Patent GB-1425972-A (GB)
Title: System for differentiating particles
IPC: G01N15/14
Filed: 1973-07-16
Abstract: 1425972 Photo-electric cell-shape analysis BLOCK ENG Inc 16 July 1973 [14 Aug 1972] 33786/73 Heading G1A The shape of a cellular body relative to a sphere is determined by making optical measurements giving outputs related to two of the surface area, the volume, the mean thickness, and the mean cross sectional area, determining the value of a relationship between these two measurements, and comparing this value with the corresponding value produced by a spherical body. As shown, Fig. 1, a blood sample is mixed with a nuclear dye, the excess dye is dialyzed, and the sample is diluted and pumped through a flow tube where it is surrounded by a sheath fluid whose composition is selected to produce desired values of refractive index, viscosity and osmotic pressure. The cells pass singly through the flow tube 52 where they are examined photo-electrically to produce at least two shape dependent functions X, Y which are combined in such a way that the resulting signal is independent of the actual size of the cell. This signal is applied to comparators 90, 92, 94 together with reference signals and the outputs counted, these counts representing the number of cell nuclei having better than a specified degree of sphericity. The device 86 for producing a given power of the signal X may be a diode function generator. Since the signals X and Y may be produced sequentially a suitable delay (not shown) may be inserted in the line 84 or sample-and-hold techniques may be used, Instead of being counted the outputs of the comparators may be correlated with other measurements made on the cells. If when diluted the concentration of the dye in solution is sufficiently low the dialysis may be omitted. The photo-electric system, Fig. 2, comprises a Xenon lamp 62A and lenses 66 and 68 which cooperate with a mask 70 having three parallel slits 72 to illuminate the centre of flow tube 52 at three closely spaced points. The transmitted light is focused by a lens 74 on to a second mask 76 having corresponding apertures 78 behind which are located three different filters 80 and associated detectors 82, the beams through the outer apertures being deflected by mirrors or prisms 79. To measure scattered light an annular stop may be placed in front of a detector to eliminate the directly transmitted beam. Fig. 3 (not shown). The mean thickness is determined by making measurements at a first wavelength at which the nuclei of white cells are optically dense so that the transmission is affected by self shadowing, and at a second wavelength at which the nuclei are optically thin to produce a volume dependent signal. The ratio of these measurements gives the desired output (Fig. 4). Since the apparent thickness depends on the orientation of the cells  relative to the beam it is preferable to use wide angle illumination and this may be achieved by filling the space between the lens and the flow tube with a fluid of high refractive index. The volume of the nuclei may be determined by transmission as mentioned under measurement of mean thickness, or by measuring fluorescence. The surface area is determined from the light scattered by the microstructure of the nuclei. Spurious scattering due to erythrocytes can be eliminated by gating the signal in response to a haemoglobin detector, and colloid scattering is concentrated in the forward direction so this can be avoided.