• flow cytometry;
  • lymphatics;
  • photoacoustic effects;
  • nanoparticles;
  • disease diagnosis

Since blood flows throughout the whole body, acting as a medium for providing oxygen and other nutrients, and drawing waste products back to the excretory systems for disposal, the state of the bloodstream is affected by many medical conditions. High or low blood cell numbers (erythrocytes, leukocytes, thrombocytes, etc.) or the presence of abnormal objects such as metastatic cells or infections correlates with stages of many diseases. Hence blood counts are amongst the most commonly performed blood tests in medicine. Compared with blood, cell composition in lymph is paid much less attention (1–3). The report by Galanzha et al. (4) in a previous issue of Cytometry Part A is the first demonstration of a new technique for counting cells in the peripheral lymphatics based on the principle of flow cytometry and photoacoustic single-cell detection.

The significance of the lymphatic system for diagnosis and therapy was already emphasized for many diseases including cancer, inflammation, lymphedema and infections (1, 2, 5–8). Advanced technologies such as MRI, PET, lymphoscintigraphy or fluorescent lymphography are capable of providing lymphatic mapping (e.g., vessels and sentinel nodes), lymphedema assessment, molecular detection of lymphatic endothelium, lymphangiogenesis and lymphangiogenetic factors (e.g., VEGFs). Unfortunately our insight into lymphatics as a multi-cell flow and cell composition under healthy and pathological conditions is dramatically insufficient. This is despite the fact that prenodal (afferent) lymph carries immune-related cells (e.g., T and B lymphocytes, dendritic cells, macrophages) and rare abnormal cells (e.g., tumor metastatic cells, bacteria, viruses), which are exclusively derivates of surrounding tissues. They are, thus, the earliest, most accurate indicators of physiological and pathological processes. Professional immune cells could provide diagnostic information about local immune status (2, 8). The amount of lymphocytes in lymph also correlates with the degree of lymphatic obstruction and inflammatory changes in skin at the lymphedema (2). The understanding of how immune and tissue-specific precursor cells extravasate from blood capillaries, get into contact with tissue cells and matrix, stay there or migrate toward lymphatics and enter them, as well as how different cell populations cooperate (intralymphatic cluster formation) or attach to lymphatic endothelium remains mainly in the realm of our imagination. This applies also to the intranodal events. Which node cells are catchers and removers of infected or cancer cells, and which are educators and regulators is a matter of multiple hypotheses. Another example is the early detection of rare tumor cells before they spread from the original tumor via the lymphatic system to colonize in distant metastases is crucial for patient survival (5, 6). Although the appearance and role of erythrocytes in afferent lymph remains unknown, their concentration might be important for early diagnosis of pathologies related to disturbances of blood vessel permeability (e.g., at diabetes or venous insufficiency).

The list of questions of how migrating cells behave in tissue, lymphatics, and nodes is long and in order to get answers to them we need methods for direct insight into the blood extravascular space events. Studies of cells harvested from blood circulation would never give us enough information to construct hypotheses on what is or may proceed in tissues.

Thus, lymph testing in addition to blood testing may have significant diagnostic value. Of course, there are numerous published data originating from the static situations that elucidate some of the mechanisms. However, what we need are dynamic pictures that would enable qualitative and quantitative evaluation of the intercellular processes in tissue fluid and lymph. The realization of this approach, however, requires overcoming many technical problems such as difficulties with lymph sampling, a relatively small lymph volume (few μL at the direct puncture of skin lymph vessels) insufficient for early detection of rare abnormal cells, or low sensitivity and low speed of existing methods that prevent their application in vivo for detection of individual cells in unstable, complex forward–backward lymph flow in poor optical conditions.

That is why the novel approach from the Zharov group gives us so much hope for at least partly solving the above problems. With this method we could “hear” cells like the astronomers “hear” new planets, though not seeing them in images. Of course, in our case confrontation of sounds and pictures can take place as shown in this article. They applied a novel, in vivo flow cytometer with photoacoustic detection of individual cells in lymph flow. Photoacoustic (also called as optoacoustic) methods and their application in spectroscopy, analytical chemistry, and microscopy have a very old history [see in (9)] and recently have demonstrated clinical significance for in vivo noninvasive imaging of vessels and tumors at a depth up to 3 cm using laser energy (10). The basic idea of the method is very straightforward: a laser-generated sound in a single cell is detected with an acoustic transducer. The physical processes are also simple (9, 10): light-induced heating of a biological cell due to excitation of its molecular components into excited states by incident photons (as in a fluorescent method) and follow up conversion of the absorbed optical energy via nonradiative relaxation into heat (instead of re-emitting fluorescence light). Transient thermal expansion of local absorbing inhomogeneities of an object causes the generation of acoustic (ultrasound) waves. Currently, photoacoustic methods provide the highest absorption sensitivity (five orders of magnitude better than in conventional absorption spectroscopy) at low sensitivity to light scattering and autofluorescence. The technique requires robust nanosecond pulse lasers broadly used in the biomedical field and a conventional miniature ultrasound technique.

Previously, a photoacoustic method and its modifications were applied for imaging and spectroscopy of samples containing many cells in the measuring volume (9, 10). The authors of the discussed paper extended this method to the single cell analysis in in vivo flow conditions. The key innovation is the optimal combination of the basic principles of flow cytometry and photoacoustics within one technical platform. The improvement in sensitivity is achieved by using new schematics and advanced optical, electronic and ultrasound components. This provides a label-free detection of individual erythrocytes, lymphocytes, and melanoma cells in lymph flow in living animals with the currently best concentration threshold limit of one cancer cell on the background of millions of normal white blood cells in vivo. For some particular applications, strongly absorbing nanoparticles such as gold nanorods and nanoshells, or carbon nanotubes may serve as excellent multicolor photoacoustic probes on the subcellular level. Using such nanoparticles in this pilot experimental study, the authors have demonstrated simultaneous identification and counting of healthy, apoptotic and necrotic immune-related fast-flowing cells.

The diagnostic novelty of this study is the transformation of lymphatic colorlessness, as a commonly accepted limitation, to a diagnostic advantage. The researchers showed that lymph transparency provides an extremely low background signal compared with blood. In the future, this fact may provide label-free detection of different types of cells, even different subpopulations of white blood cells.

This study offers an elegant solution of old detection problems related to the very complex (irregular, oscillating, etc.) motion of lymph leading to temporal and spatial cell instability. It has been solved by this group on the basis of natural lymphatic functioning. As a major result of these studies, lymphatic valves, driven by phasic contractions, were suggested to be used as natural nozzles providing “hydrodynamic focusing” and thus, forming a single-file cell flow convenient for in vivo measurements. Remarkable, that the robust technical decision was done on the basis of the anatomic nature of lymphatics, while in conventional flow cytometry, the problem of artificial hydrodynamic cell focusing has been solved by significant effort.

Another interesting point presented in this article is multispectral (multicolor) cell detection. This approach may ultimately provide up to 100 colors for cell identification, which is much higher than used to date in conventional flow cytometry. As an example, two-wavelength pulse laser excitation gave spectral identification of fast flowing cells in vivo by the usage of multicolor nanoprobes having specific adsorption spectral fingerprints.

From a research point of view, newly presented technology may revolutionize lymphatic research by filling gaps in yet unclear issues such as: (a) dymanic biodistribution of living and apoptotic cells, bacteria, tumor cells; (b) interaction of metastatic cells with immune-related cells and host environment; (c) multi-correlation between quantity of metastatic cells in lymphatics, primary tumor size, distant metastasis development, and rate of CTCs in blood flow; (d) the monitoring of cell response to drugs and radiation; (e) cell “traffic” simultaneously in the lymph and blood systems, as the authors have developed not only lymph but also blood photoacoustic flow cytometry in vivo (11).

Undoubtedly the most attractive point is further clinical translation of this method. The evidence usually originates from animal studies and should not necessarily reflect human processes. For example, human granulocytes extravasate but do not enter lymphatics, whereas dogs and sheep reveal the presence of these cells even in normal lymph. Animals form lymphatic aggregates in obstructed lymphatics; this has not so far been shown in humans.

Clinical perspectives look completely possible but some questions still remain to be solved. A new method can never be universal. This is why the first steps of human studies should be directed at identification of lymph cell populations in leg superficial lymphatics located relatively close to the skin surface. The data can then be compared with those from direct sampling by cannulation. The movement of lymph cells in terms of velocity, stoppage, detachment from endothelium and effects of drugs on these processes would provide us with data necessary for manipulation with immune recognition mechanisms in tissues and nodes.

Another important medical application is lymph testing of cancer patients who have undergone surgery for tumors. Intraoperative detection of metastatic cells in afferent lymphatics integrated with the status of sentinel lymph nodes looks like the most promising for prediction and estimation of cancer metastasis development. Perhaps the presence and count of metastatic cells in afferent lymph, even at the negative status of sentinel lymph nodes, could be crucial for predicting the clinical outcome.

The pivotal application is noninvasive quantitative monitoring of cells in skin lymphatics for treatment of skin cancers (e.g., melanoma), lymphedema, inflammation, infections, or obesity-related states. Toward this goal, the authors have demonstrated the capability of a new technique to count and identify rare individual cells in skin lymphatics with the example of melanoma cells. It remains unclear how to provide a rapid finding of a lymphatic valve in skin vessels noninvasively, to achieve accurate counts of numerous cells like lymphocytes.

In general, it is our opinion that the first attempt to estimate the capability of photoacoustic lymph flow cytometry in vivo is slightly chaotic and should be carefully further verified with a focus on molecular cell targeting with functionalized nanoparticles, and methods of delivery of laser radiation to deep lymphatics. However, despite its preliminary character, this paper demonstrates that biologists and physicians may have a new powerful tool that opens new avenues in basic lymphatic research and perhaps, clinical applications including qualitative and quantitative detection of immune-related cells in normal, apoptotic, and necrotic stages, or integration of noninvasive sentinel lymph node mapping and metastasis assessment. Some of the developed approaches may have great success in conventional flow cytometry as well. In addition, this method could be easily integrated with fluorescent and light-scattering techniques.

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