In vivo flow cytometry enables highly sensitive diagnosis

An adaptation of flow cytometry principles is allowing researchers to noninvasively detect single cells directly in the bloodstream and lymph flow–even within thick tissue, and with or without labeling. Tests look promising for extremely early cancer diagnosis.

By Barbara Goode

Flow cytometry has enabled revolutionary discoveries in cell biology and medicine. In a single minute, a flow cytometer can individually quantify a few million cells at subcellular and molecular levels based on multiple parameters such as morphology, composition, proliferation, gene expression, and apoptosis.

The main substrate for flow cytometry, blood, reveals pathological processes and enables clinical diagnosis of most diseases, including cancer. Approximately 90% of all cancer deaths are caused by metastatic spread of the primary tumor. In 2007, the American Society of Clinical Oncology (ASCO) cited circulating tumor cells (CTCs) as tumor markers. And the detection of CTCs in cancer patients has been suggested as a prognostic tool, as the presence of malignant cells in the blood is necessary for the development of metastatic tumor spread.

The clinical utility of CTCs for metastasis prevention remains unclear, however, because at the time of initial diagnosis, incurable metastasis can be already established. In vitro techniques have an ultimate threshold of 1 CTC per sample volume–and thus most advanced assays have sensitivity levels of 1 to 5 CTCs/mL (equivalent to between 5000 and 25,000 CTCs per approximately five liters of an adult patient’s blood). Recent data indicate that this CTC level may be sufficient for the development of overt metastases. Besides, the invasive extraction of cells from a living system may lead to artifacts and prevent the long-term study of cells in their native biological environment.


FIGURE 1. in vivo photoacoustic flow cytometry uses a diode laser to detect individual cells, either without labels or using gold nanoparticle labels, in blood and lymph flow or in sentinel lymph nodes (left). Results are derived from the photoacoustic signal (upper trace) and boxcar gate (lower trace; a), and compressed photoacoustic signals from single melanoma cells (b). Averaging 36 signals from one melanoma cell produces a composite reading (c). The clinical prototype system uses optical fiber to deliver laser radiation to vessels in the wrist, and a focused cylindrical ultrasound transducer (inset).
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So, as valuable as flow cytometry is, this example highlights some of its limitations.

Noninvasive, in vivo cytometry

Greater sensitivity would be possible with in vivo flow cytometry–that is, by using the vessels in a living organism as channels through which native cells flow. The advantage of this approach is the possibility to noninvasively detect rare CTCs within a large volume of blood with potential sensitivity better than in currently available assays.

Adapting the principles of flow cytometry from in vitro to in vivo, however, involves considering many factors that are not part of the ex vivo environment. Among these are light scattering and absorption by vessel walls and surrounding tissues, the presence of many cells in multiple flow streams in vessel cross sections, and the fluctuations of cell velocity and position within a vessel. As a result, tracking small numbers of cells in the body in vivo is technically challenging.

Further, while cell biologists often use fluorescent labeling in flow cytometry, in vivo research involves concerns about cytotoxicity of available fluorescent tags, immune response to tags, and the influence of light scattering and the autofluorescent background, which allow the assessment of only superficial microvessels having a relatively slow flow velocity and, thus, may significant lengthen the examination time of a large blood volume.


FIGURE 2. A time-resolved multicolor photoacoustic flow-cytometry system is able to identify pigmented melanoma cells in blood flow by using short laser pulses of different wavelengths (λ) interspersed with time delay (T).
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A team at the University of Arkansas for Medical Sciences has developed an alternative, clinically relevant approach: in vivo photoacoustic flow cytometry based on photoacoustic spectroscopy technology developed by Vladimir P. Zharov, professor of biomedical engineering, radiology, and otolaryngology at the University of Arkansas for Medical Sciences and director of the Phillips Classic Laser and Nanomedicine Laboratories at the Winthrop P. Rockefeller Cancer Instititue (Little Rock, AR).

The approach uses photoacoustic spectroscopy without labeling (using intrinsic cell markers such as cytochromes, hemoglobin, or melanin) or with labeling via novel substances such as gold nanoparticles.

The technique involves irradiating vessels with lasers to induce transient thermal expansion of light-absorbing biomolecules or nanoparticles into individual cells. The light produces ultrasonic waves, which an external ultrasound transducer detects (see Fig. 1). The method takes advantage of both optical spectroscopy, which provides high sensitivity, and ultrasound, which provides a high spatial resolution for detecting signals from deep within tissue (up to a few centimeters). These characteristics enable assessment of individual cells in large, deep vessels through which the majority of blood (for example, 1 to 3 liters) passes every 5 to 10 minutes.

The photoacoustic scanning cytometer applies photoacoustic spectroscopy for the first time to analyze single, static cells. It uses a tunable multiwavelength laser and nanoparticles with different absorption spectra as multicolor cellular labels.

Conventional imaging modalities such MRI, CT, and PET cannot diagnose small tumors containing less than a million tumor cells. However small they are, though, such tumors can produce CTCs that are detectable with photoacoustics.

System in action

The researchers proved the principle using natural melanin nanoparticles in melanoma cells as endogenous contrast agents (see Fig. 2). A two-wavelength laser was used, with either no labeling (for example, using natural light absorption by red blood cells and melanoma cells), or nanoparticles (gold nanorods, gold-plated carbon nanotubes, and gold nanoshells–each of which has a different absorption spectra for labeling low-absorbing white blood cells and tumor cells).

A fixed 639 nm wavelength was used because it is close to the so-called “window of transparency” for most biological tissue; the diameter and length of the gold nanorods were adjusted to correlate with the wavelength. For the second wavelength, the researchers chose 850 nm–within the broad spectral range from 420 to 2300 nm of a tunable optical parametric oscillator–to coincide with the maximum absorption of gold nanoshells, noting that most relevant studies use that wavelength.

Individual cells in the bloodstream were irradiated with laser pulses interspersed with short (10 µs) time delays; the signals were then read using time-resolved photoacoustic ultrasound wave detection. The photoacoustic waves revealed the target absorption spectra. Both melanin and carbon-nanotube particles provide strong photoacoustic spectra that decrease from the visible to the near-infrared range, providing specific signal ratios at 639 and 850 nm that are distinct from the corresponding ratio for red blood cells. Thus, the researchers were able to identify melanoma cells from among red blood cells in the bloodstream.

Systems, tests, and implications

Pilot clinical trails on humans are scheduled, focusing first on early diagnosis and treatment of melanoma without labeling. After completing the safety evaluation of novel gold-based labels, the group will test diagnosis for other cancers.


FIGURE 3. This single live red blood cell, captured with a high-speed camera in lymph flow, has a telltale shape.
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Next steps involve working to develop a system with the sensitivity of one metastatic CTC per billion red blood cells. With further improvements the group hopes to raise the sensitivity level to one CTC per hundred billion normal cells by interrogating larger blood vessels (with their high flow rates, these vessels provide more frequent appearance of rare CTCs).

Detection on lymph flow

In addition, the group has has used the same principles to develop a system for detection of individual cells in natural lymph flow (see Fig. 3).1, 2 This system’s sensitivity threshold is currently one tumor cell per million white blood cells in lymph flow. (Red blood cell concentration in lymphatics is extremely low.)

The researchers believe the eventual integration of the blood and lymph targets for in vivo flow cytometry may enable discovery of a correlation between them, which would represent a breakthrough and be extremely useful for the basic study of metastasis development and for early cancer diagnosis. Such early detection would enable therapy before metastasis progresses to incurable levels.

Dr. Ekaterina Galanzha, co-investigator of the lymph project, notes that the proposed strategy may apply broadly beyond cancer detection. Potential applications involve the study of any specific cell (immune-related, apoptotic, and so on), as well as bacteria and viruses, in blood and lymph flow, and implications include an increased understanding of physiological processes (such as immunity) to diagnose disease, to monitor cell response to drugs and radiation in vivo, and to determine pharmacokinetics of drugs and nanoparticles.

In another advance, the researchers recently demonstrated the technique’s ability to detect quantum dots. The photoacoustic method requires far fewer quantum dots for labeling than do fluorescence techniques–about 50 to 100 per cell are sufficient for single-cell detection, compared to millions per fluorophore molecule required for fluorescent detection. Photoacoustic signal amplification occurs organically in nanoparticle clusters when naturally clustered cancer biomarkers on the CTC membrane are stimulated. The researchers concluded that CTC labeling will require subpicomolar and potentially femtomolar concentration of gold nanoparticles, which may further reduce toxicity concerns.‹‹

REFERENCES

  1. W. Olszewski and A. Tarnok, J. Int’l. Society for the Advancement of Cytometry (December 2008)
  2. E. I. Galanzha et al, J. Int’l. Society for the Advancement of Cytometry (October 2008)

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