Creating Biomedical Technologies to Improve Health

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Science Highlight: February 25, 2009

Listening for One Cell in a Billion

Alexander Graham Bell’s first telephone was a success because Bell was able to generate sound from light. That was 1880. Today, researchers at the University of Arkansas for Medical Sciences, Little Rock, are using the same principle to create a novel, highly sensitive diagnostic tool that may detect cancer and other diseases before they become life-threatening.

The researchers’ new “phone” combines a laser and an ultrasound transducer, and allows them to listen for both normal and abnormal cells flowing through the lymphatic system, an intricate network of lymph vessels, nodes, and organs. In addition to its other functions, this second circulatory system works closely with the body’s primary circulatory system to transport cell waste products and nutrients throughout the body. Although researchers know the lymphatic system plays a role in cancer, inflammation, and fighting infections, little is understood about the cellular composition of lymph and how cells move through this network.

This novel technique, in vivo photoacoustic flow cytometry, was developed by Vladimir Zharov, director of the Philips Classic Laser and Nanomedicine Laboratories at Winthrop P. Rockefeller Cancer Institute and professor of otolaryngology and radiology at University of Arkansas for Medical Sciences, and colleague Ekaterina Galanzha. The technique will allow researchers to identify and count a wide range of cell types, including those related to infection, cancer, and the body’s immune system. The research is based on Zharov and Galanzha’s previous experiments with in vivo blood testing in which they were able to find one cluster of skin cancer cells in a billion blood cells.

 Photoacoustic flow cytometry is based on an optical effect discovered by Alexander Graham Bell and conventional flow cytometry (left), which simultaneously measures and analyzes single cells as they stream through a beam of light in an artificial tube. The technique takes advantage of lymphatic anatomy and physiology to count and identify individual cells in lymph flow (right).

 

Finding the Lymph Channel

At the start of the lymph project, Zharov and Galanzha knew that lymph would be a challenging substance to work with. Unlike blood flow, the colorless lymph has an unstable, slower flow of two-way traffic. Current approaches to sample lymph capture just a few microliters of the sticky substance, making lymph sampling impractical for disease screening. “Collecting lymph from a site is an art,” says Zharov, but “there is a huge amount of information in the lymphatic system.”

One common laboratory technique, flow cytometry, counts and identifies individual cells as they stream one-by-one through a beam of light. This invasive technique removes cells in small quantities from their native environment and may alter cell properties. Turning art into science required researchers to find a noninvasive way to manipulate lymph cells within their native environment so they could be counted and examined.

Zharov and Galanzha’s approach to solving this problem is to use the traffic control network in the lymph system created by Mother Nature. A series of tiny tubes called prenodal lymphatic vessels lie just beneath the skin’s surface and extend to nearly every tissue in the body. Valves in these tubes maintain one-way cell flow within the lymph system.

“The cells move as a multi-file flow with different velocities and may change directions frequently, which could make assessing them very complicated,” says Zharov, who pioneered the use of lasers to generate sound for medical applications in the early 1980s. “We found that lymphatic valves may work as natural nozzles periodically channeling each cell in a straight line.” By coordinating this natural phenomenon with individual laser pulses, Zharov and Galanzha can count and identify flowing individual cells in their natural environment. This approach permits long-term monitoring of a larger volume of lymph fluid compared with one-time sampling of a small amount of lymph.

 

Hearing Lymph

To examine lymph cells, Zharov and Galanzha first apply a biologically safe level of laser energy to the skin. The light penetrates a few millimeters to a few centimeters to the lymph vessels filled with flowing lymph cells. By carefully choosing the right wavelength, the light beam heats up only cells containing light-absorbing biomolecules called chromophores, such as hemoglobin or melanin. Rapid expansion of heated chromophores causes a change in local pressure. This action creates a particular kind of sound wave, a flash acoustic wave, which travels through the tissue and is captured by an ultrasound transducer on the skin’s surface. Flash acoustic waves travel at specific speeds, making them easier to detect than generic waves coming from surrounding tissue.

In their experiments, Zharov and Galanzha found that highly pigmented cells, such as red blood cells and melanoma (skin cancer) cells, produced the strongest sound waves and were easily located without additional imaging agents. To find nonpigmented cells, such as different kinds of white blood cells, the researchers applied different-colored gold nanoparticles to target white blood cells in the lymph flow and created double sound waves using two laser pulses at different wavelengths. Because each white blood cell has an individual color signature, this approach identified fast-flowing cells whose signatures matched the gold labels.

In a preliminary animal study to detect metastatic melanoma (skin cancer cells that have spread from the original tumor site), the technique was so sensitive that it detected a single cluster of melanoma cells among a million white blood cells in lymph. This promising result could mean increased cancer survival rates in humans because 90% of all cancer deaths result from metastasis. “Early detection of tumor cells before they colonize into detectable metastases could prevent metastases or, at least, decrease their development rate, and may provide the opportunity to eliminate tumor cells from lymph and blood flow with conventional or new laser therapies,” says Galanzha.

 

Sounds of the Future

Down the road, the technique may be used deep in the body to assess deep lymph and blood vessels in organs at risk for disease. The Arkansas researchers are exploring both noninvasive and minimally invasive approaches for early detection of stroke, heart attack, infections, and inflammation. The challenge is matching the right imaging tags, or nanoparticles, to target cells so that abnormal cells are clearly heard within the noisy cell background.

Although recent experiments have focused on preclinical studies with animals, the team plans to move to clinical trials in humans in the next year or two. They will first assess cells in blood flow and then move to lymph assessment. Zharov estimates that evaluating the entire blood volume of an adult, about 5 liters of fluid, will take about an hour. Future refinements should reduce that time to about 5–10 minutes. Lymph assessments will occur at extremities such as the leg, where lymphatics are located relatively close to the skin.

When the technique becomes clinically available, Zharov hopes to provide a combination diagnostic/therapy system. “We should be able to kill disseminating metastatic cells with the same laser that detects the abnormal cells by increasing the energy a little bit,” he says. Other applications may include cleansing body fluids of bacteria and viruses and tracking cell reaction to drug therapy and radiation.

“The assessment of lymph may provide a completely new form of medical diagnostics,” says Zharov. “It won’t be as universal as a blood test, but for some [applications] it will be more specific.”

This work was supported in part by the National Institute of Biomedical Imaging and Bioengineering.

 


Galanzha EI, Shashkov EV, Tuchin VV, Zharov VP. In vivo multispectral, multiparameter, photoacoustic lymph flow cytometry with natural cell focusing, label-free detection and multicolor nanoparticle probes. Cytometry A. 2008 Oct;73(10):884–94.

Olszewski WL, Tárnok A. Photoacoustic listening of cells in lymphatics: research art or novel clinical noninvasive lymph test. Cytometry A. 2008 Dec;73A(12):1111–3.

Zharov V, Galanzha E, Shashkov E, Khlebtsov N, Tuchin V. In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents, Opt Lett. 2006 Dec 15;31:3623–5.

Zharov VP, Galanzha EI, Shashkov EV, Kim J-W, Khlebtsov NG, Tuchin VV. Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivo. J Biomed Opt. 2007 Sep-Oct;12:051503.

Zharov VP, Galanzha EI, Tuchin VV. In vivo photothermal flow cytometry: imaging and detection of individual cells in blood and lymph flow. J Cell Biochem. 2006 Apr 1;97:916–32. Review.

Zharov VP, Letokhov VS. Laser Optoacoustic Spectroscopy. Springer-Verlag. Berlin, Heidelberg, New York. 1986.

Health Terms: 
Cancer,
Cardiovascular,
Diagnostic,
Heart Disease,
Infectious Diseases,
Stroke