Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
2002-08-23
2004-06-01
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S008000, C436S010000, C436S164000, C436S149000, C436S150000, C422S073000, C422S082010, C422S082020, C422S082050, C422S082090, C435S002000
Reexamination Certificate
active
06743634
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and apparatus far differentiating various types of blood cells on the basis of their respective light-scatter signature. More particularly, It relates to a method and apparatus for differentiating blood cells, and especially platelets and basophils, on the basis of their back-scatter signature.
BACKGROUND OF THE INVENTION
The use of light scattering measurements as a means for differentiating various types of small particles is well known. For example, in virtually all sophisticated hematology instruments, it is common to measure the forward light scattering properties of blood cells by passing the cells, one at a time, through the interrogation zone of an optical flow cell. While in the interrogation zone, each cell is irradiated by a laser beam, and one or more photodetectors, strategically positioned forward of the interrogation zone, operate to sense the level of forward scattered radiation, often within several different predetermined angular ranges. In addition to measuring forward light scatter, some hematology instruments measure side scatter as well, using a separate photodetector located orthogonally of the irradiated cell. These light scattering measurements are often combined with other simultaneously made measurements to better differentiate cell types of particular interest from other cells and other particulate material within the sample that have similar light-scattering properties within the angular ranges measured. These other simultaneously-made measurements include those representing the cell's physical volume, its electrical conductivity, and its effectiveness in attenuating the irradiating beam by virtue of its presence in the beam (sometimes referred to as the axial light loss (ALL) of a cell). Having made various cell parameter measurements, the instrument then produces scattergrams in which the different parameters measured are plotted against each other. Ideally, each sub-population of cells of the same type appears in these scattergrams as a tight cluster of data points, each point representing an individual cell, and each cluster being readily identifiable from other clusters by a clearly identified spacing between the clusters. In such case, it is a simple matter to “gate” cells of one cluster from those of another cluster and to enumerate the cells of each type within the gate. Unfortunately, this ideal is sometimes difficult to realize since, for many reasons, a small percentage of cells of one type invariably invade the spatial domain of cells of other types, thereby making the cell count of a cell type of interest somewhat imprecise. As noted below, this is especially true in the case of platelet and basophil differentiation.
In an article entitled “Flow Cytometric Measurement of Platelet Function and Reticulated Platelets,” by Kenneth A. Ault, Annals New York Academy of Sciences, 677:293-308 (1993), the importance of platelet analyses for clinical applications is discussed. This article also discusses the problem of identifying and enumerating platelets using conventional light-scattering techniques. It is noted that the concentration of platelets in a normal whole blood sample is relatively high, being second only to the concentration of red blood cells (erythrocytes). One milliliter of blood normally contains about 250 million platelets. Thus, while they are about 20 times less frequent than red cells, platelets are about 25 times more frequent than all types of white cells (leukocytes) combined. While their normal size range (1 to 4 microns) enables platelets to be readily identified from other types of normal cells in a blood sample, their cluster in a scattergram usually contains a large amount of cellular debris (fragments of all cells) that “look like” platelets in terms of their normally measured volume and forward light scattering properties. Ault notes an uncertainty in differentiating platelets from cell debris on the basis of forward and side-scatter measurements alone, and he describes a more reliable technique based on both forward light scatter and fluorescence measurements. While the light scatter/fluorescence technique described by Ault does provide a more positive identification of platelets than the noted light scatter alone technique, this technique requires the additional step of selectively tagging or labeling platelets with fluorescent dye molecules, either directly or via suitable monoclonal antibodies that have been tagged with a fluorescent marker. This tagging step, of course, is both time-consuming and costly. Further, this tagging of platelets subjects the platelets to considerable agitation or manipulation, which has an undesired effect on platelet activation.
In U.S. Pat. No. 6,025,201 to D. Zelmanovic et al., several different techniques for differentiating and counting platelets are noted. The patent disclosure is directed to a method for assessing the activation state of platelets by determining their respective dry mass and refractive index. A preferred method comprises the steps of measuring the forward light-scatter of platelets irradiated by a laser beam within two different light scatter ranges, a low range of between 1 and 7 degrees, and a high range of between 5 and 20 degrees. Alternatively, the forward light-scatter measured within one of the two preferred scatter ranges is combined with a DC volume measurement, and a Mie Scattering Theory-based analysis is performed to provide platelet counting and analysis. This platelet analysis scheme is considered advantageous over the above-noted Ault technique in that it lends itself to full automation, requiring no off-line sample preparation for fluorescent tagging purposes. The same can be said, of course, for the many different fully-automated platelet analysis techniques used commercially to date. These techniques include (a) the total impedance scheme (used in the Model STKS™ and Model GEN•S™ blood analyzers manufactured and sold by the assignee hereof) where platelet volume is determined by monitoring the change in electrical impedance of a restricted aperture caused by a platelet passing through it; (b) the total light-scatter scheme (used in the TECHNICON H* System instrument made and sold by Bayer, and in the ORTHO ELT-8 instrument made and sold by Ortho Diagnostics) where the intensity of forward scattered light is monitored within one or more angular ranges, such scatter intensity being proportional to platelet volume; and (c) the combined impedance and light scatter scheme used in the Cell-Dyn® 4000 blood analyzer made and sold by Abbott Laboratories) where cell volume is determined both electrically (via aperture impedance) and optically (via forward light scatter) simultaneously. As indicated above, however, all of these schemes are susceptible to cross-contamination by non-platelets, introducing a certain degree of uncertainty in the data reported. The impedance approach is problematic in that any cell debris having a volume similar to that of a normal platelet will be displayed and counted as a platelet. The forward scatter approach is problematic in that cellular debris of the same size or volume as a platelet will produce a forward-scatter signature similar to a platelet and thereby be counted as a platelet. Further, in making such light scatter measurements, it is common to use relatively large surface area photodetectors (typically pin diodes) to collect the scattered light. Unfortunately, such large detectors are problematic in that they also collect stray light reflected by various surfaces within the optical system and thereby produce signals having a relatively low signal-to-noise ratio. Even when shaped to exclude the collection of stray light (e.g., shaped as a circular ring centered about the irradiating beam axis), these photodetectors still require a substantial surface area to achieve the gain necessary to sense the scattered radiation, and the larger the surface area, the slower the electrical response time. Schemes that combine the electrical and optical
Alter Mitchell E.
Coulter International Corp.
Kurz Warren W.
Wallenhorst Maureen M.
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