Locating marker/tracer elements detectable by neutron activated analysis within or on carrier microspheres, including microspheres used in biological experimentation
Inactive Publication Date: 2002-09-12
RHEINHARDT CHRISTOPHER +1
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Benefits of technology
0124] Not only do the microspheres carry (mechanically, or chemically, or both chemically and mechanically) the preferred marker(s), the carrier microspheres may readily be tailored (chemically, or mechanically, or both chemically and mechanically) to the intended applications environment.
Problems solved by technology
Therefore, stable isotope labeled products will have an indefinitely long shelf life: significantly longer than competing labeling methods.
The major disadvantage to assay by neutron activation technology is the required access to a neutron source.
Stable-isotope labeled microspheres serving as identifying markers cannot be assured to be emplaced in explosives save those legitimately produced, and then only under mandate of law or regulation.
Of course, to remove the microspheres, and markers, requires destruction of the compound.
Moreover, such explosion and attendant molecular dissociation does not truly get rid of the elemental markers, which remain (and will remain, short of atomic transmutation) as residue.
However, direct measurement of fluid(s) flow(s), such as within the blood stream of a living animal, is often impossible.
Moreover, direct quantitative analysis of the constituent components of complex, or extensive, mixtures of fluids is often prohibitively difficult or expensive.
The expense is magnified if many samples must be taken, and analyzed, over time.
The blood, and the organs and tissues receiving blood, within a living animal present a system that is very complex in its fluid flow patterns and dynamics, and that is difficult of direct access and measurement.
Previous systems developed for medical blood flow analysis--discussed in greater detail hereafter--have proven to be both complex and expensive.
Because of their cost and complexity, such systems have not been found suitable for use in routine industrial or environmental fluid flow and mixing measurement problems.
However, it should be recognized that the flow of blood, or blood components, within the arteries and veins of a living animal is only different in complexity, and not in the essential nature of fluid flow dynamics, from the flows of fluids occurring within factories, ecosystems, and the like.
The main (but not the only) problem with radioisotope-labeled microspheres is shelf life.
Radioactive microspheres thus have a time limited shelf life, which adds a cost factor to their use.
Care must also be taken not to let too much time go by before harvesting and analyzing the tissue samples or there may be insufficient activity to determine low fluid flows due to the `noise` threshold of a typical gamma counter used for measurement of radioactivity.
If not performed diligently experimental schedules may be disrupted.
Although due precautions are taken in transport and storage of radioactive microspheres, the constant flux of newly produced radioisotopes from manufacturer to laboratory, the controlled storage of radioisotopes still suitable for experimental use, and the long term of radioisotopes no longer suitable for use but still sufficiently radioactive so as to be unsuitably released into the environment, all involve a biohazard.
There is typically a high minimum "per order" cost of equipments from manufacturers.
These high costs severely limit the use of radioisotope-labeled microspheres in blood flow measurement, generally restricting its use to large laboratories and medical centers.
Second, because of the half-lives of their contained radioisotopes, radioactively-labeled microspheres have a limited shelf life typically ranging from weeks to several months.
Third, because of the short half-lives of many radioisotopes, radioactively-labeled microspheres are typically usable only in experiments of durations that are no more than a few weeks or months.
Sodium-iodine (NaI) crystals provide a low-cost, sensitive gamma-ray counting system with intrinsically poor spacial resolution.
As a result, researchers are limited in the number of different radioactive microspheres that can be accurately measured per sample, due to overlap between the emission energies of available radiolabels.
Increasing the number of radiolabels measurements is done only at a significant loss in sensitivity and specificity.
These techniques are similar to the spectrographic analysis of a palette of dye-colored microspheres.) p Fifth, laboratory workers using radioactively-labeled microspheres are exposed to radiation danger.
The radioactively-labeled microspheres are especially dangerous if they enter into the human body by ingestion, respiration, or accidental injection.
Finally, and perhaps most critically, disposal of the experimental animals poses significant problems, both logistically and financially.
The cost of disposal is becoming prohibitively high, recently ranging to as high as $750 U.S. or more per animal.
However, due to the precautionary measures needed to minimize radiation exposure, use of R M is restricted to specially licensed laboratories.
As mentioned above, storage of the radioactive microspheres, as well as disposal of radioactive waste, is expensive and presents a health and environmental hazard.
There are, however, significant limitations to these previous counting techniques.
First, RMBF is extrapolated from only a small aliquot of the dye-colored microspheres (CM) actually trapped within the sample, thereby entailing a substantial statistical error in RMBF calculations.
Third, there was considerable variation in the diameter of the CM used in previous studies, as admitted by Hale et al.
Fourth, the prior methods require substantial time for the tedious counting of individual dye-colored microspheres.
Automation for optical counting is expensive, typically $40-50K U.S. circa 1993 .
The method of Morita, et al. could be hampered by leaching of the label from the microspheres over time.
Another disadvantage is the need of a sophisticated and extremely expensive equipment for X-ray excitation and fluorescence detection which is not commercially available.
Because the numbers of microspheres introduced within the blood [typically five to ten million (5-10.times.10.sup.6)], and captured within the capillaries of the tissue, are large in the counting techniques, the actual numbers are commonly only estimated by statistical sampling, which induces measurement error.
Worse, even the determination of the numbers of dye-colored microspheres that are within minute sub-samples is tedious and expensive, involving in the methods of Shell, et al., and of Hale, et al., manual or semi automated observations through a microscope.
However, tissue digestion is still required.
However, with existing spectrometer sensitivities, and with analytical software programs of tractable size and execution times, which, most importantly, produce accurate quantitative results in the analysis of the individual spectral outputs of several different dyes (derived from species of CM) mixed together, it has, to date simply not been possible, to identify compatible dyes, and families of dyes, that number more than approximately one dozen.
Of these three additional steps, the second is by far the most labor intensive, and is therefore the most expensive.
The recovery of the CM by centrifugation or filtration is, however, a time-consuming and attention-demanding process that must be carefully performed.
Method used
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[0170] 1. Non-Radioactive, Stable, Isotope-Labeled Microspheres
[0171] 1.1 General Sequence of Making and Using Isotope-Labeled Microspheres
[0172] In one of its aspects the present invention is expressed in non-radioactive, stable, isotope-labeled microspheres. Preferably some eight or more different, stable, isotopically-labeled microspheres are to be made available to experimenters.
[0173] It is intended that isotope-labeled microspheres shall typically come in 2 ml glass serum bottles containing approximately 5 million spheres. The microspheres are 15 .mu.m in diameter and are suspended in normal saline containing 0.05% Tween 80 and 0.01% Thimerosal as a bacteriostat.
[0174] The tissue sample vials in which the tissue samples are to be placed are contaminant-free. The preferred sample vials are made from polypropylene, cleaned to remove trace contamination and calibrated for use in the particular activation and counting system of the assayist, which quite reasonably requires the use...
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Microspheres are permanently marked with non-radioactive stable isotopes of elements suitably detected by neutron activation analysis. The marked microspheres are suitable to permanently label diverse things. For example, families of stable-multiple-isotope-marked microspheres injected into an animal become lodged by the circulating blood within selected tissues of an animal during blood flow analysis experimentation. Absolute and relative abundances of these stable-isotope-marked microspheres residing within harvested tissues are readily accurately automatically measured in situ within the harvested tissue samples by neutron activation analysis. The quantitatively measured abundance of the isotopes, and associated microspheres, are accurately indicative of the former flow of blood containing the microspheres to the tissue. Microspheres are preferably marked with stable isotopes of gold, antimony, lanthanum, samarium, europium, terbium, holmium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, scandium and / or bromide.
Description
BACKGROUND OF THE INVENTION[0001] 1. Field of the Invention[0002] The present invention generally pertains to chemical or elemental markers or tracers that, when combined with other chemical admixtures or compounds, or when inserted into or upon objects or devices, thereafter serve to permanently identify such admixtures, compounds, objects or devices, including after such change(s) and gross change(s) to the compounds, objects or devices in form and / or in composition as may be occasioned by lapse of time, dissipation, wear, deterioration, oxidation, or explosion.[0003] The present invention particularly concerns (i) elemental or chemical markers that are indefinitely long lasting, and detectable at the level of a single atom or molecule over a fast range of densities; (ii) the use of neutron activation in the detection of elemental and chemical markers, and the elemental or chemical markers so detectable; and (iii) the packaging of, and / or carriers for, neutron-activation-detec- ta...
Claims
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Login to View More IPC IPC(8): A61K51/12A61M36/06
CPCA61K51/1255
InventorRHEINHARDT, CHRISTOPHERKEMPER, W. SCOTT
OwnerRHEINHARDT CHRISTOPHER