Method for testing a cell sample

Details Method for testing a cell sample Method for testing a cell sample

1998-08-04

2002-07-23

Williams, Hezron (Department: 2856)

Measuring and testing

Liquid analysis or analysis of the suspension of solids in a...

C073S061710, C073S865500, C324S071100, C324S071400, C702S021000

Reexamination Certificate

active

06422065

ABSTRACT:

TECHNICAL FIELD
This invention relates to a method for testing a cell sample or a fluid sample.
Many types of cells as well as artificial cells may be tested according to the method of the present invention, although the test is especially suitable for testing red and white blood cells.
BACKGROUND ART
It is well known that certain diseases give rise to changes in the condition of blood and especially of the red blood cells. Blood characteristics such as red blood cell count, mean cell volume, haemoglobin content and haematocrits are commonly used in the diagnosis of disease.
The measurement of cell volume is one of the most informative investigations in clinical medicine. Cell size is an important indicator of pathology and in conjunction with haemoglobin, forms the basic classification of anemias. It is the basis of the differentiation of white blood cells and leukaemias, the assessment of prognosis in scores of diseases and is an invaluable screening device for the thalassaemias, the most common genetic disease in the world.
Existing methodology achieves good cell size measurements in the majority of patients, failing only in those whose red cell shape differs significantly from normal and in patients with abnormal serum osmolality. As cell shape is normally distributed, 4.56% of the population have red cells that deviate from normal cell shape by more than two standard deviations. These abnormal samples are over represented in hospitals because the deviations are associated with illness more often than those within normal limits. Consequently, clinical laboratories generate incorrect cell volume values in at least 4.56% of reported results, representing about 100,000 incorrect cell volume measurements in the United States each day. In the majority of patients, these results do not cause significant problems but occasionally the erroneous measurements result in misdiagnosis and patient's death. However, this error is rarely recognised because existing automated methods cannot detect it.
A normal human blood sample is isotonic with a solution having an osmolality of about 290 mosm Kg
−1
, and at this osmolality the average red blood cell in the average individual will have a biconcave shape. It is well known that reducing the osmolality of the solution surrounding a red blood cell below a critical level will cause that cell to swell, then rupture, forming a ghost cell which slowly releases its contents, almost entirely haemoglobin, into the surrounding medium. This process, called haemolysis, can be induced using water (osmotically) or by detergents, venoms or other chemicals, thermal, mechanical or electrical agents. Tests to determine cell volume are typically carried out at isotonic osmolality.
Automated measures of cells depend upon both size and shape of the cells being tested. Since existing instruments cannot determine cell shape, instruments such as the particle counter sold under the Trade Mark Coulter Counter by Coulter Electronics Inc., compensate in the calculation of isotonic cell volume with a term that is a fixed average estimate that is in error whenever the cell shape in a sample deviates from the normal population. It is these abnormal samples that indicate pathology where accuracy is most needed that the method most often fails.
In order to estimate the size and the count of the number and properties of red blood cells in a sample, there are several commercial particle counters available which may be used. These particle counters act either by measuring the electrical or optical properties of a stream of cells that pass along a narrow tube. The property measured is usually the current flowing through the suspension in the tube or the electrical field within the tube. The signal generated depends upon several factors including cell size, cell shape and the properties of the cell membrane the difference in the electrical property of the cell and the suspending medium.
DISCLOSURE OF INVENTION
According to a first aspect of 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 by a method including the steps of:
(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 the first or at least one other aliquot of the sample cell suspension to an alteration in at least one parameter of the cell environment which has the potential to alter the shape of the cells to a known or identifiable extent to create an altered cell suspension,
(e) passing said altered cell suspension through a sensor,
(f) measuring said at least one property of the altered cell suspension,
(g) recording the measurement of said at least one property for said altered suspension,
(h) comparing the data from steps (c) and (g) and determining a shape compensation factor to be applied to the measurement of said at least one property of the first aliquot of cells in step (c) in the calculation of a cell parameter to take account of a variation in shape between the first aliquot of cells in step (c) and said altered cell suspension in step (g).
In the present invention, a cell parameter, for example cell volume, is determined by subjecting one or more aliquots of a sample cell suspension to one or more alterations of at least one parameter of the cell environment to identify a point at which the cells achieve a particular shape to obtain a sample specific shape compensation factor.
All existing automated methods include a fixed shape correction in the treatment of sensor readings taken from a single cell suspension in which the cell environment is not altered during the course of the test, which compensates for the deviation of the cells from spherical shape particles commonly used to calibrate the instruments. However, in a calculation of cell volume, as the cell shape is unknown, a fixed correction of approximately 1.5 is entered into the calculation on the assumption that a sample cell has the shape of a biconcave disc. This correction is correct for the average cell in the average person at isotonic osmolality, but it is incorrect for many categories of illness where the assumed fixed correction may induce an error of up to 60% in the estimate of cell volume. In the method of the present invention, an estimate is made of the in vivo cell shape so that a true estimate of cell volume or other cell parameter at all shapes is obtained. In the preferred embodiment of the present invention, a shape correction function is determined which is used to generate a shape correction factor which is a measure of the shape of the cell specific for that cell sample. The value of the shape correction factor generated by this function then replaces the conventional fixed shape correction of 1.5 to obtain a true measure of cell volume and other cell parameters.
According to a second aspect of the present invention, an apparatus for testing a sample cell suspension in a liquid medium in accordance with the method of the first aspect of the present invention comprises data processing means programmed to compare data from said steps (c) and (g) to determine a shape compensation factor to be applied to the measurement of said at least one property of the first aliquot of cells in the calculation of a cell parameter to take account of a variation in shape between the first aliquot of cells and said altered cell suspension.
Preferably, the data processing means comprises the internal microprocessor of a personal computer.
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, and this is measured as in the normal Coulter Counter by determining the flow of electrical current through the cell suspension as it passes through a sen

Affiliated with

Also associated with

Rating

Method for testing a cell sample does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method for testing a cell sample, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for testing a cell sample will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2894598