Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1998-08-04
2001-04-17
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S008000, C436S018000, C436S174000, C436S179000, C435S002000, C435S372000
Reexamination Certificate
active
06218190
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method in which a sample of cell suspension is analysed to determine a parameter of the cells relating to size, for instance volume, diameter or shape. In the method the cells are tested after dilution and the size measurement is corrected to the environmental conditions, especially osmolality, of the sample as supplied.
BACKGROUND ART
The automated testing of blood samples has become an important part of many medical investigations. Automated instruments' speed and ease of use has made them a preferred method of producing a complete blood count (CBC) in hospital laboratories. As well as producing values of the number of cells in a sample, the apparatus is used to measure the average size of the cells.
Electronic particle counters typically use a sensor which detects particles in a restricted flow, producing a measure of particle size and count for each particular type of particle. The sensor usually detects a change in an electrical field, an alteration in the light scatter from a laser, a change in the magnetic field density or magnetic flux, or changes in the optical, acoustic or other physical properties of the cells or cell suspension and/or suspending liquid. Whatever type of sensor is used, it produces a signal which is a product of a particle's size, shape, trajectory, number and other properties, some of which may be measured concomitantly. Electronic particle counters which use a direct or alternating current as a method to detect particles can be referred to as electronic particle sizing devices (hereinafter referred to as EPS), and produce a characteristic change in voltage or current, usually recorded as a voltage pulse as a particle passes through a restriction (aperture).
Electronic particle sizing relies on two electrodes suspended in a conducting solution which are isolated from each other except for a single conducting channel which is traversed by cells or other small particles in suspension. As a particle passes through the channel (aperture) the physical characteristics of the channel temporarily alters in proportion to the particle's size. By measuring the properties of these changes, the size and concentration of particles is determined. This is performed on red cells, white cells and platelets and any free cell suspension and may be combined with stains or other techniques to further is differentiate the cells by any means (i.e. optically, NMR etc).
Commercial EPS instruments do not use an isotonic diluent to produce a cell suspension, but instead use a hypertonic diluent typically phosphate buffered saline (PBS) fixed at 30 to 50 mOsm/Kg above the population's mean plasma osmolality. It is well known that there is a natural range of plasma osmolalities amongst individuals. It is also well known that for all permeable cells with non-rigid walls, cell size varies with the osmolality of the buffer in which it is suspended. When performing an automated cell test, such as a CBC, manufacturers test cells in a single fixed hypertonic osmolality that has no known relationship to the osmolality of the plasma of the patient being tested. This causes errors in the size estimates produced because cells tend to swell upon standing in hypotonic buffers and shrink in hypertonic buffers and because the amount the cell swells in any buffer is patient specific. Manufacturers reduce this source of error by using hypertonic buffers which induce the least change in cell size over time (ie aging before testing) at the cost of accuracy and usually results in smaller cell size.
The method of the invention overcomes the above problems by testing cells at their in vivo osmolality, thereby minimizing the possibility of swelling over time and mimicking the cell's in vivo condition in respect of its osmotic environment. The most accurate in vitro measures of in vivo cell size is possible if cells are tested in a buffer of the same osmolality as their plasma, as this removes the need for “correction factors” to adjust for incorrect buffer osmolality. In existing technology these correction factors are always fixed because the individual's plasma osmolality is unknown or is not used in existing tests. Since it is reported that patients sometimes have a plasma osmolality 50 to 100 mOsm/Kg below the population mean and since manufacturers use buffers up to 50 mOsm/Kg above the mean, gross errors are sometimes induced. Although these large deviations are uncommon, they tend to occur in patient with severe illness in whom errors can easily have serious consequences. The present method circumvents this problem in two ways; either by measuring the patient's plasma osmolality and adjusting the buffer osmolality to match, or by testing the cells in their own plasma.
It is known that cells, especially blood cells, act as osmotic devices. Reducing the osmolality of the solution surrounding a red blood cell below a critical level will cause that cell first to swell, then become spherical, and form a ghost cell which slowly loses its contents, almost entirely haemoglobin, into the surrounding medium. This process, called haemolysis, can be induced osmotically using water, or by detergents (eg soap), venoms or other chemical, thermal, mechanical or electrical agents.
It is known that the critical osmolality at which haemolysis occurs can be determined by subjecting aliquots of a red blood cell suspension to a concentration gradient which may include stepped or continuous changes in concentration, for instance as described in DE-A-3103792.
It has been recognised that the osmolality of the plasma may vary from individual to individual and, occasionally, within an individual over time. When a sample of blood of an individual having plasma with abnormal osmolality is subjecting to particle sizing, the value which is obtained for the cell size when diluted in isotonic saline will be different to the in vivo cell size. The present invention seeks to correct such errors.
DISCLOSURE OF INVENTION
In a new method according to a first aspect of the invention a cell suspension is subjected to the following steps:
1) a portion of the cell suspension is diluted with a diluent which has the same osmolality as the extracellular liquid of the cell suspension (plasma), and
2) the cells in the diluted suspension formed in step 1 are subjected to a cell size measurement while suspended in the said diluted suspension.
In this aspect of the invention the diluent used in step 1 may be constituted by plasma separated from the starting cell suspension.3 Separation may, for instance, be carried out manually or by other means. In this case it is unnecessary to determine the osmolality of the plasma to produce an accurate osmotically correct cell size. It may, however, be preferred in addition for a reference measurement of cell size to be conducted using, in place of the plasma, a diluent of known composition having an osmolality equal to that of the plasma, which requires a determination of that osmolality. Alternatively the reference measurement of size may consist of a series of measurements in varying osmolality diluents as described below in step 5. This allows a determination of the size measurement by interpolation. These reference tests allow confirmation to be obtained that the plasma used as diluent in step 1 is not having an effect on the values obtained in the measurement step 2. If the values are different further investigations have to be made.
Alternatively for step 1 the osmolality of the starting cell suspension is determined and a diluent is then made up to that osmolality. The diluent may be made up using appropriate buffers of known concentrations in suitable amounts (eg using suitable amounts of hypotonic and hypertonic fluids). The osmolality is determined, for instance, by measuring the osmolality of the plasma of the starting cell suspension, usually after separating a portion of the cell suspension into cells and plasma. The osmolality can alternatively be calculated using standard blood chemistry analysis which
Shine Ian Basil
Shine Thomas Adam
Burns Doane Swecker & Mathis L.L.P.
Wallenhorst Maureen M.
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