|Publication number||US3893767 A|
|Publication date||8 Jul 1975|
|Filing date||23 Oct 1973|
|Priority date||23 Oct 1973|
|Also published as||CA1018796A, CA1018796A1, DE2450112A1, DE2450112B2, DE2450112C3|
|Publication number||US 3893767 A, US 3893767A, US-A-3893767, US3893767 A, US3893767A|
|Inventors||Fulwyler Mack J, Gilmore Michael Thomas, Paquette Gerard Adelard, Ziffer Garret Francis|
|Original Assignee||Coulter Electronics|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (5), Referenced by (8), Classifications (17)|
|External Links: USPTO, USPTO Assignment, Espacenet|
United States Patent Fulwyler et al.
Assignee: Coulter Electronics, Inc., Hialeah,
Filed: Oct. 23, 1973 Appl. No.: 408,817
July 8, 1975  ABSTRACT Method and apparatus for differentially analyzing biological cells, especially white blood cells, in a sample that includes red blood cells, without destroying or removing the red blood cells from the sample, such as a blood smear. Apparatus for illuminating the cell sample with light including that of approximately 415 nanometers in wavelength. White blood cells are almost transparent at this color; however, whenever the light of 415 nanometers wavelength passes though a portion of the illuminated sample that includes a red cell, a sufficient quantity of light energy is absorbed by  U-S- C 356/ 356/1 356/201 the red cell to cause the resulting detection of light en- [5 l Int. Cl. G0lm 1/00 ergy to be a tag signal below a chosen threshold value. Field of Search 356/39. Tag signals thus representative of the red blood cells 356/180, 184, 186, 188, 189, l, 204, 205, can be stored in a memory such that the location of 206; l78/DlG. 36 the red blood cells can be utilized for subsequent analysis of the sample when the sample is exposed to light  References Cited of different wavelengths. In one such analysis, normal UNITED STATES PATENTS mature red blood cells can be distinguished from retic- 2 875 666 3,1959 Parker et al I I I 356/39 ulocytes. Also, the signals representative of the red 3:315:22) 4/1967 smithnnemx" 356/39 blood cells can be removed from an electrical repre- 3,413,4s4 11/1968 Kamentsky 356/39 Semalion Of the entire Sample. 3,503,684 3/1970 Preston, Jr. et al... 356/39 3,560,754 2/1971 Kamentsky 356/39 Clams 4 Drawmg LlGHT SOURCE IO 38 ELECTRONIC ANALOG I} 'l SWITCH l TO VIDEO 40 4 44 46 PROCESSOR :1 f ELEcTRorhc A A ANALOG SWITCH l 22)H 2B COMPAR- A ATO 1 ELECTROOPTICAL SYSTEM FOR CANCELLATION OF THE EFFECTS OF RED BLOOD CELLS IN A SAMPLE OF BIOLOGICAL CELLS BACKGROUND OF THE INVENTION The present invention relates to the automatic analysis of the biological cells, such as white blood cells, and more particularly to method and apparatus for effectively removing, non-destructively, from the sample the presence of red blood cells for the analysis of white blood cells.
In blood cell analysis, a typical blood smear comprises many red cells in the vicinity of white cells; therefore, accurate automatic analysis of particular white cells becomes difficult.
Red cells can be eliminated destructively by lysing a liquid blood sample and also can be delineated by differential staining. Such techniques either generally can alter all the blood cells and negate any continued analysis of the blood cells according to accepted procedures and make the red cells totally unavailable for subsequent analysis; or, leave all cells available for further analysis, but do not materially lessen the problem of red cells masking the white cells.
In the analysis of red blood cells, tests are performed to detect the presence and number of reticulocytes. Reticulocytes are immature red blood cells and are char acterized by retaining some of basophilic ribonucleic acid (RNA) containing material from the cytoplasm of the immature nucleated red cell. This material can be detected or differentiated from the normal red blood cells by staining the cells with a supra-vital stain which precipitates this material into a series of dark filaments.
SUMMARY OF THE INVENTION The invention seeks to overcome the problems of the prior art by effectively eliminating red blood cells in a biological sample, without destruction or even alteration of any of the sample. If desired. the sample can be stained in a conventional manner, as by Wrights stain. The invention also seeks to provide a means for automatically determining the percentage of reticulocytes in the sample.
The invention includes illuminating a sample, such as a blood smear, with light within a first wavelength range, such as MS nanometers, detecting absorbence of such light by the sample, and generating an output tag signal when the quantum of light from the sample is below a chosen threshold value. In this manner, when a red blood cell is interposed into the illuminating beam, it absorbs a sufficient quantity of the light energy, such that the resulting output signal is below the chosen threshold value and the cell can be effectively eliminated from the output response. Signal processing circuitry can be included in the detecting step for providing video signals representative of the blood cells, wherein the signals representative of the red blood cells have been removed.
Alternatively, instead of eliminating the red blood cells from the output response, the output tag signal can be used to store the location of the red blood cells into a memory. The sample then is illuminated with light within a second wavelength range, such as 530 nanometers wavelength, and a density analysis is performed on the detected light within the second wavelength range when the output tag signal is present. In
this way only the red cells in the sample are analyzed to distinguish normal mature red blood cells from reticulocytes.
The cited wavelength of 4 l 5 nanometers can include the range of wavelengths from approximately 405 nanometers to 425 nanometers as used herein as well as in the claims. The second wavelength range excludes all wavelengths within the first wavelength range.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram of apparatus illustrating one embodiment of the invention;
FIG. 2 is a block diagram of apparatus illustrating another embodiment of the invention; and
FIGS. 3A and 3B are histograms capable of being generated by the system shown in FIG. 2.
DESCRIPTION OF THE PREFERRED EMBODIMENTS In FIG. I, the entire apparatus is for analysis of biological cells. A source of light 12, such as a flying spot scanner, provides a point of light which moves in a raster scan. The light can be broadband light but shall include purple light in the vicinity of 4l5 nanometers in wavelength.
Light from source 12 is directed to a sample holder 16, such as a slide, upon which is a biological cell sample, such as a blood smear comprising red and. for ex ample, white blood cells. The rastering light passing through the blood smear is received by a narrow band 1 filter 20. The filter has the property of passing only purple light of approximately 4i 5 nanometers in wave length; all light not passing therethrough is reflected. Light passing through filter 20 is detected by a photocell 22 which provides an electrical signal on a lead 24 proportional to the light detected. The signal on lead 24 is coupled to an amplifier 26 which provides an amplified signal to a comparator 28; the amplified signal being representative of the amount of purple light, i.e., light of 415 nanometers wavelength, that is present.
A second narrow band filter 30, one which passes only yellow light and reflects blue light, is positioned to filter the light reflected by filter 20. The light passing through filter 30 is detected by a photocell 32, which provides a yellow representative signal on a lead 34 representative of the amount of yellow light passing through filter 30. Lead 34 is coupled to an amplifier 36 which couples an amplified yellow representative signal to a variable gain amplifier 38. The blue light reflected by filter 30 is detected by a photocell 40, which provides a blue representative signal onto a lead 42 that is coupled to an amplifier 44, where the blue representative signal is amplified and subsequently coupled to a variable gain amplifier 46.
A photocell 47 is positioned to detect the brightness of light emanating from the source 12 for providing an average brightness signal on a lead 50, which is coupled to an amplifier 52, which provides a brightness control signal on a lead 54. The brightness control signal on lead 54 is commonly coupled to the control input of variable gain amplifiers 38 and 46 such that any fluctuations in brightness of the emanating light from source 12 are adequately compensated by related changing of the gains of amplifiers 38 and 46.
The brightness control signal on lead 54 also is coupled to comparator 28 to provide a threshold voltage. The comparator 28 is operative such that, when the signal from amplifier 26 falls below the threshold voltage established by the brightness control signal on lead 54, a tag signal is coupled to an amplifier 56, which couples an amplified inhibit signal to the control inputs of electronic analog switches 58 and 60.
The electronic analog switches 58 and 60 each possesses first and second analog inputs, a control input and an analog output. The outputs of variable gain amplifiers 38 and 46 are respectively coupled to the first analog inputs of switches 58 and 60. A voltage representative of background is coupled from a variable resistor 62 to the second analog inputs of switches 58 and 60. The variable resistor 62 is coupled between a source of voltage and a source of reference potential or ground. When an inhibit signal appears at the control input of the respective switches, the voltage from variable resistor 62 is coupled to the output of the respective switches. When there is no inhibit signal on the respective inhibit inputs of switches 58 and 60, the signal on the first input is coupled to the output of the respective switches thereby coupling the output of amplifiers 38 and 46 to the outputs of switches 58 and 60 respectively.
The output of switches 58 and 60 can be coupled to video processing circuitry well known in the art, wherein a black and white picture of the sample on slide 16 can be reproduced. Alternatively, the color representative signals could be processed to provide composite color representative signals representative of the sample on slide 16. When the tag signal from comparator 28 is present, the color representative signals from variable gain amplifiers 38 and 46 are decoupled from the outputs of electronic analog switches 58 and 60 respectively, and a signal representative of background is coupled to the video processing circuitry via switches 58 and 60.
The operation of the above described circuitry effectively eliminates all color representative signals from any cell from the blood smear on slide 16 which absorbs light in the vicinity of M5 nanometers in wavelength.
lt is known that the hemoglobin of red cells absorbs light in the vicinity of 415 nanometers while the white cells are effectively transparent to such light. Accordingly, when a red blood cell on slide 16 is illuminated by light from source 12, light of4l5 nanometers will be absorbed by the red cell and the light passing through filter 20 to photocell 22 will fall below a chosen threshold value. Comparator 28 then will couple the tag or inhibit signal to the control inputs of switches 58 and 60, which as described above, provide a background signal to video processing circuitry, which effectively eliminates signals representative of the red blood cells. When an image is formed on a television monitor from the processed signals from the electronic switches 58 and 60 after paassing through well known video processing circuitry, the image will be void of red blood cells, thereby allowing data to be gathered on the other biological cells, such as white cells, without introducing errors due to the presense of red cells nearby.
It now should be apparent to those versed in the art that this invention can be implemented in a variety of ways.
In FIG. 2 a second apparatus embodying the invention generally is designated by the reference numeral 64. A source of pulsed or shuttered broadband light 66 is directed through a narrow band filter 68, which allows light of approximately 415 nanometers in wavelength only to pass therethrough. Light passing through filter 68 is directed through a slide 70, upon which is a blood smear, to form an image on the target are of an image pickup device 74, which generates a signal representative of the image at a video output terminal 76. Image pickup device 74 can be a vidicon, the scanning raster of which is controlled by the deflection and synchronizing circuitry 78 which provides horizontal and vertical deflection signals to the vidicon 74.
The output terminal 76 is coupled to an amplifier 80 which couples an amplified signal to a comparator 82, which also has coupled to it a threshold representative voltage from the wiper of a variable resistor 84. The variable resistor 84 is coupled between a source of voltage supply and a source of reference potential or ground. When the amplified signal from amplifier 80 falls below the threshold voltage coupled to comparator 82, a tag signal is produced and is coupled to a memory 86 which can be a digital memory or any suitable memory device which is addressable and stores the X-Y coordinates of the raster whenever a red cell is present. The memory 86 also receives signals representative of the deflection and synchronizing signals from circuitry 78 for providing the X-Y coordinate of the red cell. The memory 86 thereby stores the exact location in the raster where the light falls below a predetermined threshold value. Accordingly, since red blood cells absorb light of approximately 415 nanometers in wavelength, memory 86 contains the location of all the red blood cells on slide that had been imaged on the vidicon.
Filter 68 is movable to a location 68' by a filter moving means 87, such as a motor. The filter 68 can be a rotating disc containing a plurality of filter segments to achieve the same result as described hereinafter. When filter 68 is moved to location 68, broadband light from source 66 passes through a different segment of the filter, illuminates slide 70 and provides a second image on image pickup device 74. A switch 88 is controlled by filter moving means 87 such that when filter 68 is in position 68', the switch 88 couples the output of amplifier to an A/D (analog to digital) converter 90, which converts the analog signal from amplifier 80 into a digital signal which appears on a lead 92. In this embodiment, the A/D converter 90 is necessary because of the use of a digital type of memory 86. Alternatively, if an analog system were to be used, memory 86 would be an analog memory and A/D converter 90 could be eliminated and switch 88 would be coupled directly to the lead 92. Lead 92 is coupled to a gate 94, which has a control terminal 96 coupled to memory 86 and an output coupled to a video processor 98. If an analog system were desired, the video processor 98 would be an analog video processor; however, in the preferred use of a digital memory 86, the video processor also could be digital.
When filter 68 is in position 68', memory 86 will produce a signal at terminal 96 whenever a red blood cell is present at the exact raster location being scanned. Memory 86 has stored the location of all the red cells on the slide 70 containing the blood smear. When a signal appears at the control terminal 96 of gate 94, lead 92 is decoupled by the gate, thereby generating a blanking signal representative of background to the video processor 98. This blanking signal will occur only when a red blood cell is present at the raster location being scanned. When there is no blanking control signal on terminal 96, gate 94 couples the output of pickup device 74 to the video processor 98, which will produce a video signal which, when coupled to a television monitor, will provide a reproduced image void of the presence of the red blood cells. At the same time, the processor 98 can store the data pertaining to the blood smear for future analysis.
Since it is known that reticulocytes can be differentiated visually by staining the cells with a supra-vital stain, the apparatus described in FIG. 2 embodying the invention can be utilized to perform a density analysis only upon the red blood cells since the location of all the red blood cells previously had been stored in mem ory 86.
Additional apparatus can be used in conjunction with the thus far described system 64 to provide a reticulocyte analysis of the red blood cells. A density analysis computer 100 is coupled to the video output of vidicon 74 via a lead 102, thereby receiving video signals of the blood smear on slide 70. A control input to the density analysis computer 100 is supplied via a lead 104. Lead 104 is provided with an enabling signal from the memory 86. Once the location of the red cells have been determined and their positions stored in memory 86, filter 68 is moved to position 68'. A second filter 106, which is coupled to filter moving means 87, is moved between light source 66 and slide 70. Filter 106 in this embodiment of the invention only allows light of approxi mately 530 nanometers in wavelength to pass therethrough. Other types of filters could be used to provide selected wavelengths of light other than 415 nanometers, to illuminate slide 70. Filter wheels could be used to rotate filter segments 106 and 68 between light source 66 and slide 70.
When the light from filter 106 strikes slide 70, the reticulocytes will present on vidicon 74 a darker image representative of the dark filaments precipitated by the supra vital stain as described previously. It should be remembered that the stained reticulocytes appear more opaque to light than normal red blood cells.
Memory 86 provides an enabling signal via the lead 104 to computer 100, which activates computer 100 when the scanning raster of vidicon 74 is at a raster location where a red cell previously has been located. Video signals representative of the cell at that location are coupled to computer 100 wherein the amplitude of that cell is stored and processed. In this way, the density of only the red cells are analyzed by computer 100. Computer 100 can be programmed to provide a histogram readout such as that shown in FIGS. 3A and 38 to be described subsequently.
It should be understood that memory 86 can be coupled to the deflection and synchronizing circuitry 78 such that only the red cells are scanned when filter 106 is positioned between source 66 and sample 70.
FIGS. 3A and 3B respectively show histograms of approximately one normal red blood cell and a reticulocyte. These histograms could be obtained from apparatus similar to that described in FIG. 2 wherein filter 106 would be chosen as allowing only light of approximately 530 nanometers in wavelength to pass therethrough.
The histograms of FIGS. 3A and 38 were made by digitizing the video signals at the output of amplifier 80 in FIG. 2. The video signal is sampled electronically and digitized, each digital sample representing a picture element of the red blood cell being scanned. By adding together the number of such sample picture elements in a series of bins, each bin representing a range of densities. which represents a range of opacity to light of 530 nanometers, a histogram can be generated. Such a histogram is a graph whose abscissa represents density bins and the ordinate represents the number of picture elements falling within the density range of each bin.
Empirically it has been determined that normal red blood cells have density histograms wherein the greatest density a red blood cell will exhibit to light of approximately 530 nanometers wavelength is shown as density A in FIG. 3A. Reticulocytes as described previously, due to their greater opacity, will exhibit a greater opacity or density to 530 nanometer light than normal red blood cells and will produce a histogram including density variations out to density range B as shown in FIG. 313. Accordingly, the range of densities between range A and B in FIG. 3B represents the presence of the denser, more opaque reticulocyte cell. Numerical computations can be performed by computer in FIG. 2 to provide information as to the percentage of reticulocytes among the red blood cells.
To those skilled in the art other embodiments of the invention easily can be obtained, i.e., single tube color cameras utilizing color encoding filters can be used to form color representative signals of the blood smear on a slide. These color representative signals can be put through an appropriate matrix to determine the pres ence of light of4l5 nanometers in wavelength and light of 530 nanometers in wavelength. Appropriate circuitry can be provided to perform amplitude analysis of the two signals simultaneously or sequentially to determine the presence of red blood cells and the percentage of red blood cells which are reticulocytes. Similarly, threshold circuitry can be provided such that when light transmitted from the sample falls below a chosen threshold value, proper circuitry can be provided to eliminate all signals at the raster location, thereby providing a picture which would be void of red blood cells. Alternatively, apparatus could be built utilizing the teachings of this invention which would provide for non-video outputs with or without histogram printouts. Such systems could provide a digital printout of the data and/or could store the data received for subsequent computer analysis without the need for a visual display of the sample on the slide or the resultant data.
What is desired to be secured by Letters Patent of the United States is:
1. In a method for nondestructively analyzing biological cells located in a sample including cells of at least two types supported in fixed locations relative to each other and which differentially absorb light of known wavelength, the steps of:
illuminating discretely at least parts of each of the cells at their fixed locations in the sample with first light within a first wavelength range;
detecting discretely the amount of said first light from each cell part after absorption thereby; and generating a tag signal each time when the thus detected cell part light is difierent from a chosen value, said tag signal useable for indicating the fixed location in the sample of that cell part.
2. The method according to claim 1 including the steps of:
processing the thus detected first light and thereby providing electrical signals representative of each illuminated cell;
correlating the tag signals with the cell representative signals; and
blanking said cell representative signals in response to a correlation with said tag signals, thereby to eliminate electrically the electrical presence of a cell.
3. The method according to claim 2 in which said correlating includes:
storing at least one of said tag signals and said cell representative signals prior to said correlating.
4. The method according to claim 1 in which said chosen value is a threshold value; and
said generating is enabled when the detected first light falls below the threshold value.
5. The method according to claim 1 in which said first wavelength range includes 4l5 nanometers.
6. In the method according to claim I the steps of:
repeating said steps of illuminating and detecting in which said illuminating the cells is with second light within a second range of wavelengths, exclusive of at least part of said first range.
7. The method according to claim 6 which includes the steps of:
processing the thus detected second light and thereby providing electrical signals representative of each illuminated cell; and
correlating the tag signals with the cell representative signals obtained from said second light, thereby to identify electrically a specific type of biological cell.
8. The method according to claim 7 which includes the step of performing a density analysis on the detected second light and thereby differentiating at least one form of a specific type of cell.
9. The method according to claim 8 in which said first light is highly absorbed by red blood cells, which :ells define a specific type of cell.
10. The method according to claim 9 in which said second light is one to which normal red cells are more :ransparent than are reticulocytes, which define one brm of a specific cell type.
ll. ln apparatus for nondistructively analyzing bioogical cells located in a sample including cells of at east two types supported in fixed locations relative to :ach other and which differentially absorb light of :nown wavelength;
illuminating means for illuminating discretely at least parts of each of the cells at their fixed locations in the sample with first light within a first wavelength range;
light detecting means for detecting discretely the amount of said first light from each cell part after absorption thereby; and
tag signal generating means coupled to said detecting means for generating a tag signal each time when the thus detected cell part light is different from a chosen value, said tag signal useable for indicating the fixed location in the sample of that cell part.
12. Apparatus according to claim I! in which said ght detecting means includes:
processing means coupled for processing the thus detected first light and thereby providing electrical signals representative of each illuminated cell; and said apparatus further includes signal correlating means coupled to both said tag signal generating and said processing means for correlating the tag signals with the cell representative signals; and
signal blanking means coupled to said signal correlating means for blanking said cell representative signals in response to a correlation with said tag signals, thereby to eliminate electrically the electrical presence of a cell.
13. Apparatus according to claim 12 in which said signal correlating means includes output means coupled to receive said cell representative signals and be controlled by said signal blanking means so as to receive said cell representative signals only in the absence of a blanking signal from said signal blanking means.
14. Apparatus according to claim 11 in which said illuminating means includes means which define said first wavelength range to include light of approximately 415 nanometers.
15. Apparatus according to claim 12 in which said signal correlating means includes:
signal storing means for storing at least one of said tag signals and said cell representative signals prior to their correlation.
16. Apparatus according to claim I] which includes:
threshold circuit means for defining said chosen value as a threshold value; and
said tag signal generating means is enabled when the detected first light falls below the threshold value.
17. Apparatus according to claim ll in which said illuminating means includes light filtering means;
sample holding means is interposed between said illu minating means and said light detecting means, such that the light passes through said sample holding means and the cells; and
said light detecting means includes a vidicon, the
rastering of which scans each cell by a plurality of scan lines directed toward said sample holding means.
18. Apparatus according to claim II in which said illuminating means includes means for illuminating the cells with second light within a second range of wavelengths. exclusive of at least part of said first range, and said light detecting means is arranged also to detect discretely the amount of said second light from each cell part after absorption thereby.
19. Apparatus according to claim 18 in which said processing means is coupled also for processing the thus detected second light and thereby providing additional electrical signals representative of each illuminated cell; and
said signal correlating means is coupled to both said tag signal generating means and said processing means for correlating the tag signals with the cell representative signals obtained from said second light, thereby to identify electrically a specific type of biological cell.
20. Apparatus according to claim 19 which includes:
density analysis means for performing a density analysis on the detected second light;
thereby enabling differentiating of at least one form of a specific type of cell.
21. Apparatus according to claim in which said illuminating means includes light filtering means;
sample holding means is interposed between said illuminating means and said light detecting means, such that the light passes through said sample holding means and the cells; and
said light detecting means includes a vidicon, the rastering of which scans each cell by a plurality of scan lines directed toward said sample holding means.
22. Apparatus according to claim 21 in which said illuminating means includes means which defines said first light to be highly absorbed by red blood cells. which cells define said specific type of cell.
23. Apparatus according to claim 22 in which said illuminating means includes means which defines said second light to be light to which normal red blood cells are more transparent than are reticulocytes. which define one form of said specific cell tecting means comprises:
a first filter having a narrow band pass within said first wavelength range, said first filter reflecting all light not passing therethrough;
a first photodetector positioned to detect light passing through said first filter;
a second filter positioned to receive said reflected light from said first filter, said second filter having a band pass of a first chosen wavelength of light and reflecting all other light landing thereon;
a second photodetector positioned to detect light passing through said second filter; and
a third photodetector positioned to detect all light refiected from said second filter.
25. Apparatus according to claim 24 wherein said illuminating means comprises a flying spot scanner.
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|U.S. Classification||356/39, 356/416, 356/432|
|International Classification||G06T1/00, G01N21/31, H04N7/18, G01N35/00, G01N15/14, G01N15/00, G01N21/25, G01N21/27, G01N33/483, G01N33/49|
|Cooperative Classification||G01N15/1468, G01N21/314|
|European Classification||G01N21/31D, G01N15/14H|