully enumerate and in some examples sort-out various viruses including adenovirus, respiratory syncytial virus, and influenza A virus (Ferris et. al. 2004), herpes simplex virus 1 (Loret et. al, 2012, El. Bilali et. al., 2017), human immunodeficiency virus (Arakelyan et. al. 2017 a and b; Bonar and Tilton. 2017)) and vaccinia virus (Tang, V. A., et. al. 2016). The processes use fluorescent nucleotide-binding dyes specific for the single-stranded viral DNA, coupled with viral antigen-specific monoclonal antibodies. 

The labeling processes that can be employed for viral labeling and Flow Cytometry Detection are summarized in Fig. 1   

Virometry Instrumentation.

There are 3 main adjustments that enable flow cytometry to be applied for the examination of viral particles: i. the power of the lasers, ii. the position and type of detectors used in the system, iii. the sheath fluid preparation. 

The power of the laser systems and the sensitivity and types of detectors used for standard flow cytometry are more optimized towards the interrogation of larger structures such as cells and microbeads rather than viruses. For the detection of small particles such as viruses, laser systems of 300mW or more are employed. These are around 10 times the wattage of standard instrumentation with typical powers of 10-20mW.

Similarly, in traditional flow cytometers, the detectors for forward light scatter are used to determine the particle size. The detectors are placed at angles of 0.5˚ to 1.5˚ allowing the differentiation between small and large cells &/or beads (Lippe, 2018). However, for virometry, the flow cytometer side scatter detectors are set to capture signal within a 15˚ to 70˚ range and block out the signal from 0˚ to 14.9˚ to markedly reduce the background noise signal. The type of detector system is also a key factor, high performance multiplier tubes provide better resolution of signal compared with photodiodes, however, with the adoption of avalanche photodiodes, even higher levels of detection are possible.

With greater particle detection and resolution, it is necessary to prepare the sheath fluid appropriately to reduce background noise. Tradition flow cytometry requires the sheath fluid to be filtrated through 0.22µm filters, flow virometry requires filtration through 0.1µm filters to remove any particulates. Additionally, the viral sample should be filtered through a 0.45µm filter before analysis. 

What virometry is currently lacking is standardization and reference standards, however, there is no doubt that it will bring great value to research into Covid-19 therapeutics and vaccines. 

Flow virometry has already proven its value to vaccine quality control in the case of vaccinia virus (Tang et. al. 2016) by enabling the characterization of diverse vaccinia virus preparations generated for their oncolytic activity and cancer vaccine potential. In this study, fluorescently labeled virion particles were analyzed and sorted by flow virometry to reveal an unexpected a high level of heterogeneity in viral particle size and ratios of infectious to noninfectious particles. It was also found that the virus tended to aggregate over time in these preparations, a characteristic that was influenced by the storage conditions. This demonstrated how flow virometry can be applied to the formulation and preservation of vaccines and represents and legitimate platform to evaluate the quality of vaccine preparations. 

Other applications of flow virology include the characterization of virus replication as part of the pathogen’s infectivity. Flow virology was able to achieve high purity preparations of C-capsids than traditional biochemical processes, and this was critical in determining the role of C-capsid production in the viral production and maturation, and how this relates to virus infectivity.

Applications of Flow Cytometry in Infectious Disease Epidemiology.

Accurate and rapid infectious disease diagnosis is the cornerstone of effective epidemiological management. One platform showing the most potential for addressing global infections is flow cytometry-based multiplexed immunoassays. Not only can this type of assay diagnose co-infections and distinguish different infections but has the capability of coupling this with immune monitoring, all with high sensitivity and specificity. Developments in instrumentation and reagents will soon enable us to perform these types of tests in the field, in a methodical and robust manner. What is now needed is standardization of the flow cytometry instrumentation, methods and reagents, along with the development of reference materials to ensure assay performance and a commitment by the global health organizations to invest in the development of flow cytometry-based solutions for a growing number of unmet medical needs. Examples of these types of developments are outlined in table 2 with the development of flow cytometry panels designed to distinguish infections with common symptoms. This is important for field medicine applications where certain symptoms such as rash or fever, may be caused by various underlying factors. Rapid diagnosis is critical for the control of infectious disease spread and support effective resource utilization in resource-limited regions. 

Table 2. Diagnosis and surveillance of infectious disease – targeted applications using flow cytometry (adapted from Lancet Infectious Disease Vol. 2 April 2002). 

Final Thoughts

Flow Cytometry as a technology has really come full circle; from the original impedance-based flow cytometry devices that employed the Coulter principal to count cells, flow cytometry rapidly evolved towards high complexity cellular characterization and cell sorting. Now researchers have recognized the value of distilling flow cytometry down to its core scientific principals as a way to expand its applications out of the hospital clinical testing lab and into more immediate, mainstream medical applications. Flow cytometry’s ability to provide plug and play screening solutions, within a robust and sensitive format, makes it ideal for field medicine and mobile testing. And now with technological solutions providing increased sensitivity and expanded detection ranges, flow cytometry holds huge potential for the enhancement and acceleration of microbial detection and screening across many applications of modern medicine. 

Now, with the current SARS-CoV-2 pandemic still very much a part of our lives, flow cytometry analysis is front and center of many drug and vaccine development initiatives. From serological testing, to cytokine screening of patients, the ability of flow cytometry to provide near real-time clinical profiles of the immune response to SARS-CoV-2 has proved critical in our understanding the disease. And now, with accelerated vaccine and therapeutic drug trials underway, flow cytometry analysis will continue to be at the frontline of these decisive safety and efficacy studies. 

Ryan, C., Nguyen, B.-T., Sullivan, S. J. (1995) Rapid assay for mycobacterial growth and antibiotic susceptibility using gel microdrop encapsulation. J. Clin. Microbiol. 33, 1720–1726.

Swaminathan, B., Barrett, T. J., Hunter, S. B. Tauxe, R. V. (2001) the CDC PulseNet Task Force PulseNet : The molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg. Infect. Dis. 7, 382–389.

Srikumar R, Chin A C, Vachon V, Richardson C D, Ratcliffe M J, Saarinen L, Kayhty H, Makela P H, Coulton J W. (1992) Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities. Mol Microbiol. 6:665–676.

McClelland R G, Pinder A C. (1994) Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry. J Appl Bacteriol. 77:440–447

Hughes E E, Matthews-Greer J M, Gilleland H E J. (1996) Analysis by flow cytometry of surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa. Can J Microbiol. 42:859–862.

Ferris, M. M., Yan, X., Habbersett, R. C., Shou, Y., Lemanski, C. L., Jett, J. H., Yoshida, T. M., Marrone, B. L. (2004) Performance Assessment of DNA Fragment Sizing by High Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis. J. Clin. Microbiol. 42(5): 1965-1976.

Larson, E. J., J. R. Hakovirta, H. Cai, J. H. Jett, S. Burde, R. A. Keller, and B. L. Marrone. (2000). Rapid DNA fingerprinting of pathogens by flow cytometry. Cytometry 41:203-208.

Haridas, V., Ranjbar, S., Vorobjev, I. A., Goldfeld, A. E., Barteneva, N. S. (2017) Imaging flow cytometry analysis of intracellular pathogens. Methods. Vol. 112: 91-104. PMCID: PMC5857943.

Buzatu, D. A., Moskal, T. J., Williams, A. J., Cooper, W. M., Mattes, W. B., Wilkes, J. G. (2014) An integrated flow cytometry-based system for real time, high sensitivity bacterial detection. PLoS One. 9(4) : e94254.

Loret S, El Bilali N, Lippé R. (2012). Analysis of herpes simplex virus type I nuclear particles by flow cytometry. Cytometry A 81:950–959. doi:10.1002/cyto.a.22107.

El Bilali N, Duron J, Gingras D, Lippé R. (2017). Quantitative evaluation of protein heterogeneity within herpes simplex virus 1 particles. J Virol 91:e00320-17. doi:10.1128/JVI.00320-17.

Arakelyan A, Fitzgerald W, King DF, Rogers P, Cheeseman HM, Grivel JC, Shattock RJ, Margolis L. (2017). Flow virometry analysis of envelope glycoprotein conformations on individual HIV virions. Sci Rep 7:948. doi:10.1038/s41598-017-00935-w

Arakelyan A, Fitzgerald W, Zicari S, Vagida M, Grivel JC, Margolis L. 25 January (2017). Flow virometry to analyze antigenic spectra of virions and extracellular vesicles. J Vis Exp doi:10.3791/55020.

Bonar MM, Tilton JC. (2017). High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry. Virology 505:80–90. doi:10.1016/j.virol.2017.02.016

Tang VA, Renner TM, Varette O, Le Boeuf F, Wang J, Diallo JS, Bell JC, Langlois MA. (2016). Single-particle characterization of oncolytic vaccinia virus by flow virometry. Vaccine 34:5082–5089. doi:10.1016/j.vaccine.2016.08.074.C

Lippé R. Flow Virometry(2018) A Powerful Tool to Functionally Characterize Viruses. J Virol 92(3)-17.

Mejuto, P., Luengo, M., Diaz-Gigante, J. (2017) Automated Flow Cytometry: An alternative to urine culture in a routine clinical microbiology laboratory? Int. J. Microbiology 8532736. PMID:29090008.

Authored by: Dr. Julie Bick

Dr Julie Bick is a medicinal biochemist who has spent close to 7 years with FlowMetric Life Sciences. After receiving her doctorate in Biochemistry at Southampton University in the UK, she began her career as Associate Professor at Rutgers University, NJ, before moving to the west coast to perform biomedical research with Syngenta and Novartis at the Tory Mesa Research Institute in San Diego. Dr. Bick specializes in biomedical engineering of cells and proteins in order to provide innovative therapeutic and diagnostic solutions. She brings to FlowMetric a clinical expertise across a wide range of therapeutic areas from autoimmunity to oncology and chronic inflammatory conditions, acquired over 25 years of research experience in academic, biotechnology and pharmaceutical laboratories. In leading FlowMetric Life Sciences’ innovation initiatives, Dr. Bick has been collaborating with BurstIQ to implement Block Chain solutions into the company’s Contract Research Organization division, with a focus on enhanced big data analytics and process control solutions in the regulated clinical environment. Dr. Bick is committed to working with local Community Colleges to support STEM programs for the next generation of scientists.