Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1998-11-19
2001-01-16
Jones, Dameron (Department: 1619)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S007200
Reexamination Certificate
active
06174698
ABSTRACT:
BACKGROUND OF THE INVENTION
Under various cell environments, most biological cells can drastically change their shape (called cell “deformability”) without structural damage to the cell membrane, without a loss in cell contents to the cell environment, and without a change in the chemical function of the cell membrane. Under other cell environments, the cell membrane can lose chemical function, can suffer structural damage, and can rupture to lose cell contents to the cell environment (called cell fragility). Thus, cell deformability and cell fragility are two independent characteristics of a cell membrane. Cells can have any combination of a high-to-low deformability and a high-to-low fragility. However, most normal cells have high deformability and moderate-to-low fragility.
Deformability is one important characteristic for some cells such as “sensory receptors” which are normally stationary in the cell environment and for other cells such as blood and lymph cells which normally move in the cell environment. Cell fragility is an important characteristic for almost all normally stationary and normally moving cells because changes in the cell environment can cause considerable amounts of water to move into cells to rupture more fragile cells. Changes in cell fragility can change the ability of a cell to perform its normal function in a normal or abnormal cell environment. For example, an abnormally high fragility of red blood cells will lead to premature rupture of many red blood cells in a normal blood environment which will reduce the circulating pool of red blood cells and will reduce the red blood cell transport of oxygen to tissues. Yet, these highly fragile cells can have normal cell deformability. Thus, fragility might clinically be a more important cell characteristic than deformability.
Cell fragility is altered by many conditions such as cell age, duration of blood-bank storage, treatment with a variety of membrane-binding drugs, and progression of membrane or hemoglobin-related diseases such as diabetes and sickle-cell anemia, respectively. Thus, a rapid, highly accurate, and easily applied method is needed for clinical measurements to assess cell fragility as an index of “cell strength” which is defined as the degree to which the cell membrane can maintain its structural integrity and its chemical function in a normal and an altered cell environment. Most current methods for assessing cell strength primarily create mechanical forces on cells to assess either the deformability or the fragility of these cells. These methods include Osmotic-Gradient Ektacytometry, and Cell Filtration, Micropipette Suction, and Osmotic Fragility tests.
Osmotic-Gradient Ektacytometry is one method that applies mechanical forces to measure a combination of cell deformability and cell fragility. This method uses a viscometer device to measure shape changes which are induced in red blood cells by various speeds of rotation (called applied shear stress) of the test cells with different osmotic solutions in the cell environment. The osmotic-spectrum curves which are produced by Ektacytometry are highly variable for the same test-cell sample due to small changes in sample ambient temperature, pH, and plasma osmolality. Thus, these osmotic-spectrum curves are complex and very difficult to use for interpretation of changes in cell deformability. As a result, Ektacytometry currently requires very sophisticated equipment, extensive operator training, and highly controlled test conditions to obtain one measurement value for the combination of cell deformability and fragility. This makes Ektacytometry usable only in a few research laboratories and not in a clinical setting.
Other methods such as Cell Filtration and Micropipette Suction also require the external application of a mechanical force to cells but these methods primarily measure cell deformability by forcing (filtering) cells through various size pores or by mechanical aspiration of these cells into micropipettes of fixed tip size and taper. These methods also require very sophisticated equipment and substantial operator training; and they are extremely time consuming to obtain an analysis of only a relatively few cells in each test-cell sample. Thus, these methods have also not been accepted into general clinical use.
Assessment of cell deformability in the Cell Filtration method has been improved by measurements of electrical impedance (Hanss et al, U.S. Pat. No. 4,835,457) and measurements of time (David D. Paterson, U.S. Pat. No. 4,491,012) during the application of external mechanical force (pressure) to force red blood cells to pass through either an artificial membrane filter or a foil system (Helmut Jahn, U.S. Pat. No. 4,797,606). These measurement methods are extremely sensitive to manufacturing tolerances on the filter or foil and both of these measurement methods primarily assess only cell deformability and not cell fragility. Thus, these measurement additions to the Cell Filtration method have also not found their way into common clinical use.
The Osmotic Fragility method was one of the earliest techniques that was developed for assessment primarily of red blood cell fragility rather than deformability, and it is one of the few methods currently in clinical use. The Osmotic Fragility method is time-consuming, and it requires multiple blood handling steps, and relatively large volumes of blood samples. The Osmotic Fragility method uses exposure of blood samples to a large range of salt concentration to poroduce a large range in the osmotic pressure for the cell environment, the osmotic pressure mechanically forces water into cells to swell cells to the point of cell membrane rupture.
The osmotic pressure method has been adapted by Groves and Rodriguez (U.S. Pat. No. 4,535,284) to apply high frequency and low frequency electrical currents to detect the percent of red blood cells that are altered during application of osmotic mechanical forces over a large range of osmotic cell environments. This use of high frequency and low frequency electrical currents is combined with Coulter Counter® to classify individual red blood cells as normal or abnormal.
Overall, the osmotic mechanical gradient method and various refinements to this method can only detect relatively large changes in cell membrane fragility because this osmotic-based machanical method provides no information about the rate of cell lysis (which occurs when the cell membrane ruptures). The lack of method sensitivity to mild or moderate changes in cell fragility has led to the clinical use of the osmotic mechanical method only for diagnosis of one disease called hereditary spherocytosis.
In contrast to methods that apply external mechanical forces mostly to measure cell deformability or in one case (osmotic gradients) to measure cell fragility, there is a chemical method which changes the mechanical characteristics of cell membranes to assess cell membrane fragility. It was first shown some fifty years ago that some chemicals can be changed by light (called photoactivation) to induce the rupture or breakup of red blood cell membranes (called hemolysis) in a test tube. Since then, this basic process (called photohemolysis) has been extensively studied in a variety of test-tube experiments.
The mechanism for photohemolysis is oxygen dependent and is thought to involve the generation of singlet oxygen with the subsequent oxidation of proteins in the red blood cell membrane. It has been suggested that this protein oxidation leads to the creation of extra water channels in the cell membrane. This would increase passive cationic exchange across the cell membrane to give a subsequent influx of water into the cell to swell the cell to the point of hemolysis. It has been suggested that photohemolysis could also involve peroxidation of the lipid layers in the cell membrane. This peroxidation appears to limit the ability of molecules to move (called membrane fluidity) in the lipid bilayer of the cell membrane, which then appears to limit the ability of the cell to undergo shape cha
Carrithers David W.
Carrithers Law Office
Jones Dameron
Micro-Med, Inc.
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