Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
1998-08-04
2003-12-23
Moran, Marjorie (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S002000
Reexamination Certificate
active
06668229
ABSTRACT:
This application is a §371 application of PCT/GB96/03256, filed Dec. 27, 1996.
TECHNICAL FIELD
The present invention relates to a method of measuring cell membrane permeability and is applicable to all types of cells, including red cells, white cells, platelets, fibroblasts, tissue cells, amoebae, fungi, bacteria, all eucaryotic and procaryotic cells as well as synthesized cells or particles.
BACKGROUND ART
Permeability is the passage of matter in a fluid or gaseous state through another material, usually in a solid state, measured as a rate or total volume transferred across a membrane per unit time per unit surface area at standard temperature and pressure. Biologically, many membranes, especially cell membranes, are selectively permeable enabling cells to transfer nutrients, hormones, gases, sugars, proteins or water across their membranes. This transport may be passive, depending solely upon the partial pressures or concentrations of the substances on either side of the membrane or it may be active, requiring energy to counter existing concentrations. Different cells have different molecule specific rates of permeability which are closely related to the cell's function.
Current tests of red cell permeability produce a single value for permeability, typically by measuring the change in concentration of a radio labelled molecule (often water) in or around a cell (or a population of cells).
DISCLOSURE OF INVENTION
According to the present invention there is provided a new method in which a sample of cells suspended in a liquid medium, wherein the cells have at least one measurable property distinct from that of the liquid medium, is subjected to analysis to determine a measure of cell permeability of the sample of cells by a method including the steps:
(a) passing a first aliquot of the sample cell suspension through a sensor,
(b) measuring said at least one property of the cell suspension,
(c) recording the measurement of said property for the first aliquot of cells,
(d) subjecting a second aliquot of the sample cell suspension to an alteration in at least one parameter of the cell environment which has the potential to induce a flow of fluid across the cell membranes and thereby alter the said at least one property of the cells,
(e) passing said second aliquot through a sensor,
(f) measuring said at least one property of the cell suspension under the altered environment,
(g) recording the measurement of said at least one property for the second aliquot of cells,
(h) comparing the data from steps (c) and (g) as a function of the extent of said alteration of said parameter of the cell environment and change in the recorded measurements of said at least one property to determine a measure of cell permeability of the sample.
Blood cells travel through the entire body once a minute continually transporting gases and metabolites. Blood cells also act as messengers or surrogate hormones, transmitting information around the body. It has been discovered that this peripatetic existence allows the blood cells to signal distant pathology. For example, when the brain dies, when a limb has an occluded blood supply or the kidney fails to remove essential toxins, the blood cell's membrane permeability changes. Cell membrane permeability has never been measured routinely and only rarely measured experimentally. Until now, there have been no rapid or reliable methods of performing such measurements. It has also been discovered that red cell permeability is complex, dynamically changing as molecules cross the cell's membrane depending on, for example, the shape and structure of the cell and membrane pump activity. The method of the present invention produces existing measures of permeability, but more usefully it produces more sensitive, accurate and descriptive measures of cell permeability within sixty seconds with no sample preparation.
Preferably, the property of the cells which differs from the liquid medium is one which is directly related to the volume of the cell. Such a property is electrical resistance or impedance which may be measured using conventional particle counters such as the commercially available instrument sold under the trade name Coulter Counter by Coulter Instruments Inc. Preferably, the sensor used to detect cells and measure a change in the cells' property is that described in our co-pending International application (Agent's reference 62/2681/03). In this apparatus the cell suspension is caused to flow through an aperture where it distorts an electrical field. The response of the electrical field to the passage of the cells is recorded as a series of voltage pulses, the amplitude of each pulse being proportional to cell size.
In the preferred method of the present invention, a measurement of cell permeability is determined by obtaining a measure of the volume of fluid which crosses a sample cell membrane in response to an altered environment. The environmental parameter which is changed in the method may be any change which results in a measurable property of the cells being altered. Preferably, a lytic agent is used to drive fluid across the cell membranes and thereby cause a change in cell volume. Preferably therefore, the environmental parameter change is an alteration in osmolality, most preferably a reduction in osmolality. Typically, the environment of the first aliquot is isotonic and thus the environment of the second aliquot is rendered hypotonic. Other suitable lytic agents include soap, alcohols, poisons, salts, and an applied shear stress.
It is possible to subject only a single aliguot of sample suspension to one or more alterations in osmolality to achieve this effect, although is preferred to use two or more different aliquots of the same sample suspension. Most preferably, the sample suspension is subjected to a continuous osmotic gradient, and in particular an osmotic gradient generated in accordance with the method of our co-pending International application (Agent's reference 80/4936/03).
In the preferred method of our co-pending International application (Agent's reference 62/2734/03), a number of measurements of particular cell parameters are made over a continuous series of osmolalities, including cell volume and cell surface area, which takes account of the deviation of the cells from spherical shape particles commonly used to calibrate the instruments. An estimate of in vivo cell shape made so that an accurate measurement of cell volume and cell surface area at all shapes is obtained. A sample suspension is fed continuously into a solution the osmolality of which is changed continuously to produce a continuous concentration gradient. Reducing the osmolality of the solution surrounding a red blood cell below a critical level causes the cell first to swell, then rupture, forming a ghost cell which slowly releases its contents, almost entirely haemoglobin, into the surrounding medium. The surface area of the each cell remains virtually unchanged on an increase in cell volume due to a reduction in osmolality of the cell's environment as the cell membrane is substantially inelastic. The time between initiation of the alteration of the environment in each aliquot to the passage of the cells through the sensing zone is kept constant so that time is not a factor in any calculation in cell permeability. An effect of feeding the sample under test into a continuously changing osmolality gradient, is to obtain measurements which are equivalent to treating one particular cell sample with that continuously changing gradient.
Preferably, the measurements are recorded on a cell-by-cell basis in accordance with the method of our co-pending International application (Agent's reference 62/2734/03). The number of blood cells within each aliquot which are counted is typically at least 1000 and the cell-by-cell data is then used to produce an exact frequency distribution of cell permeability. Suitably this density can be displayed more visibly by using different colours to give a three dimensional effect, similar to t
Shine Ian Basil
Shine Thomas Adam
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