Surgery – Diagnostic testing – Detecting nuclear – electromagnetic – or ultrasonic radiation
Reexamination Certificate
2001-05-08
2003-01-28
Lateef, Marvin M. (Department: 3737)
Surgery
Diagnostic testing
Detecting nuclear, electromagnetic, or ultrasonic radiation
C600S437000, C601S002000, C601S003000
Reexamination Certificate
active
06511430
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a non-invasive method for detecting and monitoring apoptosis. In particular, the present invention relates to a method for detecting apoptosis using ultrasound imaging.
BACKGROUND OF THE INVENTION
Ultrasound imaging is one of the commonest radiological modalities presently used in clinical medicine. It is used to image the developing fetus, to image organs and vascular components, and to image tumours. Recently, high frequency ultrasound based systems have been developed to produce high resolution images of biological specimens such as spheroids or human tissues in vivo.
High frequency ultrasound imaging has been used to investigate the living and hypoxic regions of cell spheroids indicating the ability of this imaging modality to differentiate different types of cellular morphologies (Sherar et al., Ultrasound backscatter microscopy images the internal structure of living tumour spheroids,
Nature
1987; 330: 493-495; Bérubé et al., Use of a high frequency ultrasound microscope to image the action of 2-nitroimidazoles in multicellular spheroids,
Br J Cancer,
1992; 65: 633-640).
Apoptosis is one mechanism by which biological cells undergo cell death. It plays a significant role in both normal and disease-related biological processes (Hockenbery, D. Defining apoptosis.
Am J Pathol,
1995; 146: 16-19; Majno G., Joris I. Apoptosis, oncosis, and necrosis.
Am. J Pathol,
1995; 146: 3-15; Fraser A., Evan G. A view to a kill.
Cell,
1996;85: 781-784). In addition, cells undergo apoptosis in response to a variety of stresses including chemotherapy, radiation therapy, photodynamic therapy and heat. It is useful in both experimental and clinical applications to know whether cells are undergoing apoptosis. This is currently determined by taking samples of the cells or tissues of interest and observing, using histological and DNA measurement methods, whether the cells exhibit the morphological changes that are indicative of apoptosis. These changes include membrane blebbing, DNA condensation and DNA fragmentation. However, these methods are not only invasive, but also time-consuming, requiring processing of a cell or tissue sample before data relating to apoptosis can be obtained.
The ability to differentiate apoptotic cells from living or otherwise dead cells non-invasively in-vitro and in vivo would potentiate clinical diagnoses, the understanding of disease processes and normal biological processes that involve apoptosis, and provide a more efficient way of studying apoptosis in response to therapeutic agents.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a non-invasive method of monitoring apoptosis in cell culture, ex-vivo tissues and in-vivo tissues using high frequency ultrasound imaging, which comprises the steps of
1.) imaging a selected site of the cell culture or tissues using high frequency (above 20 MHz) ultrasound imaging (before image);
2.) exposing the selected site to an apoptosis-inducing stress;
3.) imaging the selected site or a portion thereof, using ultrasound imaging at subsequent timed intervals (after image(s));
4.) measuring the signal amplitude of a region of interest of the selected site in the before and after images;
5.) comparing the signal amplitude measurements for the regions of interest in the before and after images and determining whether the after image regions. exhibit an increase in amplitude as compared to the before image regions which is an indication that apoptosis has begun; and
6.) measuring the change in the frequency spectrum of the radiofrequency ultrasound backscatter signal in the region of interest in the before and after images and confirming that apoptosis has begun when the slope of the frequency spectrum has increased.
In another aspect of the invention, further confirmation of the occurrence of apoptosis is achieved by the step of calculating the average scatterer size in the region of interest by applying an ultrasound scattering model to the radiofrequency ultrasound signals from the regions of interest. Apoptosis is confirmed when the average scatterer size has reduced significantly according to this analysis. The decrease in scatterer size reflects the fragmentation of nuclear material that occurs during apoptosis.
More simply, the method of the invention is to use ultrasound imaging to monitor and measure the apoptotic process in cell culture, ex-vivo tissues and in-vivo tissues using the three-step process of:
1) Imaging of the treated sample or region of tissue before and during treatment and/or imaging treated and untreated regions in the sample or tissue;
2) Measurement of the change in signal amplitude or intensity in the treated and untreated samples or regions of tissue; and
3) Measurement of the change in frequency spectrum of ultrasound scatter from the treated and untreated regions.
An optional fourth step to this process is:
4) Calculation of average scatterer size from the frequency spectra collected.
In essence, the present invention offers a new use of ultrasound imaging to monitor the process of apoptosis. We have discovered that the processes of nuclear condensation and fragmentation that are indicative of apoptosis result in an approximately 3-6-fold increase in the amplitude of ultrasound scattered from cells in culture, ex-vivo tissues and in-vivo tissues as compared to normal cells not undergoing apoptosis. Also, we have discovered that the frequency spectrum of the ultrasound scattered from biological samples and tissues containing cells undergoing apoptosis is different from untreated samples not undergoing apoptosis. The specific subcellular features which permit the apoptotic phenomenon to be visualized have been investigated and are shown to be related to the changes in the cellular nuclear material cells undergo during apoptosis.
Apoptosis is indicated in the sample or region of tissue if both the signal intensity increases and the slope of the frequency spectrum increases. Further confirmation is indicated by a decrease in the average scatterer size in the treated regions. The regions of tissue that satisfy these criteria could be colour coded on the original ultrasound image, for example. There are various methods known in the art for calculating the signal amplitude, slopes of the frequency spectra and the average scatterer size parameters, and any of these may be used, with the choice being one that a person skilled in the art can select readily.
DETAILED DESCRIPTION OF THE INVENTION
The present method is a non-invasive method utilizing high frequency ultrasound imaging to detect and monitor apoptosis in cells or tissues, in vitro, in vivo or ex vivo. As will be appreciated by those of skill in the art, the term “high frequency ultrasound imaging” is meant to refer to ultrasound imaging at frequencies of greater than 20 MHz. The method involves taking high frequency ultrasound images of the cells or tissues of interest prior to the application of an apoptosis-inducing stress, herein referred to as a “before image”, as well as taking a high frequency ultrasound image following application of the stress, referred to herein as an “after image”. Alternatively, high frequency ultrasound images of treated and untreated regions of the sample or tissue can simultaneously be taken. In this case, the ultrasound image of the untreated region would be equivalent to the “before image” and the ultrasound image of the treated region would be equivalent to the “after image”. The terms “before image” and “after image” as used herein encompass both of the foregoing alternatives.
“Apoptosis-inducing stress”, as referred to herein, is meant to encompass any stress which will result in the initiation of apoptosis. Examples of apoptosis-inducing stresses include chemotherapeutic agents, drugs, photodynamic therapy, chemical modifiers aimed at protecting tissues from radiations such as sunscreens, radiations including X-rays, gamma rays and ultraviolet radiations, oxygen and/or nutrient deprivation that can occur after organ removal for tr
Czarnota Gregory J.
Hunt John
Kolios Michael
Sherar Michael D.
Jain Ruby
Lateef Marvin M.
University Health Network
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