Pushing the Norms of Conventional High-Complexity Clinical Cytometry

by Dr Carsten Lange

Flow cytometry is a powerful technique for the detailed analysis of complex populations which, over the last two decades, has evolved from a staple technique of the research laboratory into an essential part of the modern clinical laboratory.

Some of the current ‘norms’ for Clinical Flow Cytometry include its critical use for phenotyping hematological malignancies, as well as playing a vital role — along with other testing methods — in diagnosing disease, informing treatment plans and monitoring patients. Only time can tell how this powerful analytical technology will contribute to the clinical lab of the future. We can, however, anticipate that it will only continue to increase in importance, based on technical innovations that have driven the evolution of flow cytometry over the last decade.

In addition to today’s applications in disease diagnostics, the power of this technology continues to be used in cell biology research and pharmaceutical discovery. This evolution has been made possible by a higher number of analytical parameters to measure cells in suspension. The first cytometers were systems capable of merely three or four parameters, using a single laser and four detectors, and were the size of a small car. Today, however, Flow Cytometers (including cell sorters) can analyse more than 30 parameters, and new technology in benchtop analysers can deliver exponentially better performance in a smaller footprint.

Shifting paradigms

This paradigm shift, toward higher performance in a small instrument, is driven by clinical laboratories that want to capture the power of flow cytometric analysis, but don’t want to invest a significant amount of time in learning the instrumentation. The democratization of flow cytometry is enabled by key advances in technology. Advantage is being taken of prominent concepts in other scientific fields, such as the telecommunications industry, to allow the subsystems to be miniaturized while at the same time providing even better performance. These compact high-performance systems not only deliver better performance than historically expensive systems, but they are also easy to set up, operate and maintain, enabling a greater number of clinical laboratories to maximize the power of flow cytometry.

The Power To See More

Performance of flow cytometers is typically measured by their capacity to resolve and their sensitivity to detect dim and/or rare populations. In this regard, efficient light management for optimal excitation and emission of fluorochrome-tagged cells is critical to performance.

One hallmark of the APD is the high quantum efficiency in excess of 80%, especially for wavelengths greater than 800 nm.

Figure 1. The wavelength division multiplexer (WDM) uses avalanche photodiode detectors (APD), versus photomultiplier tubes (PMT). One hallmark of the APD is the high quantum efficiency in excess of 80%, especially for wavelengths greater than 800 nm.

With conventional flow cytometers, laser excitation sources are optimized by shaping and focusing light through a series of lenses and filters onto a flow cell where cells are hydrodynamically focused. However, newer flow cytometers use unique laser designs that are focused onto a flow cell with integrated optics. These systems can ensure increased excitation of the dyes not only on (and within) cells, but also increased collection of the emitted light for integration and measurement. When designing a compact clinical cytometer, the use of fibre optics to carry light is an efficient way of transmission, providing flexibility in laying out system components. These cables capture emitted light to deliver it onto a unique detector array, reducing crosstalk between channels, which improves performance.

Another recent development is a key concept borrowed from the telecommunications industry, the wavelength division multiplexer (WDM), which is used for light detection and measurement. Wavelength division multiplexing is a method used to deconstruct and measure multiple wavelengths of light as signals that relate to analytical parameters. The detectors used to measure each parameter are avalanche photodiodes (APDs), which are highly sensitive semiconductor devices. By contrast, conventional clinical cytometers to date have (and continue to use) photomultiplier tubes (PMTs).

The major advantages of using APDs over PMTs include but are not limited to:

  1. Enhanced linearity;
  2. 4 — 5 times the quantum efficiency (Fig. 1);
  3. Higher dynamic range, 106 versus 103;
  4. Smaller size and about one-tenth the cost.

Figure 2 shows the WDM of the first commercially available clinical cytometer to use compact APDs which reduce the overall instrument footprint (DxFLEX, Beckman Coulter). Each WDM contains optical and detector components to selectively measure specific wavelengths. This improves light collection for higher sensitivity to detect dim populations.

The WDM uses fibre optics and bandpass filters to separate the light wavelengths

Figure 2. The WDM uses fibre optics and bandpass filters to separate the light wavelengths

The WDM’s innovative and simple design uses a single bandpass filter to select the various colours of light. This contrasts with traditional clinical cytometers, which use a series of dichroic steering filters and bandpass filters that bounce the light along an array, leading to successively less available light, resulting in diminishing light collection efficiency, and ultimately compromising fluorescence sensitivity and resolution (Fig. 3).

Multiple dichroic filters to direct the light path are not required with the WDM Light efficiency is increased as light loss due to refraction is minimized.

Figure 3. Multiple dichroic filters to direct the light path are not required with the WDM. Light efficiency is increased as light loss due to refraction is minimized.

Simplifying High Complexity

Leveraging the linearity of detection systems that use APDs in the operation of the cytometer can be dramatically simplified owing to the predictability of the signals. The linear gain and the normalization performed during the daily quality control routine takes care of the relative variations during instrument set-up commonly seen in instruments. Further, setting up a highcomplexity assay is simplified by using a software gain-only adjustment. The linearity of gain adjustment also simplifies the typically arduous task of spectral compensation which has been the barrier for many to push to a higher number of colours/parameters. To maximize the benefit of the APD linearity, new software algorithms have been developed that facilitate set-up and analysis of high-complexity experiments by simplifying compensation.

It is now possible to create a compensation library that stores the APD gain settings and spectral spill-over coefficients for every parameter and multicolour combination. This allows users to make a virtual spectral compensation matrix selecting various single colours from the library. In addition, the library can intelligently adjust the compensation values when gains are adjusted owing to the predictive responses of linear APDs. The result is a dramatically simplified and intuitive method of setting up high-complexity applications.

The Size Factor

For most cytometers, measuring size of particles less than 300 nm is difficult because they deliver relative sizing information using forward scattered light from the 488 nm blue laser. For these systems, particles of less than 1 µm (1000 nm) usually fall below the noise threshold of the laser and detector subsystems. In contrast, newer systems use principles of Mie scattering, which predicts that with lower wavelengths of excitation there will be an increased amount of scattered light and improved resolution.

Therefore, measuring scattered light from a shorter-wavelength 405 nm violet laser versus a longer-wavelength 488 nm blue laser will allow the system to resolve smaller particles. The use of the violet side scatter parameter enables systems to detect particles of less than 0.2 µm (200 nm) in size, enabling excellent resolution of microparticles.

The future is now

Combining powerful performance and innovative design and technology, it is possible to deliver a compact, easy-to-use flow cytometer. Pushing the ‘norms’ of conventional flow cytometry, today’s — and tomorrow’s — cytometers simplify high-complexity applications in the clinical laboratory, as well as a deeper understanding in the frontier applications of hematopoietic cancers. Flow cytometry remains a powerful tool for interrogating complex questions. Today’s clinical laboratories want to harness that power and are demanding smaller and more powerful flow cytometers that are more affordable and easier to use. Using innovation, engineers can deliver solutions to meet the challenge.

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