Method and Apparatus for Automatically, Identifying and Counting Various Cells in Body Fluids

• US 3,864,571 A
• Filed: 02/10/1971
• Issued: 02/04/1975
• Est. Priority Date: 02/10/1971
• Status: Expired due to Term

• Current Assignee: Thomas R. Tornese
• Original Assignee: Irving M. Stillman, Jules R. Clemens, Neil M. Trowe
• Inventors: Stillman, Irving M., Trowe, Neil M., Clemens, Jules R.
• Primary Examiner(s): Brochelt, Archie R.
• Application Number:
  US05/114,297
• Time in Patent Office: 1,455 Days
• Field of Search: 250/71R,83.3UV,208,211R,237R,304,302,373 235/92PC
• US Class Current: 250/302
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G06M 11/04 with provision for distinguishing between different sizes of objects investigating particle size in general G01N15/00

Abstract

The various types of cells in body fluids are automatically identified by measuring the area of each cell that is occupied by nucleus and cytoplasm and automatically comparing these measurements to each other and also to the total cell area. In the preferred embodiment, the fluid containing the cells is treated with a stain capable of fluorescing when complexed and then excited by an ultraviolet source. The nuclear and cytoplasmic portions of each corpuscle will fluoresce with a radiation characteristic of the type of material. The sample to be counted is caused to flow past a counting station which is in the same plane as the source of ultraviolet radiation and the measurements are taken from the secondary fluorescent emissions of these materials. This information is fed to logic circuits and ultimately to counters which output the results.

Claims

1. The process of differentiating leukocytes in a sample of body fluid comprising the steps of:

a. Obtaining at least a one cubic millimeter sample of the body fluid;

b. Treating said sample with a fluorochrome material that will cause radiation of a characteristic wavelength to be emitted from at least one of the sub-cellular materials when subjected to radiation in the range of 0.30 to 30 microns;

c. Initiating relative movement between the sample and a radiation source which has at least one associated co-planar radiation detection means;

d. Continuing the relative motion until the complete sample has passed the radiation source and the at least one co-planar radiation detector in a manner so that the leukocytes in a substantial portion of the sample are in a single cellular array as they pass said plane;

e. Causing said radiation source to irradiate the sample as the leukocytes pass in said single cellular array;

f. Measuring the overall cell sizes of the cells passing said plane by detecting the initiation of the secondary fluorescent emissions with said radiation detection means and continuing said detection until each cell has passed said plane;

g. Simultaneously determining the amount of each sub-cellular material present in each cell as it passes said plane by detecting the characteristic radiations being emitted by each sub-cellular material in each cell;

h. Feeding these determinations for each cell as it passes said plane to a logic means wherein the cell is identified on the basis of total cell size and the relative amounts of subcellular material present;

i. Causing a separate counter for each type of leukocyte to be advanced when a leukocyte of that type is identified by steps;

j. Repeating steps (e) to (i) to until substantially all of the leukocytes is said sample have passed said plane and have been identified.

2. The method of differentiating cells on the basis of the overall size of the cell and the relative portion of the cell occupied by nuclear and cytoplasmic material comprising the steps of:

a. Subjecting the cellular material to a treatment that will enhance at least one characteristic of either the nuclear or cytoplasmic material whereby the portion of the cell occupied by the at least one material having an enhanced characteristic can be clearly differientiated from the remaining portions of the cell;

b. Automatically determining the proportion of the material present that has been enhanced by said treatment;

c. Determining the total size of the cellular bOdy from the enhanced characteristic thereof;

d. Automatically identifying the cells by the relationship between the total size as determined in step C and the portion of the cell occupied by the at least one material having an enhanced property;

e. Displaying at least the cumulative count of each cell type so identified.

3. In the method of claim 2 wherein the step of subjecting the cellular material to a treatment that will enhance at least one physical characteristic of either nuclear of cytoplasmic material comprises the step of staining the cellular material.

4. The method of claim 3 wherein the stain is acridine orange.

5. The method of claim 4 wherein one or more drops of a solution consisting of 100 milligrams of acridine orange per 50 milliliters of ethyl alcohol is added to a one milliliter sample of blood mixed with an anticoagulant.

6. The method of differentiating cells on a basis of maximum cross-sectional area of the cell and the relative proportion of the cell occupied by nuclear or cytoplasmic material comprising the steps of:

a. Subjecting the sample to be studied to a treatment that will enhance at least one characteristic of either the nuclear or cytoplasmic sub-cellular material whereby an area coupled by the at least one material having an enhanced characteristic can be clearly differentiated from other sub-cellular materials of the cell;

b. Automatically determining the relative areas of the materials that have been enhanced by said treatment;

c. Determining the overall size of the cellular body from the enchanced characteristic thereof;

d. Automatically identifying the cells by the relationship between the areas occupied by the nuclear material, the cytoplasmic material and the total cell area;

e. Indicating at least the number of each cell type identified.

7. The method of identifying white cells comprising the steps of:

a. Staining a sample of body fluid containing white cells with a stain which will cause characteristic secondary fluoresence from the sub-cellular materials of the white cells;

b. Directing ultraviolet energy onto a portion of a sample consisting of substantially a single cell;

c. At a co-planar radiation detection position detecting the characteristic secondary fluorescent emissions from each of said stained sub-cellular materials;

d. Determining the overall size of the cell being identified;

e. Supplying this information to a logic circuit wherein, on the basis of these determinations, the cell is identified.

8. Apparatus for identifying and counting different types of corpuscles which have been stained with a fluorochrome in a sample of body fluid comprising:

means for irradiating at least a portion of the sample of body fluid thereby causing a characteristic secondary emission from each different type of subcellular material in the corpuscles of the fluid sample, said irradiating means being a source of ultraviolet energy;

means for detecting the characteristic secondary emissions from the sub-cellular materials in the fluid sample at a plurality of discrete wavelengths whereby the amount of each sub-cellular material in substantially each cell of the fluid sample can be determined while a simultaneous determination of the overall size of the corpuscle is being made; and

means for identifying the type of corpuscles on the basis of overall size and the relative amounts of each sub-cellular material present in the corpuscle being identified.

9. The apparatus of claim 8 in which the means for detecting the characteristic emissions from each sub-cellular material is co-planar with said means for irradiating said sample.

10. The apparatus of claim 9 wherein the detection means consists of a photocell.

11. The apparatus of claim 10 wherein the photocell has associated therewith a plurality of filters to permit serial determinations of the discrete wavelengths being emitted by said sample.

12. The aPparatus of claim 11 wherein the associated filters are mounted so that they can rotate relative to the single photodetector.

13. The apparatus of claim 12 wherein the single photodetector is a multiaperatured photocell.

14. The apparatus of claim 12 wherein the single photodetector is a solid state device containing a plurality of photosensitive regions.

15. The apparatus of claim 8 wherein the detection means consists of more than one photocell.

16. The apparatus of claim 8 where the detection means consists of multiaperatured photocell.

17. The apparatus of claim 8 wherein the detection means consists of a multiaperatured N-Window photocell.

18. The apparatus of claim 8 wherein the means for detecting the characteristic emissions from the sub-cellular material consists of a plurality of detection means which are mounted relative to each other to permit determination of volumetric relationships between the sub-cellular materials.

19. The apparatus of claim 18 wherein there are three detection means and they are orthogonally mounted.

20. The apparatus of claim 19 wherein each detection means consists of a group of three horizontally disposed slits that are each positioned to receive emissions from the irradiated sample.

21. The apparatus of claim 19 wherein the detection means consists of multiaperatured photocells.

22. The apparatus of claim 21 wherein the detection means consists of a multiaperatured N-Window photocell.

23. The apparatus of claim 18 wherein the detection means consists of a plurality of horizontally disposed slits.

24. The apparatus of claim 23 wherein the detection means comprises a single group of three slits positioned to receive emissions from the irradiated sample.

25. The apparatus of claim 8 wherein the detection means consists of at least one horizontally disposed slit.

26. The apparatus of claim 25 wherein the detection means consists three horizontal slits vertically aligned in the same plane and positioned to receive emissions from the irradiated sample.

27. The apparatus of claim 25 wherein between the irradiated sample and the detection means is disposed a refractive device for separating the characteristic wavelengths being emitted by the sub-cellular material.

28. The apparatus of claim 25 wherein the emission detection means are located at a plurality of levels relative to the plane of the irradiation means.

29. The apparatus of claim 28 wherein the plurality of levels are each located within a band whose center lies in the plane of the irradiation means and the detection means, the width of said band being no greater than the average size of the smallest leukocyte cell.

30. The apparatus of claim 8 including means to cause said sample of body fluid to move in relation to said irradiating means.

31. The apparatus of claim 30 wherein said means moves the fluid sample through a flow chamber that is normally disposed to a plane that passes through the irradiation means.

32. The method of differentiating corpuscles in a sample of body fluid basis of the overall size of the corpuscles and the relative portion of the corpuscle occupied by sub-cellular material comprising the steps of:

a. Subjecting the corpuscles to a treatment that will enhance at least one characteristic of at least one sub-cellular material whereby the portion of the corpuscle occupied by the at least one sub-cellular material having an enhanced characteristic can be clearly differentiated from the remaining portions of the corpuscle;

b. Automatically determining the proportion of the sub-cellular material present that has been enhanced by said treatment;

c. Determining the total size of the corpuscle;

d. Automatically identifying the corpuscles by the relationship between the total size as determined in step (c) and the portion of the corpuscle occupied by the at least one material having an enhanced property;

e. Displaying at least the cumulative count of each corpuscle type sO identified.

33. The process of differentiating leukocytes in a sample of body fluid comprising the steps of:

a. obtaining at least a one cubic millimeter sample of the body fluid;

b. treating said sample with a fluorochrome material that will cause radiation of a characteristic wavelength to be emitted from at least one of the sub-cellular materials when subjected to radiation;

c. initiating relative movement between the sample and a radiation source which has at least one associated coplanar radiation detection means;

d. continuing the relative motion until the complete sample has passed the radiation source and the at least one co-planar radiation detector in a manner so that the leukocytes in a substantial portion of the sample are in a single cellular array as they pass said plane;

e. causing said radiation source to irradiate the sample as the leukocytes pass in said single cellular array;

f. measuring the overall cell sizes of the cells passing said plane by detecting the initiation of the secondary fluorescent emissions with said radiation detection means and continuing said detection until each cell has passed said plane, the detected duration of secondary emissions then being correlated with the rate of relative movement of the cell to determine cell size;

g. simultaneously determining the amount of each sub-cellular material present in each cell as it passes said plane by detecting the characteristic radiations being emitted by each sub-cellular material in each cell;

h. feeding these determinations for each cell as it passes said plane to a logic means wherein the cell is identified on the basis of total cell size and the relative amounts of sub-cellular material present;

i. causing a separate counter for each type of leukocyte to be advanced when a leukocyte of that type is identified by steps (e) to (h);

j. repeating steps (e) to (i) until substantially all of the leukocytes in said sample have passed said plane and have been identified.

34. The method of identifying white cells comprising the steps of:

a. staining a sample of body fluid containing white cells with a stain which will cause at least three different bands of characteristic secondary fluorescence from different sub-cellular materials of the white cells;

b. directing ultraviolet energy onto a portion of a sample consisting of substantially a single cell;

c. at a co-planar radiation detection position detecting the characteristic secondary fluorescent emissions from each of said stained sub-cellular materials;

d. determining the overall size of the cell being identified by measuring the time duration of occurence of the secondary fluorescence;

e. supplying this information to a logic circuit wherein, on the basis of these determinations, the cell is identified.

35. Apparatus for identifying and counting corpuscles in a sample of body fluid comprising:

a. means for irradiating at least a portion of the sample of body fluid thereby causing a characteristic emission having at least three different wavelength bands from different types of sub-cellular material in the corpuscules of the fluid sample;

b. means for detecting the characteristic emissions from the sub-cellular materials in the fluid sample and the relative percentage amounts of the three different wavelength bands whereby the amount of each sub-cellular material in substantially each cell of the fluid sample can be determined while a simultaneous determination of the overall size of the corpuscule is being made; and

c. means for identifying the corpuscules on the basis of overall size and the relative amounts of each sub-cellular material present in the corpuscule being identified.

36. The apparatus of claim 35, wherein said body fluid is moving at a known rate, said corpuscules are stained with a fluorochrome and said means for irradiating is a source of ultraviolet energy, and wherein the three different wavelength bands are red, gReen and yellow, said cell sixe being determined by said detecting means by measuring the duration of emissions from each cell while said cell is moving at said known rate.

37. The apparatus of claim 35, including a means for moving the fluid sample through a flow chamber that is normally disposed to a plane that passes through the irradiation means.

38. The method of identifying types of cells in a sample of body fluid comprising the steps of:

a. staining a sample of body fluid containing cells with a stain which will cause characteristic secondary fluorescence from different sub-cellular materials of the cells;

b. directing ultraviolet energy onto a portion of a sample consisting of substantially a single cell;

c. detecting the characteristic secondary fluorescent emissions from each of said stained sub-cellular materials;

d. determining the overall size of the cell being identified; and

e. identifying the cell type on the basis of percentage of green, yellow, and red secondary fluorescent emission bands detected, and cell size.

39. The method as defined in claim 38, wherein the sample of body fluid contains white blood cells, and wherein step (e) comprises identifying monochromatic and polychromatic cells by determining whether only one of the colors emitted exceeds about 25% of the sum of the emitted colors, or whether two or more of the colors emitted exceed about 25 percent of the sum of the colors emitted, respectively.

40. The method as defined in claim 39, wherein step (e) further comprises additionally identifying specific cell types within the monochromatic and polychromatic groups on the basis of the specific color and colors which predominate, as well as cell size.

41. The method as defined in claim 39, wherein monochromatic cells are identified as a lymphocyte, monocyte, reticulocyte or platelet, and wherein polychromatic cells are identified as a neutrophil, eosinophil, or basophil.

42. The method as defined in claim 41, wherein the specific cell type is identified substantially in accordance with the following characteristics:

43. The method as defined in claim 38, wherein the cells are moved at a known rate, and wherein the overall size of the cell being identified is determined by measuring the time duration of occurrence of the secondary fluorescence.