METHOD AND APPARATUS FOR DETERMINING THE COUNT PER UNIT VOLUME OF PARTICLES

Abstract: A method and apparatus for accurately determining the count per unit volume of particles, such as red blood cells, suspended in a fluid containing at least one undesirable interference particle, such as air bubbles. A predetermined volume of fluid containing the first and second particles is passed through a flow cytometer which produces a forward light scattering pattern that is detected and measured. The volume and index refraction of each of the first and second particles is determined. The first and second particles are discriminated in accordance with their respective indices of refraction and the total count and volume of particles is corrected to provide the count per unit volume of red blood cells.

Description: This invention is directed to a method and apparatus for accurately determining the count per unit volume of particles suspended in a fluid containing one or more undesirable particles. More particularly, the invention pertains to determining the count per unit volume of blood cells in a flow cytometric system wherein the count is corrected for air bubbles present in the blood suspension medium. The use of flow cytometry for counting blood cells has been increasing over the past several years. A fundamental consideration in the accuracy of blood cell counting is that the suspension of blood cells, if allowed to sit unmixed, will become spatially inhomogenous due to sedimentation or sticking to the container walls. This occurs due to the fact that the blood cells are in general denser than the fluid they are suspended in and may also have an electrostatic interaction with the walls of the container. This inhomogeneity may interfere with the accuracy of the blood cell counting. In order to solve this problem, the suspension of blood cells is mixed prior to the cells being passed through the counting mechanism. However, if the suspension is mixed, air bubbles will be introduced into the fluid which may interfere with the accuracy of the blood cell count. This interference is generally caused by the trapped air increasing the fluid volume measured by the system which erroneously lowers the cell count and also by small air bubbles which may be counted as cells erroneously increasing the cell count. Heretofore, there has not been a system for solving the substantial problems in counting blood cells in a flow cytometer. At present, if the suspension is sampled during or shortly after mixing, error due to air is introduced and if counting is delayed to remove the air, cell loss due to sedimentation or sticking to the container walls is observed. There is described in EP-A-0 140 616 a method and apparatus for determining the volume and index of refraction of particles. Object of EP-A-0 140 616 is to obtain the index of refraction as well as the volume of individual particles of the same type simultaneously. The present invention is directed to a method for accurately determining the count per unit volume of fluid of first particles suspended in a fluid containing said first particles having a first index of refraction and at least one second particle having a second index of refraction different from the first index of refraction. The apparatus of the present invention accurately determines the count per unit volume of fluid of first particles suspended in a fluid containing said first particles having a first index of refraction and at least one second particle having a second index of refraction different from the first index of refraction. The present invention is directed to a method and apparatus for accurately determining the count per unit volume of particles that overcomes the sources of potential error in a system utilizing flow cytometric techniques. The invention discriminates between undesirable particles and desired particles on the basis of a difference in relative index of refraction. The count of desired particles is then corrected to take into account the undesirable particles to provide an accurate count per unit volume. The particles desired to be counted, such as red blood cells, are suspended in a fluid. The suspension fluid may contain one or more air bubbles that become introduced as a result of mixing of the fluid. The red blood cells have an index of refraction different from the index of refraction of the air bubbles. A predetermined volume of fluid containing both cells and air bubbles is aspirated from a mixing chamber and each particle, in turn, is passed through a light beam. Each particle produces at least one forward light scattering pattern. The above-mentioned application utilizes a multiple angle light scattering measurement technique from which there can be determined the volume and index of refraction of particles passing through a flow cytometer. The intensity of light from at least a portion of the scattering pattern is detected and measured and a signal representative of such intensity if generated. All the particles in the aspirated volume of fluid are passed through the light beam and the combined total number of particles is counted. The volume and index of refraction of each of the particles is thereafter determined using the multiple angle technique. A discriminator separates the cells and air bubbles by their respective indices of refraction. Thereafter, the count and volume of particles is corrected by the count and volume of air bubbles to determine the count per unit volume of red blood cells. The intensity of light from the scattering pattern is detected within a plurality of angular intervals and a signal is generated from each angular interval. The angular intervals are uniquely selected such that the light contains sufficient information to determine the volume and index of refraction of the particles. It is preferred that two angular intervals be selected. A two-dimensional graphical representation may be provided which shows a first region of particles having the index of refraction of blood cells and second region of particles having the index of refraction of air. In one embodiment, a threshold technique is utilized to separate the blood cells from air. A threshold value is set in accordance with the indices of refraction of the blood cells and air so that only those particles above or below the threshold are counted. Since the number of cells will be much more than the number of air bubbles, only the number and volume of air bubbles are determined and then the total count initially measured and detected can be corrected for the amount of air to provide an accurate count per unit volume of red blood cells. The present invention will be described in more detail in connection with the following drawings in which Figure 1 is a block diagram of a preferred embodiment of the present invention. Figure 2 is a schematic diagram of the flow cell and detection optics used in the present invention. Figure 3 is a graphical representation of the separation of particles by their indices of refraction and Figure 4 is a block diagram showing separate counters between the discriminator and the microprocessor. Referring now to the drawings, Fig. 1 shows a block diagram of the system of the present invention wherein blood cells are introduced through conduit 10 into a mixing and incubation chamber 12. The conduit 10 comprises a conventional sampling apparatus, not shown, for introducing blood cells into the chamber 12. A suspension fluid is introduced through conduit 14 to suspend the cells therein. An isovolumetric sphering reagent may also be introduced along conduit 14 to sphere the red blood cells contained in the sample. The isovolumetric sphering of the red blood cells ensures that the measurement is fully independent of the orientation of the cell during optical measurement in a flow cytometer. The individual cells are first mixed in the chamber 12 to mix with the suspension fluid and to react with the sphering agent. The cells are then permitted to incubate during which a number of cells may stick to the chamber walls and/or settle on the chamber bottom. The sedimentation will occur as mentioned, due to the blood cells being denser than the suspension fluid. In addition, the cells may have an electrostatic interaction with the walls of the container 12 thereby causing them to be attracted to the walls. Thus, if allowed to sit unmixed prior to passage through the flow cytometer, the cells will become spatially inhomogenous which will interfere with the accuracy of the blood count. Following incubation, the cells are again mixed in the chamber 12 prior to being passed to a flow cell 16 in order to avoid the problem of spatial inhomogeneity. As noted previously, mixing generally will cause a plurality of air bubbles to be formed in the suspension fluid. In accordance with the inventive method, a predetermined volume of suspension fluid having cells and air bubbles is aspirated from the mixing chamber 12. Therefore, each cell is passed through the flow cell 16 for detection. In addition, any air bubbles in the aspirated volume will also be detected. A conventional aspirating device, not shown, may be used to pass the predetermined volume of fluid from the mixing chamber 12 to the flow cell 16. The flow cell 16 can be of conventional design, for example, as described in U.S. Patent No. 3,661,460, assigned to a common assignee which discloses a sheath-stream flow cell. As shown in Figure 2, the fluid stream 40 is passed through the flow cell 16 which is designed so that individual cells flow successively through an optically defined view volume. The stream 40 is encased in a sheath 42 having an index of refraction the same as the stream 40. The red blood cells 44 flow through the passageway 46 which has a narrowing internal diameter thereby constricting the stream 40 to cause individual cells to flow through the view volume. Illumination optics 18 directs a light beam through the view volume to illuminate, in turn, each particle as it passes through the cell 16. The illumination comprises a laser 48, precision slit aperture 50 and a lens system 52 for condensing the light beam into the center of the view volume, as shown in Figure 2 while maintaining the substantially collimated light from the laser 48. As each individual particle passes, in turn, through the view volume of flow cell 16, it interrupts the light beam. Accordingly, light is scattered mainly in the forward direction and in an angular intensity pattern from which the volume and index of refraction can be measured. The intensity of light in the scattering pattern is detected and measured in the detection optics 20 which generates an electrical signal. The signal is then processed to determine the desired information as will be hereinafter described. The detection optics utilizes a multiple angle light scattering measurement technique. As shown in Figure 2, the detection optics includes a lens 54 which collects the forward scattered light within two critical angular intervals which are seperately detected and measured from which the volume and index of refraction of each individual particle is determined. The light from the lens is directed to a beam splitter 56 which transmits approximately half the light and reflects the remaining light which is directed to and reflected from a mirror 58. The light passed through the beam splitter 56 is directed through a dark field stop 60 adapted to pass light scattered within a low angular interval. Similarly, light reflected from mirror 58 is directed through a dark field stop 62 adapted to pass light scattered within a high angular interval. The light from the two different angles is collected by lenses 64 and 66 then detected by two separate detectors 68 and 70, respectively. Returning now to Figure 1, a counter 22 counts the combined total of particles passing through the flow cell 16. The signal is then transmitted to index of refraction processor 24 where the volume and index of refraction of each particle is determined. A discriminator 26 distinguishes between the blood cells and air bubbles by their respective indices of refraction. The index of refraction of air is approximately 1.00, while the index of refraction of red blood cells is approximately in the range of 1.38 to 1.44. The discriminator 26 separates the signals in accordance with the measured index of refraction. A graphical representation may be obtained by plotting the results for each particle. The graph will depict one region of the particles having one index of refraction and a second region of particles having a second index of refraction. An example of such a graph is shown in Figure 3. Region A includes the concentration of red blood cells while region B includes the air bubbles. Region C shows the effect of occassional coincidence of red cells in the view volume. Errors due to coincidence may be corrected by standard techniques commonly used in cell counters. Thereafter, processor 28 is utilized to correct the count and volume for the detected air bubbles to provide an accurate determination of the count per unit volume of red blood cells. The total count of particles passed through the flow cell is inputted to the processor 28 together with the predetermined volume of fluid aspirated. Processor 28 is programmed with an algorithm that will take the total number of particles counted and subtract the number of air bubbles to provide an accurate count of red blood cells. In addition processor 28 will subtract the volume of air bubbles from the total volume of fluid aspirated to determine the accurate volume of fluid. The number of cells is then divided by the accurate volume of fluid to provide an accurate measure of the total count of red blood cells per unit volume of fluid. Moreover, the processor 28 can be a microprocessor programmed with an algorithm that sets a threshold value at either of the measured indices of refraction. Processor 28 can include a counter to count only those particles above or below the threshold value. An adder will then determine the total volume of particles counted. In addition, the mean volume per air bubbles can be calculated and multiplied by the number of air bubbles to determine the total volume of air in the sample stream. Figure 4 illustrates the use of separate counting means 30 and 32 coupled between the discriminator 26 and processor 28. After discriminator 26 separates signals in accordance with the indices of refraction, counters 30 and 32 then individually count each of the signals in the two regions. This information is fed to the processor 28 together with the combined count and volume originally measured and detected to determine the accurate count per unit volume of blood cells. While illustrative embodiments of the subject invention have been described and illustrated, it is obvious that various changes and modifications can be made therein without departing from the present invention which is limited only by the scope of the appended claims.

Claims: A method for accurately determining the count per unit volume of first particles suspended in a fluid containing said first particles having a first index of refraction and at least one second particle having a second index of refraction different from the first index of refraction by passing the fluid containing both particle types through a light beam, each of said first type or second type of particles producing, in turn, at least one forward light scattering pattern, wherein the combined total number of said first and second particles is detected by measuring the intensity of light of at least a portion of each of said light scattering patterns, characterized by the following steps: detecting (20) and thereafter counting (22) the combined total number of said first and second particles passing through said light beam from said light scattering pattern; determining (24) the volume and index of refraction of each of said first and second particles passing through said light beam from said at least one forward light scattering pattern; discriminating (26) between said first and second particles by their respective first and second indices of refraction; determining the total number and total volume of said second particles; determining (28) the count and volume of said first particles passing through said light beam and a corrected volume of fluid by correcting the combined total count of particles and the predetermined volume of fluid with the number and volume of second particles respectively; and determining (28) the count per unit volume of fluid of said first particles from the corrected count and corrected volume. The method of Claim 1, wherein said first particles are red blood cells and said second particles are air bubbles, and further including the steps of: aspirating a volume of said fluid from a mixing chamber (12) prior to passing the fluid through the light beam; and mixing the fluid containing red blood cells causing air bubbles to be formed in said fluid prior to aspiration. The method of Claims 1 or 2, wherein the step of determining the volume and index of refraction of each of said first and second particles includes the steps of: detecting and measuring the intensity of light within each of a plurality of angular intervals of said scattering pattern to generate a signal from each angular interval, wherein said angular intervals are selected such that the light contained therein contains sufficient information for the precise determination of the volume and index of refraction of said first and second particles; and determining the volume and index of refraction of said first and second particles from the magnitudes of said signals. The method of any one of Claims 1 to 3, wherein the discriminating step includes the step of providing a two-dimensional graphical representation depicting a first region of particles having said first index of refraction and a second region of particles having said second index of refraction. The method of Claim 4, wherein the discriminating step includes setting a threshold at either of said first or second index of refraction and counting only those particles above or below said threshold and the count per unit volume of fluid of first particles is determined by dividing the count of first particles by the volume of predetermined fluid less the volume of second particles, and further including the step of calculating the mean volume per second particle and multiplying said mean volume by the number of said second particles to determine the total volume of said second particles. An apparatus for accurately determining the count per unit volume of first particles suspended in a fluid containing said first particles having a first index of refraction and at least one second particle having a second index of refraction different from the first index of refraction by passing the fluid containing both particle types through a light beam, each of said first type or second type of particles producing, in turn, at least one forward light scattering pattern, wherein the combined total number of said first and second particles is detected by measuring the intensity of light of at least a portion of each of said light scattering patterns, comprising: means (20,22) for detecting and thereafter counting the combined total number of said first and second particles passing through said light beam from said light scattering pattern; means (24) for determining the volume and index of refraction of each of said first and second particles passing through said light beam from said at least one forward light scattering pattern; means (26) for discriminating between said first and second particles by their respective first and second indices of refraction; means for determining the total number and total volume of said second particles; means (28) for determining the count and volume of said first particles passing through said light beam and a corrected volume of fluid by correcting the combined total count of particles and the predetermined volume of fluid with the number and volume of second particles respectively; and means for determining the count per unit volume of fluid of said first particles from the corrected count and corrected volume. The apparatus of Claim 6, wherein the means (20,22) for detecting the combined total number of said first and second particles includes means for measuring the intensitiy of light of at least a portion of each of said light scattering patterns, and further including: means (24) for determining the volume and index of refraction of each of said first and second particles passing through said light beam from said at least one forward light scattering pattern; and means (26) for discriminating between said first and second particles by their respective first and second indices of refraction. The apparatus of Claims 6 or 7, wherein the correcting means (28) includes: means for determining the total number and total volume of said second particles; means for determining the count and volume of said first particles passing through said light beam and corrected volume of fluid by modifying the combined total number of particles and the predetermined volume of fluid with the number and volume of second particles respectively; and means for determining the count per unit volume of fluid of said first particles from the modified count and volume.

Inventors: GRONER, WARREN
Applicants: TECHNICON INSTRUMENTS CORPORATION(A DELAWARE CORPORATION)
Application year: 1987
Main classification: G01N15
IPC codes: G06M11, G01N15