Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2000-08-29
2004-03-02
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C356S337000, C422S073000
Reexamination Certificate
active
06699680
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of analysing a sample of free cells, in particular blood cells.
BACKGROUND TO THE INVENTION
Automated block analysers which count and size blood cells, represent a huge advance in the field of chemical medicine, but retain some drawbacks. They are inherently incapable of differentiating like sized particles. While the automated count may be correct in terms of the total number of particles, traditional methods do not count small red cells, parasites or bits of cells as platelets. Anyone in need of good quality platelet counts is well advised to travel to Africa or Asia which, having few automated blood analysers, provide better quality complete blood counts because manual methods of counting avoid several errors which are common concomitants of automated sizing and counting instruments. Errors in counting platelets have serious consequences for the patient as they may result in unnecessary tests, inappropriate treatment, or mis-diagnosis. The known causes of spurious low platelet counts are EDTA dependent clumping, cold platelet agglutination, platelet satellitism and the presence of like sized particles which are not platelets.
All haematology texts, laboratories and manufacturers have been aware of the inaccuracies of automated platelet counts for many years and advise a manual inspection of a blood film for every patient with an abnormal platelet count, which is the practice in most haematology laboratories. Currently manufacturers are attempting to reduce errors by detecting light refraction (since platelets are more refractile) or by detecting stains applied to platelets before they reach the sensor. The practice of manual verification whenever the platelet measurements fall outside the normal range is comforting to the patient but expensive for the laboratories. However, hitherto, there was no method of alerting labs of the need for manual verification for patients with high platelet counts that erroneously fell inside the normal range. Thus automated platelet counts are of limited value when platelet counts are low, they are of uncertain value when the counts are normal, and are not entirely secure when the counts are elevated (thrombocytosis).
As will be discussed below, the applicants have discovered that there are other small particles, cell fragments, which are also not detected and properly distinguished by existing automated blood cell analyzers. The importance of cell friability or the generation of cell fragments has hitherto not been recognised.
SUMMARY OF THE INVENTION
According to one aspect of the present invention, there is provided a method of determining a measure of the number of platelets in a cell suspension containing platelets, the method comprising the steps of: counting the number of small particles in the suspension; agitating the suspension in the presence of a gas; counting the number of small particles in the suspension after agitation; and comparing the two counts to obtain a measure of the number of platelets.
The method of the invention differentiates platelets from other small particles exploiting a simple physiological property and removes spuriously low automated platelet counts. The accurate measurement of platelet number and function is of great consequence to a patient's health. Aside from bleeding to death from thrombocytopenia (insufficient platelets), platelets are risk components of strokes, heart attacks, and inflammation, and are important elements in the growth of epithelial malignancies and metastases.
All red cells are very sensitive to osmotic stress, and most cells are sensitive to mechanical stress. It has been found that platelets are relatively insensitive to mechanical and osmotic stress except when exposed to contact with air. Platelet suspensions alter their properties upon exposure to air, or its component gases to a greater extent if they are simultaneously stressed. By carefully controlling the handling of platelets before and during testing, by eliminating or regulating a suspension's exposure to air, more accurate platelet counts can be obtained. By intentionally exposing platelets to air while subjecting them to stress any induced change in the platelet population can be recorded. Because existing methods ignore the effect of air, they are inducing errors in their platelet counts.
The methods described in WO 97/24598, WO 97/24599 and WO 97/24601 provide a way to measure the size, shape, and number of particles while the particles are simultaneously exposed to a variety of osmotic gradients. However, further information may be derived from this test by combining it with the present invention. For example, by testing a whole blood suspension under osmotic stress, and comparing the results to the same sample after mechanical agitation in the presence of air, the components of the sample population which are altered by mechanical agitation in the presence of air can be determined. Furthermore, the types and proportion of each type can be revealed in a single procedure. Thus, red cells, white cells, micro-spherocytes, and bacteria are uninfluenced by mechanical agitation in the presence of air whereas platelets alone disappear. The advantage of this method in that in addition to explaining a way to differentiate and count platelets, it offers existing instruments a very simple method of increasing the accuracy of their counts.
In this application we also disclose a method of measuring cell friability since mechanical stress induces fragmentation in those cells. When cells are counted in cell counters, neither lysis nor fragmentation nor ghosts are induced. When cells are subjected to osmotic stress they lyse and ghost or fragment or both. The addition of mechanical agitation enhances the effect and the addition of air into the agitated suspension further intensifies the effect. Thus, in order to differentiate between cell fragments and platelets, it has been found to be preferable to compare the count of small particles before and after agitation in the presence of air against osomolality. This process of inducing the nature of these small particles can be likened to the identification of an unknown fluid by raising its temperature; if it boils at 78° C. it is ethyl alcohol, if it boils at 100° C. it is water and at 357° C. it is mercury.
In order to provide quantative measures from the present invention, it is preferable to monitor at least one of the following: A) the air quantity in contact with the suspension, B) the intensity of agitation and C) the duration of agitation, and to relate this measured value to the difference between the two counts.
The small particles counted in the method of the invention include platelets, bacteria, cell fragments, and micro-spherocytes, which typically have a volume of 7-10 femtoliters.
According to another aspect of the present invention, a method of analysing a sample of free cells in vitro comprises applying a known or identifiable quantity of stress to the sample, measuring the sample before and after the application of stress to provide at least one reading from which quantitative information relating to the number of cell fragments in the sample caused by the applied stress can be determined, and relating this reading to the quantity of stress to provide an indication of cell friability.
When red cells die, they lose their contents, a process termed lysis. They are then transformed into either ghost cells or fragments depending, in part, on the cell's membrane properties, and in part on the provocation. Hitherto, this mechanism has been little recognised or understood.
The present invention is based on the realisation that if cells, such as blood cells, and in particular red cells, are stressed in vitro the relationship between the applied stress and fragmentation to the sample is characteristic for normal samples and for many diseases. Since some cells (platelets) produce no detectable fragments and some (red cells) produce a large number this method also provides a way of distinguishing betw
Shine Ian Basil
Shine Thomas Adam
Gitomer Ralph
Perman & Green LLP
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