U.S. patent number 3,916,205 [Application Number 05/365,460] was granted by the patent office on 1975-10-28 for differential counting of leukocytes and other cells.
This patent grant is currently assigned to Block Engineering, Inc.. Invention is credited to Marcos Kleinerman.
United States Patent |
3,916,205 |
Kleinerman |
October 28, 1975 |
Differential counting of leukocytes and other cells
Abstract
A mixture of dyes for differentially staining biological cells.
A system for the automatic absolute counting of leukocytes,
erythrocytes and reticulocytes and differential counting of
leukocytes utilizes a composition characterized by specific
fluorescent dyes for staining a blood specimen and includes means
for irradiating, counting and classifying leukocyte types. When the
stained sample is irradiated, characteristic fluorescent
intensities in specific spectral ranges uniquely identify
particular leukocyte types.
Inventors: |
Kleinerman; Marcos (Arlington,
MA) |
Assignee: |
Block Engineering, Inc.
(Cambridge, MA)
|
Family
ID: |
23439004 |
Appl.
No.: |
05/365,460 |
Filed: |
May 31, 1973 |
Current U.S.
Class: |
250/461.2;
250/302; 356/36; 435/40.51; 250/304; 356/39 |
Current CPC
Class: |
G01N
15/147 (20130101); G01N 33/5094 (20130101); G01N
15/1468 (20130101); G01N 2015/008 (20130101); G01N
2021/6441 (20130101) |
Current International
Class: |
G01N
33/50 (20060101); G01N 21/64 (20060101); G01N
15/14 (20060101); G01N 15/00 (20060101); G01N
021/38 () |
Field of
Search: |
;250/302,304,365,458,461
;356/36,39 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
3315229 |
April 1967 |
Smithline |
3497690 |
February 1970 |
Wheeless, Jr. et al. |
|
Primary Examiner: Borchelt; Archie R.
Attorney, Agent or Firm: Schiller & Pandiscio
Claims
What is claimed is:
1. Method of identifying a blood cell, and comprising the steps
of:
staining said blood cell with a plurality of dyes, at least one of
which is a fluoresecent dye;
illuminating the dyed cell with a sequence of exposures to
radiation, each of said exposures being at a limited wavelength
band corresponding to the wavelength absorption band of a
respective one of said dyes;
observing during each such exposure the characteristic wavelength
band of emission or transmission by a selected one of said dyes in
said dyed cell in response to each of said exposures; and
comparing with one another the relative intensities of the emission
or transmission observed for each sequential exposure, whereby said
cell can be characterized by such comparison.
2. Method as defined in claim 1 wherein another dye of said
plurality of dyes is a sensitizer capable of transferring energy
absorbed in its characteristic absorption wavelength band to
molecules of said fluorescent dye in proximity to said sensitizer;
and
further including the step of observing the fluorescent emission
from said dyed cell in the characteristic wavelength emission band
of said fluorescent dye in response to an exposure of said dyed
cell to radiation in the characteristic wavelength absorption band
of said sensitizer.
3. A method as defined in claim 1 wherein said plurality is at
least three, and one of said dyes differentially dyes nucleic acids
of said cell, one of said dyes stains the basic portions of said
cell and the third of said dyes dyes the acidic portions of said
cell.
4. A method as defined in claim 1 wherein at least two of said dyes
are fluorescent dyes which dye different types of blood cells so
that each said type fluoresces under each said illumination with a
different intensity.
5. A method as defined in claim 4 wherein said cell types are
leukocytes, erythrocytes and reticulocytes.
6. A method as defined in claim 4 wherein said cell types are
different types of leukocytes.
7. The method for identifying leukocyte types as claimed in claim 6
wherein said plurality of dyes includes sulfonated triazinyl
derivatives of 4,4' diamino stilbene for imparting a blue
fluorescence to the protein of leukocytes and to neutrophil and
eosinophil granules.
8. The method for identifying leukocytes types as claimed in claim
6 wherein said plurality of dyes includes 8-p-toluidino
1-naphthalene sulfonic acid for imparting a blue-green fluorescence
to eosinophil granules.
9. The method for identifying leukocytes distributed as claimed in
claim 6 wherein said plurality of dyes includes brilliant
sulfalfavine for imparting a blue-green fluorescence to eosinophil
granules.
10. The method for identifying leukocyte types as claimed in claim
6 wherein said plurality of dyes includes ethidium bromide for
imparting a red fluorescence to the nuclei of all leukocytes.
11. A system for identifying leukocytes, erythrocytes and
reticulocytes randomly distributed in a medium, said system
comprising:
a. means for staining said leukocytes, erythrocytes and
reticulocytes with a dye composition including sulfonated triazinyl
derivatives of 4,4' diamino stilbene, sulfonated fluorescent
derivatives of 1,8 napthalimide, and a phenanthridinium dye;
b. source means for sequentially illuminating the stained
leukocytes, erythrocytes and reticulocytes with optical radiation
in the characteristic absorption wavelength band of each said dye
and
c. detector means optically coupled to said irradiated leukocytes,
erythrocytes and reticulocytes for identifying each said type cell
according to its fluorescent intensity.
12. The system as claimed in claim 11 wherein said sulfonated
fluorescent derivatives of 1,8 naphthalimide is brilliant
sulfaflavine.
13. The system as claimed in claim 11 wherein said phenanthridinium
dye is ethidium bromide.
14. The system as claimed in claim 11 wherein said detector means
includes photodetector means for measuring light scattered by said
leukocytes for differentiating said leukocytes according to their
size.
15. A system for differentially classifying and counting leukocyte
types, particularly eosinophils, neutrophils, monocytes and
lymphocytes stained with at least a pair of compatible fluorescent
dyes and fixed on slide means, said system comprising:
means for illuminating said slide means with a sequence of at least
two exposures, each of said exposures being at a different
wavelength band corresponding to the wavelength absorption band of
a corresponding one of said dyes;
detector means optically coupled to said slide means for detecting,
during each of said exposures, the characteristic wavelength band
emitted by one of said dyes in said leukocytes in response to a
corresponding one of said exposuresbut not the characteristic
wavelength band emitted by any of the other dyes responsively to
said one of said exposures; and
means responsive to said detector means for classifying and
counting said leukocyte types according to a comparison of the
fluorescent intensities emitted by each said leukocyte type during
a sequence of exposures.
16. A system for differentially classifying and counting leukocyte
types randomly distributed in a blood sample, said system
comprising:
means for staining the leukocyte types in said sample with a dye
composition comprising a plurality of fluorescent dyes, at least
one of said fluorescent dyes imparting a first characteristic
fluorescence to the nuclei of all said leukocytes, said other
fluorescent dyes imparting a second characteristic fluorescence to
the cytoplasm of eosinophils, neutrophils, monocytes and
lymphocytes;
means for flowing the stained leukocytes in said sample in single
file;
source means for illuminating said stained leukocytes passing in
single file through said flow means with a sequence of at least two
exposures, each of said exposures being at a different wavelength
band corresponding to the wavelength absorption band of a
corresponding one of said dyes;
detector means optically coupled to said flow means for detecting,
during each of said exposures, only the characteristic wavelength
band emitted by one of said dyes in said leukocytes in response to
a corresponding one of said exposures but not any wavelength band
emitted by any other of said dyes in response to said one of said
exposures; and
means responsive to said detector means for classifying and
counting said leukocyte types according to a comparison of the
fluorescent intensities emitted by each said leukocyte type during
a sequence of exposures.
17. System for identifying a blood cell stained with a plurality of
compatible dyes, at least one of which is a fluorescent dye, said
system comprising, in combination:
means for illuminating said cell with a sequence of exposures to
radiation, each exposure of said sequence being at a different
wavelength band corresponding to the wavelength absorption band of
a corresponding one of said dyes;
means for observing during each of said exposures only the
characteristic wavelength band emitted or transmitted by one of
said dyes in said cell in response to a corresponding one of said
exposures;
means for generating a signal in response to each emission or
transmission from said cell resulting from each of said exposures;
and
means for comparing said signals to obtain a characteristic value
for said cell.
18. System as defined in claim 17 wherein said means for
illuminating includes means for providing at least two of said
exposures at different wavelength bands corresponding to the
absorption bands of respective ones of said dyes; and
said means for observing includes means for observing fluorescent
emission in the wavelength band of emission from said fluorescent
dye due only to exposure of said cell to radiation in the
wavelength absorption band of said fluorescent dye, and means for
observing fluorescent emission in the wavelength band of emission
from said fluorescent dye due only to exposure of said cell to
radiation in the wavelength absorption band of another of said
dyes.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention:
The present invention relates to cytology and, more particularly is
directed towards a composition for staining specific components of
cells particularly blood cells, and a method and apparatus for
differentially counting and classifying leukocyte types.
2. Description of the Prior Art:
Presently, there are several methods and systems for counting and
classifying leukocytes. In the usual method, a cytotechnician
microscopically views a blood smear prepared on an ordinary
microscope slide that has been stained with one of the Romanowsky
stains, such as the Wright or Giemsa stains. The cytotechnician
sequentially examines 100 leukocytes and classifies each
accordingly to its type. Not only is this method time consuming,
but it suffers also from the disadvantage of limited reliability
relative to counting and classifying of the less abundant cells
such as monocytes, eosinophils and basophils. In a recently
developed automated system, the leukocytes are made to flow through
three or four different channels, each channel provided with means
for staining the leukocytes flowing therein with a particular dye.
Due to the fact that several channels and different chemical
treatments are required for each cell type, such a system suffers
from the disadvantages that it is complex in design and costly to
manufacture.
In another automatic system, a selected region of a blood smear,
stained in the usual manner with a Romanowsky stain, is scanned
mechanically under a microscope provided with an electronic image
tube. When a leukocyte is in the field of view of the image tube,
the slide is stopped. An image analyzing computer connected to the
image tube classifies the leukocyte according to its cell profile
and cytoplasm color. Since this system requires the services of a
technician to select a region of the smear suitable for automatic
scanning, such systems have suffered from the disadvantages of
being time consumming and costly.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a composition
of matter, a method and an apparatus involving the automatic
absolute counting of leukocytes, erythrocytes reticulocytes, and
other cells, and the differential counting and classifying of
leukocyte types which do not suffer from the heretofore mentioned
disadvantages. The present invention provides a dye composition for
distinguishing eosinophils, monocytes, lymphocytes, mature and
immature neutrophils, erythrocytes and reticulocytes randomly
distributed in a blood medium. The dye composition comprises a
plurality of compatible dyes, at least one of the dyes imparting a
characteristic fluorescence to all leukocytes stained therewith.
The dye composition is such that, when the stained leukocytes are
irradiated with optical radiation having the characteristic
absorption wavelengths of each dye, the relative intensities of the
light emitted and/or transmitted by each leukocyte type defines a
particular leukocyte type.
The term "compatible dyes" is used here to mean that no dye in the
mixture prevents any other dye in the mixture from selectively
staining characteristic structures of the cell types of interest
when irradiated in the characteristic absorption region of said
other dye, such selective staining being essentially not different
from the action of said other dye when used by itself.
The term "fluorochrome stain" is used to designate a stain which
under suitable illumination produces better differentiated optical
parameters about the cell type of interest by its fluorescence than
by its absorption properties, for instance, when the light
transmitted by the dye in its main absorption band is not much
different in intensity from that transmitted by its surroundings,
but the fluorescence intensity of the same dye is several times or
more greater than that of its surroundings. The term "differential
staining" is used to mean that the dye being used will stain a
given component of the cell type of interest for instance the
protein of said cell type to a much greater extent that other
components, for instance nucleic acids, of the same cell type. One
fluorescent dye composition, particularly adapted for slide
scanning and flow tube methods for differentially counting and
classifying leukocytes, comprises sulfonated triazinyl derivatives
of a diamino stilbene, having each amino group in each of the
phenyl rings of the stilbene molecule, preferably a sulfonated 4,4'
diamino stilbene, either of the dyes 8-p-toluidino-1-naphthalene
sulfonic acid or brilliant sulfaflavine, and ethidium bromide. In
the slide scanning method, a drop of blood is spread on a
microscope slide and, after fixation, is stained by immersion in
the dye composition. In the flow tube method, a small amount of a
blood sample, suitably diluted, is stained in a liquid solution of
the dye composition and the cells are forced to flow in single file
through a capillary tube. In both methods, the stained leukocytes
are irradiated under ultraviolet and violet light and their
fluorescence is detected through a blue-green filter. The
leukocytes are presented with a fluorescence in decreasing order to
intensity from eosiniphils to immature neutrophils to mature
neutrophills to monocytes to lymphocytes. The stained leukocytes
are then irradiated under a green light and the nuclei of the
different leukocyte types are detected through a red filter.
Leukocytes exhibit a red fluorescence of comparable intensity,
reticulocytes fluoresce with an appreciably smaller intensity and
normal erythrocytes have a negligible fluorescence. The leukocytes
are counted and classified, for example, by measuring the ratio of
the blue-green component of cell fluorescence to the red nuclear
fluorescence. In the flow tube method, excitation with ultraviolet
light produces a strong orange-red component, most pronounced in
neutrophils and eosinophils, the intensity of which, with respect
to the blue-green component, provides another parameter for
distinguishing particular leukocyte types. In addition, the
transmitted violet light is measured to distinguish erythrocytes
from leukocytes. In contrast to the negligible absorption of violet
light by leukocytes, erythrocytes and reticulocytes exhibit an
appreciable absorption of violet light.
The invention accordingly comprises the composition, method steps
and apparatus possessing the construction, combination of elements,
and arrangement of parts that are exemplified in the following
detailed disclosure, the scope of which will be indicated in the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
For a fuller understanding of the nature and objects of the present
invention, reference should be had to the following detailed
description taken in connection with the accompanying drawings
wherein:
FIG. 1 is a block and schematic diagram of a slide scanning system
for differential counting of leukocytes;
FIG. 2 is a block and schematic diagram of a flow tube system for
differential counting of leukocytes; and
FIG. 3 is a graphic representation illustrating the fluorescent
characteristics of selected leukocyte types on a methanol fixed
smear stained with one of the mixtures herein described.
DETAILED DESCRIPTION OF THE INVENTION
Generally, the process embodying the present invention involves the
staining of a blood medium with a dye composition comprising a
plurality of dyes, at least one of the dyes imparting a
characteristic fluorescence to all leukocytes. The dye composition
is such that, when the stained blood medium is illuminated with
light having the characteristic absorption wavelengths of each dye,
the relative intensities of the light emitted and/or transmitted by
each cell type at the optical wavelength region of emission and
absorption characteristic of each dye uniquely depends, in at least
one characteristic wavelength region, on the type of leukocyte. The
light emitted by the irradiated leukocytes in each of their
characteristic wavelength regions is measured and the leukocyte
types are classified according to the relative intensities of the
emitted light in the characteristic wavelength region of each
dye.
In particular, the process embodying the present invention involves
the staining of leukocytes randomly distributed in a medium with a
dye composition comprising a mixture of (1) a sulfonated triazinyl
stilbene derivative, (2) a naphthalene sulfonic acid; and (3) a
cationic dye. The stained leukocytes are irradiated with
ultraviolet and violet light and detected through a green or
blue-green filter. The green component of leukocyte fluorescence is
presented with a characteristic fluorescent intensity against a
dark background. The stained leukocytes are also irradiated with
green light and the red fluorescence of only the nuclei of the
cells are detected through a red or orange-red filter. As shown in
FIG. 3, the detected leukocytes are characterized by a fluorescence
in decreasing order of intensity from eosinophils to neutrophils to
monocytes to lymphocytes, each leukocyte type uniquely defined by
the ratio of the blue-green component of cell fluorescence to the
red nuclear fluorescence with respect to the red fluorescence from
the nucleus.
The dyes for protein, such as the cytoplasm of white blood cells
and granules of granulocytes, preferably are sulfonated triazinyl
derivatives of a diamino stilbene, for example 4,4'-bis{4-(3
sulfoanilino)-6-[bis (2-hydroxy-ethyl)-amino]-1,3,5triazin-2-yl }
amino stilbene 2,2'-disulfonic acid tetrasodium salt and their
alkyl, alkoxy or halogen substituted derivatives. For these dyes,
the optimum excitation wavelength band is in the range of 320 to
390 nanometers and the optimum fluorescent wavelength band is 440
to 550 nanometers. These dyes impart a strong blue fluorescence to
the protein of leukocytes and the neutrophil granules and a weaker
blue fluorescence to eosinophils, monocytes and lymphocytes.
The dyes for eosinophil granules preferably are the anilino or
toluidino naphthalene sulfonic acids and their alkyl, alkoxy or
halogen substituted derivatives; 4,4' diamino stilbene 2,2'
disulfonic acid, N, N, N', N' tetraacetric acid and its alkyl,
alkoxy or halogen substituted derivatives; sulfonated fluorescent
derivatives of 1,8 naphthalimide, such as brilliant sulfaflavine
and its alkyl, alkoxy or halogen substituted derivatives;
8-p-toluidino-1 naphthalene sulfonic acid and its alkyl, alkoxy or
halogen substituted derivatives; and 8-hydroxy-1, 3, 6 pyrene
trisulfonic acid and its alkyl, alkoxy and halogen derivatives. The
anilino and toluidino naphthalene sulfonic acids have an optimum
excitation wavelength band in the range of 320 to 410 nanometers
and an optimum fluorescent wavelength band in the range of 440 to
550 nanometers. Brilliant sulfoflavine has an optimum excitation
wavelength band in the range of 360 to 450 nanometers and an
optimum fluorescent wavelength band in the range of 480 to 550
nanometers. These dyes strongly stain the eosinophil granules with
a green-blue or green fluorescence.
The dyes which impart a strong red fluorescence to the nucleic
acids of all leukocytes, mainly in the nuclei, preferably are
cationic dyes, for example the phenanthridinium dyes such as
ethidium bromide and its alkyl, alkoxy or halogen derivatives; and
in dry smears, acridine orange; and rhoduline orange. Ethidium
bromide has an optimum excitation wavelength range of 480 to 550
nanometers and an optimum fluorescent wavelength range of 580 to
650 nanometers.
Referring now to FIG. 1, there is shown a slide scanning system 10
for differentially counting leukocytes. By way of example, an
alcohol-fixed blood smear 11 on a slide 12 is immersed in a
preferred dye solution comprising 1 .times. 10.sup.-.sup.4 molar
ethidium bromide; 1 .times. 10.sup.-.sup.3 to 1 .times.
10.sup.-.sup.4 molar brilliant sulfaflavine; and 1 .times.
10.sup.-.sup.4 molar 4,4' bis {4-(3-sulfoanilino)-6[bis
(2-hydroxy-ethyl)amino]-1,3,5 triazin-2-yl } amino stilbene 2,2'
disulfonic acid tetrasodium salt in a solvent including a buffer of
0.01 to 0.1 molar 2-amino- 2(hydroxymethyl)-1,3 propanediol-HC1,
commonly known as tris-HC1, at a pH concentration range of 8.0 to
10.0, for best results, the pH concentration range is 8.5 to 9.5.
In alternative embodiments, the buffer is other than tris-HC1, for
example sodium borate or sodium bicarbonate. After a staining time
of approximately 10 minutes, slide 12 is rinsed in an aqueous
solution, for example distilled water, for approximately 1 minute
and then dried.
Thereafter, slide 12 is irradiated with illumination from a source
14, for example a mercury arc lamp, which is focused thereon via a
collimating lens 16, an excitation filter wheel 18, a beam splitter
20 and a condensing lens 22. Filter wheel 18 includes a filter 32
which transmits ultraviolet and violet light, for example 320 to
440 nanometers and a filter 34 which transmits green light, for
example the 546 nanometer mercury band. In the illustrated
embodiment, by way of example, beam splitter 20 is a dichroic
mirror. In alternative embodiments, beam splitter 20 is other than
a dichroic mirror, for example a reflector such as a straight
surface mirror. The field of view containing the fluorochromed
leukocytes is imaged on a photo-electronic device 24, for example
the photosensitive surface of an image scanning tube, via a
collimating lens 26 and an emission filter wheel 28. Filter wheel
28 includes a blue-green filter 36 and a red filter 38. As
hereinafter described, filter wheels 18 and 28 are indexed by means
of a controller 30.
Initially, controller 30 indexes filter wheels 18 and 28 in such a
manner that filters 32 and 36 are positioned in the transmitted
light path. That is, slide 12 is irradiated with ultraviolet and
violet light and blue-green fluorescent images are presented at the
photosensitive surface of image scanning tube 24 via blue-green
filter 36. The green components of leukocyte fluorescence appear
against a dark background with a fluorescence in decreasing order
of intensity from eosiniphils to neutrophils to monocytes to
lymphocytes. Increasing the excitation radiation from 360 to 440
nanometers with respect to that from 320 to 360 nanometers,
maximizes the difference between the fluorescent intensities of the
eosiniphils and neutrophils. The photosensitive surface of image
tube 24 is scanned in a specified pattern determined by a control
40 which is programmed by a computer 42. The intensity of the
irradiated leukocytes are measured by computer 42 and data signals
for each measurement are stored in a memory 44 at X,Y address
location corresponding to the X,Y positions on the photosensitive
surface of image scanning tube 24.
Controller 30 then indexes filter wheels 18 and 28 in such a manner
that filters 34 and 38 are positioned in the transmitted light
path. That is, slide 12 is irradiated with green light and red
fluorescent images are presented at the photosensitive surface of
image scanning tube 24 through red filter 38. In this case, only
the red fluorescence of the nuclei of the leukocytes appear against
a dark background at the photosensitive surface of image tube 24.
In the manner hereinbefore described, the fluorescent intensities
of the nuclei at each of the X,Y positions on the photosensitive
surface of image scanning tube 24 are measured by computer 42. The
measurement data stored in memory 44, i.e., the green component of
cell fluorescence, is addressed into computer 42 for determining
leukocyte types by generating differential counting data signals
representing the ratio of the green component of cell fluorescence
to red nuclear fluorescence with respect to the red fluorescence
from the nucleus as shown in FIG. 3. Cell profile data signals,
i.e. a measurement of the time period during which a signal from a
cell is received, distinguish lymphocytes from monocytes. The
differential counting data signals generated by computer 42 are
applied to a display 46, for example a digital display of the
relative abundance of the different cell types, for visual
presentation. It is to be understood that, in alternative
embodiments display 46 is other than a digital display, for example
a cathode-ray tube, a chart recorder, or a magnetic tape
recorder.
It will be readily appreciated that systems other than that shown
in FIG. 1 can be used for differentially counting and classifying
leukocytes types on a stained blood smear. For example, a manual
system wherein the stained slide is irradiated in the manner
hereinbefore described and observed through a microscope. In
alternative embodiments, the irradiated leukocytes are detected by
means other than an image scanning tube, for example one or a
plurality of photo-detectors and a flying spot scanner.
Referring now to FIG. 2, there is shown a flow tube system 50 for
differential counting of leukocyte types. By way of example, a
blood sample is diluted in a saline solution and the cells are
fixed with formaldehyde. It is to be noted that the cells can be
fixed with formaldehyde prior to or after dilution. The white cells
are stained in a solution comprising 1 .times. 10.sup.-.sup.4 molar
4,4' bis {4-(3 sulfoanilino)-6-[bis (2-hydroxy ethyl) amino]-1,3,5
triazin -2- yl}amino stilbene; 1 .times. 10.sup.-.sup.3 to 1
.times. 10.sup.-.sup.4 molar brilliant sulfaflavine; and 1 .times.
10.sup.-.sup.4 molar ethidium bromide. The resulting suspension is
diluted and buffered with either 0.1 molar tris-HC1 or 0.05 molar
borax at a pH in the range of 9.0 to 9.2. As an example, the blood
sample has a dilution range of fifty to one hundred fold in the
suspension, the final concentration of which is approximately 1
.times. 10.sup.-.sup.4 molar or less in each of the fluorescent
dyes. The cells are made to flow single file through a narrow tube
52, for example a capillary tube, wherein each of the leukocytes is
irradiated with illumination from a source 54, for example a
mercury arc lamp. The illumination generated by mercury arc lamp 54
is directed through a collimating lens 56 to a reflecting filter
58, for example a dichroic filter. The wavelength band of light
passing through dichroic filter 58 is focused on capillary tube 52
at 62 via a condensing lens 64. In the illustrated embodiment, by
way of example, dichroic filter 58 transmits green light in the 546
nanometer mercury band. The wavelength band of light reflected by
dichroic filter 58 is directed to a reflecting filter 66, for
example a dichroic filter, and focused on capillary tube 52 at 70
via a condensing lens 72. In the illustrated embodiment, by way of
example, dichroic filter 66 reflects ultraviolet and violet light
in the 320 to 410 nanometer mercury band. It is preferred that the
distance between the locations denoted by reference characters 62
and 70 is typically in the range of 50 to 200 microns. A
photo-electric device 74, for example a photo-multiplier senses the
blue-green emission light from capillary tube 52 via a collecting
lens 76, a dichroic mirror 77 and a blue-green emission filter 78.
A photo-electric device 80, for example a photo-multiplier, senses
the orange-red emission light from capillary tube 52 via collecting
lens 76, dichroic mirror 77, a dichroic mirror 82 and an orange-red
emission filter 84. A photo-electric device 81, for example a
photodiode, senses the ultraviolet and violet light at 70 via an
emission filter 82 having a pass band in the approximate range of
410 to 430 nanometers.
Photo-multiplier 74 detects the blue-green component of cell
fluorescence, and photo-multiplier 80 detects the red fluorescence,
and photo-multiplier 81 detects the violet light. Erythrocytes and
reticulocytes are characterized by appreciable absorption of violet
light and leukocytes are characterized by negligible absorption of
violet light. Accordingly, photo-multiplier 81 detects the violet
light absorption for distinguishing red cells from white cells.
Data signals generated by photo-multipliers 74 and 80 are applied
to a precessor 86, for example a small dedicated computer, wherein
the detected leukocyte fluorescences are differentially counted and
classified as the ratio of the green component of cell fluorescence
to the red nuclear fluorescence with respect to the red
fluorescence from the nucleus for each leukocyte type. Differential
counting data signals generated by computer 86 for each leukocyte
passing through capillary tube 52 are applied to a display 88, for
example a digital display of the relative abudance of the different
cell types, for presentation. In addition, data signals generated
by photodiode 81, representing a measurement of the absorbed violet
light, are applied to computer 86 for distinguishing erythrocyte
and leukocytes. Leukocyte types can be distinguished from one
another by measuring light scattered by the leukocytes by means of
photodetectors, for example photodiodes. It is to be understood
that, in alternative embodiments, display 88 is other than a
digital display for example, a cathode-ray tube, a chart recorder
or a magnetic tape recorder. Furthermore, it is to be understood
that, in alternative embodiments, the light emitted from source 54
is directed to tube 52 by means other than a pair of beam
splitters, for example, two sources each emitting light which is
directed towards tube 52 or a source characterized by a scanning
light beam.
The following non limiting examples further illustrate the staining
of slides and suspensions hereinbefore described.
EXAMPLE I
SLIDE
A drop of whole blood is placed on a microscope slide and spread
into a thin film with the aid of another microscope slide. After
the film has dried, the blood smear is fixed by immersing the slide
in methyl alcohol for five minutes. Thereafter, the slide is
immersed in a liquid mixture comprising:
1. 100 parts per million of 4,4'-bis{4-(3 sulfoanilino)-6-[bis
(2-hydroxy-ethyl)-amino]-1,3,5 triazin-2-yl }amino stilbene
2,2'-disulfonic acid tetrasodium salt;
2. 1,000 parts per million of brilliant sulfaflavine; and
3. 30 parts per million of ethidium bromide;
4. 12,000 parts per million of 2-amino-2-(hydroxymethyl)-1,3
propanediol and enough concentrated hydrochloric acid solution to
bring the pH of the solution to 9.0.
After ten minutes in the liquid mixture, the slide is rinsed in
distilled water for one minute and then dried.
EXAMPLE II
SUSPENSION
One part of whole blood is mixed with four parts of a solution at a
pH of 9.0 comprising:
1. 1,200 parts per million of 2-amino-2-(hydroxymethyl)-1,3
propanediol-HCl buffer pH 9.0;
2. 85,000 parts per million of sodium chloride;
3. 1,000 parts per million of brilliant sulfaflavine;
4. 100 parts per million of 4,4'-bis {4-(3-sulfoanilino)-6-[bis
(2-hydroxyethyl)-amino]-1,3,5triazin-2-yl}amino stilbene
2,2'-disulfonic acid tetrasodium salt; and
5. 30 parts per million of ethidium bromide.
After three minutes, the suspension is mixed with one part of a 20%
solution of formaldehyde. Five minutes later, the mixture is
diluted 15 to 20 times with a solution comprising:
6. 12,000 parts per million of 2-amino-2-(hydroxymethyl)-1,3
propanediol-HCl buffer (pH 9.0);
7. 85,000 parts per million of sodium chloride;
8. 100 parts per million of 4,4'-bis {4-(3-sulfoanilino)-6-[bis
(2-hydroxy-ethyl)-amino]-1,3,5 triazin-2-yl}amino stilbene
2,2'-disulfonic acid tetrasodium salt; and
9. 15 parts per million of ethidium bromide.
In this example, the stained suspended cells exhibit the following
optical properties under ultraviolet illumination of approximately
365 nanometers:
a. The cytoplasm and granules of the neutrophils exhibit a visible
fluorescence with spectral peaks in the blue and orange-red
regions. The blue component results from the direct excitation of
the triazinyl dye and the red component, most pronounced in the
granules, is due to energy transfer from the triazinyl dye to
ethidium bromide, the latter being present in the granules, and
elsewhere in the cytoplasm, at concentrations which are too small
to be efficienctly excited by direct absorption of the ultraviolet
light.
b. The cytoplasm and granules of the eosinophils exhibit a stronger
visible fluorescence than the neutrophils with spectral peaks in
the blue-green and orange-red regions. The blue-green component
results mainly from the excitation of the brilliant sulfaflavine
dye and the triazinyl dye, and the red-orange component is due to
energy transfer from the triazinyl dye and the brilliant
sulfaflavine dye to ethidium bromide; the latter being present in
the granules, and elsewhere in the cytoplasm at concentrations too
small to be efficiently excited by direct absorption of the
ultraviolet light.
c. The cytoplasm and nucleus of lymphocytes and monocytes exhibit a
weaker fluorescence than that of either the neutrophils or
eosinophils. This fluorescence is characterized by a spectral
distribution having a peak in the blue region and a smaller peak or
shoulder in the red region, the fluorescence of the monocytes being
smaller than the fluorescence of the lymphocytes.
Under violet illumination of approximately 400 to 440 nanometers,
the eosinophils exhibit a strong green fluorescence which is
several times stronger than that of the other leukocytes.
Under green illumination, the nuclei, and to a lesser extent the
cytoplasm, of all the leukocytes exhibit a red fluorescence;
eosinophils having the brightest fluorescent intensity, neutrophils
and monocytes having comparable fluorescent intensities of a lesser
brilliance, and lymphocytes generally having the lowest fluorescent
intensity.
It is to be noted that red cells exhibit a negligible fluorescence
under any of the above conditions.
EXAMPLE III
SUSPENSION
One part of whole blood is mixed with one part of a 10% solution of
formaldehyde for five minutes. The suspension is then mixed with
four parts of a solution comprising:
1. 1,200 parts per million of 2-amino-2-(hydroxymethyl)-1,3
propanediol-HC-1 buffer (pH 9.0);
2. 85,000 parts per million of sodium chloride;
3. 300 parts per million of 8-p toluidino-1-naphthalene sulfonic
acid;
4. 100 parts per million of 4,4'-bis {4-(3-sulfoanilino)-6-[bis
(2-hydroxy-ethyl)-amino]-1,3,5 triazin-2-yl }amino stilbene
2,2'-sulfonic acid tetrasodium salt; and
5. 20 parts per million of ethidium bromide.
After five minutes, the mixture is diluted 15 to 20 times with a
solution comprising:
6. 12,000 parts per million of 2-amino-2-(hydroxymethyl)-1,3
propanediol-HCl buffer (pH 9.0);
7. 85,000 parts per million of sodium chloride;
8. 60 parts per million of 8 toluidino 1 naphthalene sulfonic
acid;
9. 100 parts per million of 4,4'-bis
{4-(3sulfoanilino)-6-[bis(2-hydroxy-ethyl)-amino]-1,3,5
triazin-2-yl }amino stilbene 2,2'-disulfonic acid tetrasodium salt;
and
10. 15 parts per million of ethidium bromide.
In this example, the stained suspended cells exhibit the following
optical properties under ultraviolet illumination of approximately
365 nanometers:
a. The cytoplasm and granules of the neutrophils exhibit a visible
fluorescence with spectral peaks in the blue and orange-red
regions. The blue component results from the direct excitation of
the triazinyl dye and the red component, most pronounced in the
granules, is due to energy transfer from the triazinyl dye to
ethidium bromide, the latter being present in the granules, and
elsewhere in the cytoplasm, at concentrations which are too small
to be efficiently excited by direct absorption of the ultraviolet
light.
b. The cytoplasm and granules of the eosinophils exhibit a stronger
visible fluorescence than the neutrophils with spectral peaks in
the blue-green and orange-red regions. The blue-green component
results mainly from the excitation of 8-p-toluidino-1-naphthalene
sulfonic acid and the triazinyl dye, and the red-orange component
is due to energy transfer from the triazinyl dye and the
8-p-toluidino-1-naphthalene sulfonic acid to ethidium bromide; the
latter being present in the granules, and elsewhere in the
cytoplasm at concetrations too small to be efficiently excited by
direct absorption of the ultraviolet light.
c. The cytoplasm and nuclei of lymphocytes and monocytes exhibit a
weaker fluorescence than that of either the neutrophils or
eosinophils. This fluorescence is characterized by a spectral
distribution having a peak in the blue region and a smaller peak or
shoulder in the red region, the fluorescence of the monocytes being
smaller than the fluorescence of the lymphocytes.
Under violet illumination of approximately 400 to 440 nanometers,
the eosinophils exhibit a strong green fluorescence which is
several times stronger than that of the other leukocytes.
Under green illumination, the nuclei, and to a lesser extent the
cytoplasm, of all the leukocytes exhibit a red fluorescence;
eosinophils having the brightest fluorescent intensity, neutrophils
and monocytes having comparable fluorescent intensities of a lesser
brilliance, and lymphocytes generally having the lowest fluorescent
intensity.
It is to be noted that red cells exhibit a negligible fluorescence
under any of the above conditions.
In summary, the present invention provides a single dye composition
comprising three fluorescent dyes for uniquely distinguishing
leukocytes, erythrocytes and reticulocytes randomly distributed in
a medium and a method and apparatus for absolute counting of
leukocytes and reticulocytes and differential counting of
leukocytes which have been stained with such a dye composition.
When the stained leukocytes are irradiated by light having the
characteristic absorption wavelengths of each dye, the relative
intensities of leukocyte fluorescence at the optical wavelength
region of emission of each dye depends, in at least one
characteristic wavelength region, on the type of leukocyte. The
light emitted by the irradiated leukocytes in each of their
characteristic wavelength regions is measured and the leukocyte
types are classified according to the relative intensities of the
emitted light in the characteristic wavelength region of each dye.
Transmitted violet light is measured to distinguish erythrocytes,
reticulocytes and leukocytes, erythrocytes and reticulocytes
exhibiting appreciable absorption of violet light and leukocytes
exhibiting negligible absorption of violet light.
Since certain changes may be made in the foregoing disclosure
without departing from the scope of herein involved, it is intended
that all matter contained in the above description and depicted in
the accompanying drawings be construed in an illustrative and not
in a limiting sense.
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