1. Field of the Invention
This invention relates to a particle analyzing apparatus and method for measuring the nuclear shift index of particles by suitably preparing a liquid specimen such as blood, passing particles such as white blood cells contained in the specimen through a detecting zone to detect signals corresponding to the particles, and processing the detected signals.
2. Description of the Prior Art
White blood cells present in human blood are classified into monocytes, neutrophils, eosinophiles and basophils, and determining the numbers of particles according to class or the content of these particles as a percentage is a useful tool in clinical examination. Accordingly, in order to classify white blood cells into the aforementioned particles and enumerate the same automatically, apparatus heretofore developed for this purpose are adapted to dilute blood with a diluent, supply the diluted blood to a detector to detect any electrical or optical change produced when the blood cells pass through the detector, classify the particles and count the same.
A first conventional apparatus of this kind is adapted to destroy red blood cells by using a hemolytic agent to obtain an electrolyte in which only white blood cells are suspended, pass the electrolyte through a detector provided with pores, and detect a change in electrical impedance (e.g., electrical resistance) at the porous portion, the change occurring when white blood cells pass through the pores. This apparatus enables white blood cells to be identified based on a difference in the magnitude of the detected signal.
A second conventional apparatus is adapted to pass a dilute solution of blood, which is in the form of a fine stream, through the central portion of a flow cell, and irradiate the fine stream with light to detect an optical change, such as a change in fluorescence or scattered light, produced when the blood flows through the cell. With this apparatus, white blood cells can be identified based on a difference in fluorescent intensity or intensity of scattered light detected by staining the white blood cells.
The porous portion constituting the detecting section of the first conventional apparatus covers an area that is considerably large in comparison with particle size. For this reason, particles cannot be detected on a microscopic scale. By way of example, if particles having a diameter of several microns are to be detected, the pores would have to possess a hole diameter and pass length on the order of tens of microns to 100 microns in order to prevent clogging. In addition, the only information acquired relates to particle size.
With the second conventional apparatus, the detecting zone can be made smaller than the size of the particles by narrowing down the irradiating light flux. By this reducing the size of the detecting zone, particles can be detected on a microscopic scale. In other words, various intrinsic characteristics possessed by the particles can be detected in greater detail, so that a greater amount of information can be extracted.
For example, as set forth in the "Bulletin of the Electrotechnical Laboratory" by Yoshio Nomura, Vol. 44, No. 3, pp. 185-186, and in "Flow Cytometry and Sorting" by L. L. Wheeless, et al., pp. 125-135, an apparatus is available in which irradiation is performed using a slit-shaped laser beam. Specifically, as shown in FIG. 10, a slit-shaped laser beam 124 having a width of approximately 4 .mu.m is projected in a direction perpendicular to that of cell flow, and fluorescent profile is measured when the cells cross the laser beam 124. The detection signal thus obtained is illustrated in FIG. 11. Signal widths C and N are commensurate with the diameters of cell 120 and nucleus 122. Accordingly, the ratio of nuclear diameter to cell diameter is obtained from N/C. Using the slit beam also makes it possible to take measurements to determine whether polynuclear cells are present.
Further, in "Cytometry" by L. L. Wheeless, et al., Vol. 5, pp. 1-8, an example is described in which detection error ascribable to cell orientation is prevented. To this end, an X-Y-Z slit scanning method is used in which X and Y axes are taken in a plane containing the slit beam and the direction of cell flow is taken as the Z axis, with the fluorescent profile being analyzed in the X, Y and Z directions. Both parameters, namely the N/C ratio and nuclear fluorescent intensity obtained, are used to enable cell discrimination.
A problem encountered with the first conventional apparatus is that only particle size information can be obtained, as mentioned earlier. Therefore, in order to classify and quantify white blood cells, it is required that the group of white blood cells in each class be made large enough to enable it to be distinguished from groups of white blood cells in other classes. This means that the hemolytic agent must be carefully selected, and that measurements must be taken while strictly controlling such measurement conditions as temperature. However, since the detection principle from the outset is based on particle size, this would make it impossible to detect the various characteristics possessed by the particles. For example, it would not be possible to detect the state of nuclear shift of the cells.
An advantage of the second conventional apparatus is that many characteristics can be detected from a single particle by reducing the size of the detecting zone, as mentioned above. However, nowhere does the aforementioned literature describe determining the nuclear shift index of white blood cells, which is one characteristic possessed by white blood cells, or a technique for achieving this.
Nuclear shift index rises as the maturity of white blood cell granulocytes progresses. FIG. 12 is a view for describing the nuclear shift of neutrophils cited in "Clinical Laboratory Methods" by Masamitsu Kanai, et al., 28th Edition, Vol. 6, p. 50. An increase in neutrophils with a small number of lobes is referred to as "left shift". If an increase in the total number of white blood cells appears at this time, this indicates a highly active myeloid function and the likelihood of leukemia. If a reduction in the total number of white blood cells appears, myeloid function is considered to be impaired and the patient is readily susceptible to infection. An increase in neutrophils with large number of lobes is referred to as "right shift". There is often a decrease in the total number of white blood cells in this case as well. This is considered to indicate pernicious anemia.
Thus, determining the lobe state of specific cellular nuclei in various white blood cells by examination has great clinical significance.