U.S. patent number 3,790,760 [Application Number 05/184,183] was granted by the patent office on 1974-02-05 for apparatus for counting, differentiating and sorting particles according to their microstructure variations.
This patent grant is currently assigned to Cornell Aeronautical Laboratory, Inc.. Invention is credited to Paul F. Stiller.
United States Patent |
3,790,760 |
Stiller |
February 5, 1974 |
APPARATUS FOR COUNTING, DIFFERENTIATING AND SORTING PARTICLES
ACCORDING TO THEIR MICROSTRUCTURE VARIATIONS
Abstract
An apparatus for counting and sorting the formed elements of
blood, comprising the phase illumination of sample flow through a
microcapillary passage, photodetectors responsive to the
microstructure of the formed elements, a signal analyzer responsive
to the photodetectors, a logic network for generating control
signals, a sorter responsive to the control signals for separating
and collecting the formed elements.
Inventors: |
Stiller; Paul F. (Clarence,
NY) |
Assignee: |
Cornell Aeronautical Laboratory,
Inc. (Buffalo, NY)
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Family
ID: |
22675874 |
Appl.
No.: |
05/184,183 |
Filed: |
September 27, 1971 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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877729 |
Nov 18, 1969 |
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Current U.S.
Class: |
377/10; 356/335;
377/53 |
Current CPC
Class: |
G06M
1/101 (20130101) |
Current International
Class: |
G06M
1/10 (20060101); G06M 1/00 (20060101); G06m
011/02 () |
Field of
Search: |
;235/92PC |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Wilbur; Maynard R.
Assistant Examiner: Gnuse; Robert F.
Attorney, Agent or Firm: Jaffe; Allen J.
Parent Case Text
This is a continuation of application Ser. No. 877,729, filed Nov.
18, 1969, now abandoned.
Claims
1. Apparatus of the character described, comprising;
a. first means for containing a sample of fluid, the constituents
of which include particles each having a microstructure indicative
of the identity thereof,
b. said first means having at least a portion thereof that is
transparent to electromagnetic radiation,
c. means for irradiating said portion of said first means and the
sample fluid therein with electromagnetic radiation,
d. optical means for a magnifying the image of said portion of said
first means and the sample fluid therein and for converting small
invisible optical phase differences between the microstructure
components of each particulate constituent of said sample fluid to
visible intensity differences,
e. detection means responsive to the electromagnetic radiation
passing through said first means for developing a varying output
signal indicative of the varying intensity of said electromagnetic
radiation,
f. means for analyzing and processing said varying output signal
for developing a plurality of control signals each indicative of a
specific particulate constituent of said sample fluid,
g. at least two collecting means adapted for fluid communication
with said first means downstream of said portion thereof, and
h. control means responsive to said control signals for placing
predetermined individual collecting means in fluid communication
with said
2. The apparatus according to claim 1, wherein;
i. the number of said collecting means corresponds to the number
of
3. The apparatus according to claim 1, wherein;
i. said first means is so sized relative to said particulate sizes
that the particles are substantially sequentially arranged in said
portion thereof.
4. The apparatus according to claim 1, wherein;
i. said detection means has a radiation sensitive area that is much
smaller than the image of the particle impinging thereupon whereby
only a thin cross-sectional slice of the particle image traverses
said sensitive area
5. The apparatus according to claim 4, wherein;
j. said first means is so sized relative to said particulate sizes
that the particles are substantially sequentially arranged in said
portion thereof.
6. The apparatus according to claim 5, wherein;
k. said optical means includes means for shifting the phase of the
radiation passing through said microstructure components with
respect to the radiation passing therebetween by substantially a
quarter of a
7. The apparatus according to claim 6, wherein;
l. said means for illuminating comprises a substantially
monochromatic
8. The apparatus according to claim 7, wherein;
m. said means for analyzing and processing said varying output
signal comprises a plurality of narrow band-width filters and a
binary logic
9. The apparatus according to claim 8, wherein;
n. said collecting means comprises a plurality of movable tubes,
each spaced from an end of said conduit means, and
o. said control means comprises motive devices for moving said
tubes into
10. The apparatus according to claim 9, wherein;
11. The apparatus according to claim 1, wherin;
i. said optical means includes means for shifting the phase of the
radiation passing through said microstructure components with
respect to the radiation passing therebetween by substantially a
quarter of a
12. The apparatus according to claim 11, wherein;
j. said means for illuminating comprises a substantially
monochromatic
13. The apparatus according to claim 1, wherein;
i. said collecting means comprises a plurality of movable tubes,
each spaced from an end of said conduit means, and
j. said control means comprises motive devices for moving said
tubes in
14. The apparatus according to claim 13, wherein;
15. The apparatus according to claim 1, wherein;
i. said means for analyzing and processing said varying output
signal comprises a plurality of narrow band-width filters
responsive to said
16. The apparatus according to claim 15, wherein;
j. said means for analyzing and processing further comprises a
plurality of thresholding circuits responsive to the output from
said narrow band-width
17. The apparatus according to claim 16, wherein;
k. said means for analyzing and processing further comprises a
binary logic
18. The apparatus according to claim 17, wherein;
l. said narrow band-width filters respond to frequencies contained
within said varying output signal from said detection means
substantially in the ratio of one, six and ten, and
19. The apparatus according to claim 1, further comprising;
i. a quantity of sample fluid in said first means, the constituents
of which include particles each having a microstructure indicative
of the
20. The apparatus according to claim 19, wherein;
j. said sample fluid comprises blood and said particles comprise
the formed
21. The apparatus according to claim 1, further comprising;
22. The apparatus according to claim 1, wherein;
i. said detection means has a radiation sensitive area that is much
smaller than the image of the particle impinging thereupon whereby
only a thin cross-sectional slice of the particle image traverses
said sensitive area as said particle moves with respect to said
detection means, and there is further provided;
j. second detection means located adjacent said first mentioned
detection means, said second detection means has a radiation
sensitive area so sized to span a slice of the image of said first
means that is substantially
23. The apparatus according to claim 22, wherein;
k. said second detection means is located upstream of said first
mentioned detection means with respect to the direction relative of
sample fluid
24. The apparatus according to claim 23, further comprising;
l. means responsive to predetermined output signals from said
second
25. The apparatus according to claim 24, further comprising;
m. counting means responsive to said control signals and to said
outputs
26. The apparatus according to claim 24, further comprising;
m. enabling means for rendering said first mentioned detection
means operative in response to a signal derived from said second
detection means
27. The apparatus according to claim 26, further comprising;
n. a quantity of sample fluid in said first means, the constituents
of which include particles each having a microstructure indicative
of the
28. The apparatus according to claim 27, wherein;
o. said sample fluid comprises blood and said particles comprise
the formed elements thereof, and wherein;
p. said predetermined output signals are indicative of the
erythrocytes and
29. Apparatus of the character described, comprising;
a. first means for containing a sample of fluid, the constituents
of which include particles each having a microstructure indicative
of the identity thereof,
b. said first means having at least a portion thereof that is
transparent to electromagnetic radiation,
c. means for irradiating said portion of said first means and the
sample fluid therein with electromagnetic radiation,
d. optical means for magnifying the image of said portion of said
first means and the sample fluid therein for converting small
invisible phase differences between the microstructure components
of each particulate constitutent of said sample fluid visible to
intensity differences,
e. detection means responsive to the radiation passing through said
first means for developing a varying output signal indicative of
the varying intensity of said electromagnetic radiation, and
f. means for analyzing and processing said varying output signal
for developing a plurality of output signals each indicative of a
specific
30. The apparatus according to claim 29, wherein;
g. said first means is so sized relative to said particulate sizes
that the particles are substantially sequentially arranged in said
portion thereof.
31. The apparatus according to claim 30, wherein;
h. said detection means has a radiation sensitive area that is much
smaller than the image of the particle impinging thereupon whereby
only a thin cross-sectional slice of the particle image traverses
said sensitive area
32. The apparatus according to claim 31, wherein;
i. said optical means includes means for shifting the phase of the
radiation passing through said microstructure components with
respect to the radiation passing therebetween by substantially a
quarter of a
33. The apparatus according to claim 32, wherein;
j. said means for illuminating comprises a substantially
monochromatic
34. The apparatus according to claim 33, wherein;
k. said means for anlayzing and processing said varying output
signal comprises a plurality of a narrow band-width electronic
filters and a
35. The apparatus according to claim 29, wherein;
g. said detection means has a radiation sensitive area that is much
smaller than the image of the particle impinging thereupon whereby
only a thin cross-sectional slice of the particle image traverses
said sensitive area
36. The apparatus according to claim 29, wherein;
g. said optical means includes means for shifting the phase of the
radiation passing through said microstructure components with
respect to the radiation passing therebetween by substantially a
quarter of a
37. The apparatus according to claim 36, wherein;
h. said means for illuminating comprises a substantially
monochromatic
38. The apparatus according to claim 29, further comprising;
39. The apparatus according to claim 29, wherein;
g. said detection means has a radiation sensitive area that is much
smaller than the image of the particle impinging thereupon whereby
only a thin cross-sectional slice of the particle image traverses
said sensitive area as said particle moves with respect to said
detection means, and there is further provided;
h. second detection means located adjacent said first mentioned
detection means, said second detection means has an electromagnetic
radiation sensitive area so sized to span a slice of the image of
said first means
40. The apparatus according to claim 39, further comprising:
i. means responsive to predetermined output signals from said
second
41. The apparatus according to claim 40, further comprising;
j. counting means responsive to said control signals and to said
outputs
42. The apparatus according to claim 40, further comprising;
j. enabling means for rendering said first mentioned detection
means operative in response to a signal derived from said second
detection means
43. The apparatus according to claim 29, wherein;
44. The apparatus according to claim 29, wherein;
g. said means for analyzing performs a spectrum analysis of said
varying
45. Apparatus of the character described, comprising;
a. first means for containing a sample of fluid, the constituents
of which include particles each having a microstructure indicative
of the identity thereof,
b . said first means having at least a portion thereof that is
transparent to electromagnetic radiation,
c. means for irradiating said portion of said first means and the
sample fluid therein with electromagnetic radiation,
d. optical means for magnifying the image of said portion of said
first means,
e. detection means responsive to the electromagnetic radiation
passing through said first means for developing a varying output
signal indicative of the varying intensity of said electromagnetic
radiation, said detection means having an electromagnetic radiation
sensitive area that is much smaller than the image of the particles
impinging thereupon whereby only a thin cross-sectional slice of
the particle image traverses said sensitive area as said particle
moves with respect to said detection means, and
f. means for analyzing and processing said varying output signal
for developing a plurality of output signals each indicative of a
specific particulate constituent of said sample fluid.
Description
BACKGROUND OF THE INVENTION
The present invention relates to the analysis and control of fluids
containing particles which have microstructures of varying optical
densities. More particularly, the present invention finds specific
application in the analysis of fresh whole blood to differentiate,
count, sort and collect the formed elements thereof.
Current clinical practice involves the sampling of a patient's
blood to determine the type, quantity, and characteristics of the
various formed elements thereof; which, as is well known, comprise;
red cells, five major varieties of white cells, and blood
platelets. The current technique is to smear a small quantity of
whole blood on a microscope slide, to stain it, and then, it
manually observe, differentiate and count the "blood cells."
Various devices are presently known which attempt the automation of
blood counting. These for the most part are capable of counting red
cells and, by chemical lysing, total white cells. None of these
known devices are capable of differentiating in real time the five
basic varieties of white cells, nor can they handle all of the
cells, including the platelets, without secondary procedures, and
none are capable of sorting and saving the sampled cell.
According to one of the currently known devices, a blood sample is
diluted and, to count white cells, a portion of the sample is lysed
to destroy the red cells and pumped through a dark field
illuminated observation chamber. As each particle passes through
the chamber a photo sensor records the forward scattering of light
caused by the white cells. To count red cells, the remaining
unlysed sample is further diluted and pumped through the chamber.
This device cannot distinguish between cell types and the sample is
destroyed in the test.
Another known prior device utilizes a conductive fluid to dilute
the sample. As the fluid passes through the sampling chamber the
conductivity of the fluid changes because of the presence of the
cells. Red and white cell differentiation is possible because of
the relative size of the two cells and the resulting difference in
conductivity. This system is not capable of reliable platelet
counting and the sample is also destroyed in the process.
SUMMARY OF THE INVENTION
The foregoing, as well as other, disadvantages of the prior art
devices are overcome by the principles of the present invention
which provides an apparatus which is capable of differentiating all
of the blood cell types without the necessity of chemical lysing or
dying. In addition the cells are not destroyed during the test and
can be sorted and collected for further analysis.
The present invention generally relates to a method and apparatus
for the analysis and control of fluids containing particles whch
have microstructures of varying optical path lengths. A sample of
the fluid is diluted and pumped through a microcapillary
observation chamber for substantially sequential flow of the
individual particles within the fluid. A portion of the
microstructure of each particle is observed by transilluminated
phase optics. Photosensitive devices respond to the optical path
length variations within each observed particle and generate a
varying output signal indicative thereof. The output signal is
analyzed by a suitably programmed logic network which develops
control signals that are applied to flow controllers for sorting
and collecting the different particles based on the varying
microstructures thereof.
With respect to the formed elements of whole blood, it has been
found that each blood cell type exhibits a microstructure having
its own special random pattern which does not vary with respect to
cell size or maturity. That is to say, the spatial distribution of
the optical densities for each of the microstructure patterns are
specific to each cell type. Thus, the individual cell types can be
identified by differences in their microstructures. It is not
necessary, however, to observe or scan the complete microstructure
of any given cell before an identification can be made. It has been
found, according to the present invention, that sufficient
information for cell identification can be obtained by observing
only that portion of the microstructure contained within a narrow
slice across a randomly oriented cell, as it passes through the
microcapillary tube.
Since the cell and its microstructure exhibit substantially the
same color and contrast, ordinarily incoherent illumination will
not permit visualization of the microstructure. However small
optical path length differences do exist within the cell
microstructure and, according to the present invention, detection
means are provided that are responsive to these path length
differences as the cells flow sequentially through the
microcapillary tube. The signal developed by such detection means,
suitably processed will be indicative of the identity of the
particular cell type that generated such signal. In this manner,
the apparatus can function in real time to differentiate, count,
sort and collect the formed elements of whole blood.
Once the particle passing through the microcapillary is identified
the present invention permits segregation thereof by the provision
of suitable means controlling the flow of particles to a plurality
of separate collecting vessels or containers. The containers or
collecting vessels may be controlled by piezoelectric,
electrostrictive or magnetostrictive elements connected thereto
which cause movement of preselected containers to align with the
efflux from the microcapillary in response to the control signal.
Basically, then, the present invention provides means for
identifying, counting and sorting fluidized particles according to
their microstructures, comprising, conduit means for containing the
flow of a sample of fluid, the constituents of which include
particles each having a microstructure indicative of the identity
thereof; said conduit means having at least a portion thereof that
is transparent to electromagnetic radiation; means for illuminating
said portion of said conduit means and the sample fluid flowing
therethrough; optical means for magnifying the image of said
portion of said conduit means and the sample fluid flowing
therethrough for converting optical path length or phase
differences between the microstructure components of each
particulate constitutent of said sample fluid to contrast or
intensity differences; detection means responsive to the radiation
passing through said conduit means for developing a varying output
signal indicative of the varying intensity of said radiation, and
means for analyzing and processing said varying output signal for
developing a plurality of output signals each indicative of a
specific particulate constituent of said sample fluid.
BRIEF DESCRIPTION OF THE DRAWINGS
For a fuller understanding of the present invention; reference
should be had to the following detailed description of the same
taken in conjunction with the accompanying drawings, wherein;
FIG. 1 is a schematic representation of the apparatus according to
the present invention; with parts thereof shown in block form,
FIG. 2 is a sectional view taken across line 2--2 of FIG. 1,
FIG. 3 is a sectional view taken across line 3--3 of FIG. 1,
FIG. 4 is a more detailed schematic of the signal analyzer of FIG.
1 illustrating the conventional components thereof in block
form,
FIG. 5 is an exemplary graphical representation of relative power
spectrum versus frequency for the various types of white blood
cells,
FIG. 6 is a schematic functional diagram of the sorter illustrated
in FIG. 1.
FIG. 7 is a sectional view of the sorter illustrated in FIG. 1,
and
FIG. 8 is a view similar to FIG. 1 illustrating a modification.
FIG. 9 is a view similar to FIG. 3 illustrating a detail of the
FIG. 8 modification.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the forthcoming description of the present invention, examples
of its application in the field of blood analysis will be discussed
in detail. It is to be emphasized that these examples are for
illustrative purposes only and are not necessarily intended to
limit the application of the inventive concepts to this particular
field.
There are seven formed elements in normal human blood, which are in
the form of cells or cell fragments that are released with the
circulating blood from the bore marrow and other sources. The
formed elements are suspended in a saline plasma solution. The
cells occupy approximately 50 percent of the total volume of the
circulating blood. The white cells or leukocytes are true cells
with nucleii and active motility; they are the largest of the
formed elements ranging from 12 microns to 20 microns. There are
five types of white cells; viz., neutrophils, eosinophils,
basophils, lymphocytes and monocytes. The other formed elements of
blood are the erythrocytes (red cells) and the thrombocytes
(platelets). The average number of formed elements per cubic
millimeter of blood is as follows:
"Cell" Type Number Erythrocytes 5.40 .times. 10.sup.6 Neutrophils
4.40 .times. 10.sup.3 Eosinophils 0.20 .times. 10.sup.3 Basophils
0.04 .times. 10.sup.3 Lymphocytes 2.50 .times. 10.sup.3 Monocytes
0.30 .times. 10.sup.3 Thrombocytes 250 .times. 10.sup.3
The leukocytes have a neutral optical density and are quite
flexible and easily disturbed from their generally spherical shape.
The erythrocyte (red cell) is a cell fragment containing no
nucleus. All red cells are approximately the same size and shape,
in the form of a bi-concave disc 7.2 microns in diameter by 2.4
microns in thickness. They are quite flexible and quite optically
dense because of the hemoglobin content. Thrombocytes are the
smallest formed elements. They are approximately 2 microns in
diameter and contain a rather dense granule.
Referring now to the drawings and, more particularly, to FIG. 1, a
sample container is schematically depicted at 10 as having a
conically tapered lower portion about which is located a plurality
of electrical wires forming coils 11 for coaction with an
internally mounted stirring impeller 12. Coils 11 are connected to
a suitable source of electrical power (not illustrated).
Located adjacent sample container 10 is a vessel 13 for dispensing
a measured quantity of undiluted blood to container 10. For this
purpose a piston member 14 slidably engages the interior
cylindrical wall of vessel 13 for delivering the undiluted blood
from an inlet 16 to an outlet 18 which communicates with container
10.
A dilutent supply vessel 20 is provided adjacent container 10 for
supplying a measured quantity of dilutent fluid, such as a saline
solution, to sample container 10. A piston 22 slides within the
cylindrical wall of vessel 20 and controls the delivery of dilutent
from an inlet 24 to an outlet 26 which is in fluid communication
with container 10.
A piston 28 is slidably received in container 10 for dispensing the
mixture of blood and dilutent through the lower conical portion 30
thereof into an integral or suitably attached cylindrical conduit
32. Pistons 14, 22 and 28 may be operated to provide constant rate
of flow by any number of suitable conventional actuators (not
illustrated). Alternatively, any suitable structure can be provided
for mixing and dispensing the blood and dilutent at a constant
rate.
Conduit 32 communicates with a reduced diameter microcapillary
passage 33, which as shown in FIG. 2 is of flattened rectangular
cross section. The size of passage 33 must be sufficiently small to
allow the particles to pass therethrough in substantially single
file. When the particles are the formed elements of whole blood a
size on the order of 8 by 10 microns has been found to be
appropriate. The properly sized microcapillary passage 33 can be
fabricated as illustrated in FIGS. 1 and 2 from a pair of optically
transparent plates such as glass 330 and 332 having sandwiched
therebetween a layer 334 which may have been vacuumed deposited and
processed with an appropriate mask or barrier to prevent a central
strip thereof from being deposited on the glass plate. When the
plates 330 and 332 are combined this central undeposited strip will
then form the capillary passage 33. It is to be pointed out that
any other suitable technique may be employed for achieving a
capillary passage of the required dimensions. The conduit 32 is
fitted to a funnel-shaped mouth portion 336 of the glass sandwich
by means of a resilient plug 338 or the like.
A phase optical system is provided adjacent the microcapillary
passage 33 for magnifying and observing the particle flow
therethrough, which system comprises; a source of illumination 34,
a diaphragm 36 having a central opening 38, condensing lenses 40,
objective lenses 42 and a phase or diffraction plate 44. The
optical system images the specimen passing through the
microcapillary passage 33 onto an image plane 46 at which is
located a suitable photosensitive device 48, illustrated greatly
enlarged for clarity, which may typically comprise an N-P-N silicon
planar photodetector. The size of the photosensitive device or
detector 48 is such with respect to the size of the imaged
specimen, a white blood cell for example, that only a thin
cross-sectional slice of the cell image traverses the detector as
it passes through the microcapillary 33, the image of which is
depicted at 33i in FIG. 3. The signal from detector 48 is delivered
via line 49 to and suitably amplified by a signal amplifier A, the
output from which is carried by line 49'.
As shown in FIG. 3 a second photodetector 50 is located adjacent
detector 48 and is responsive to the illumination falling outside
of the projected image 33i for compensating for changes in the
illumination levels from light source 34, as is common. The
reference illumination signal is carried by line 51 which leads to
amplifier A.
The signal from detector 48 is fed into a signal analyzer 52 via
line 49'. As shown in FIG. 4 the signal analyzer comprises a
plurality of narrow band-width filters 520, 522 and 524 which
severally deliver signals to a plurality of detectors or low pass
filters 526, 528 and 530 via lines 532, 534 and 536. Detectors 526,
528 and 530 deliver signals to circuits 538, 540 and 542,
respectively via lines 544, 546 and 548. Circuits 538 and 542 are
threshold circuits, which might typically comprise schmidt
triggers, whereas circuit 540 is a variable threshold circuit which
might typically comprise a conventional electronic comparator. The
output of detector 526 is also fed into comparator 540 via branch
from line 544, as is illustrated.
Referring again to FIG. 1, the output signals from circuits 538,
540 and 542 are delivered to a logic network 54 via lines, 56, 58
and 60, respectively. As will be discussed in greater detail
hereinbelow, network 54 might typically comprise a 3-bit binary
logic network which can yield up to eight decision outputs in
response to three inputs. The output signals from network 54 are
delivered to a counter 62 via lines 64, 66, 68, 70, 72, 74 and 76.
Branch signals from lines 64, 66, 68, 70, 72 and 74 are severally
delivered for the control of a sorter 78 via lines 80, 82, 84, 86,
88 and 90. Signals from the counter 62 may be delivered to suitable
indicators and/or recorders via lines 92, 94, 96, 98, 100, 102 and
104.
Sorter 78 is suitably fixedly attached to the glass sandwich 330,
332 such that capillary passage 33 communicates with the interior
thereof as shown in FIG. 7. With reference to FIGS. 6 and 7, the
sorter housing 78 has affixed to the bottom external surface
thereof a plurality of circularly arrayed sample collecting vessels
780, 782, 784, 786, 788 and 790 about a central collecting vessel
792. A plurality of motive devices 794, 796, 798, 800, 802 and 804
are fixed at one end to the interior of the sorter housing 79 and
severally at their other end to a plurality of flexible collecting
tubes 806, 808, 810, 812, 814 and 816, which are circularly arrayed
at one end about the longitudinal axis of capillary passage 33. The
tubes pass through openings in the base of sorter 78 with the lower
ends of each communicating with the interior of their respective
collecting vessel 780, 782, 784, 786, 788 and 790. A central tube
818 having a flared upper end is coaxial with capillary passage 33
and is in communication with central collecting vessel 792 as is
illustrated in FIG. 7. The signals from logic network 54 via lines
80, 82, 84, 86, 88 and 90 are delivered respectively to motive
devices 794, 796, 798, 800, 802 and 804 for the actuation
thereof.
Motive devices 794, 796, 798, 800, 802 and 804 may either be of the
piezoelectric, electrostrictive or magnetostrictive type which, as
is well known, elongate in response to an applied signal.
A circulating pump 820 is provided for maintaining a flow of inert
gas through the collecting tubes to the collecting vessels. To this
end, a plurality of inlet passages are provided (only one of which
is shown) between each of the collecting vessels and the pump
inlet.
In the operation of the above described apparatus, as applied to
the counting, identification and sorting of the formed elements of
blood, the blood sample is mixed with the dilutent in container 10
by means of the magnetic or other suitable stirrer 12. The mixture
is preferably treated with an anti-coagulant to prevent clotting
and may be maintained at a constant temperature by suitable means,
not illustrated. The dilutent functions to provide adequate
separation of the cells to insure that individual cells will pass
through the system in serial fashion. Since the response time of
the apparatus will depend upon the spacing between cells as they
pass through the microcapillary 33, which in turn is dependent upon
the amount of dilutent, the proportion of dilutent employed can
function to keep the flow of cells within a reasonable response
time for the apparatus. For example, at a 10:1 dilution rate in
order to observe 1,000 cells per second a velocity of cells through
the microcapillary of 0.024 meters per second would be
required.
If the cells are observed under ordinary incoherent optical
illumination their microstructures would not be descernable. This
is because there is only a very small difference in optical path
length between the microstructure and the surrounding portions of
the cells, with essentially no difference in color or contrast.
There exists only a phase difference which is not visible under
ordinary illumination. However, with the phase illumination of the
present invention, the substantially invisible grain structure or
microstructure of the cells, as they file through capillary 33 in
serial fashion, is made visible in the image plane 46.
The opening 38 is diaphragm 36 creates substantially a point source
of monochromatic light which falls upon the microstructures of the
cell as well as the surrounding medium therebetween. Since the
microstructures have refractive indicies which result in optical
path lengths that differ from that of the surrounding medium, the
radiation that passes therethrough experiences a shift in phase
with respect to the remainder of the radiation. In other words, the
microstructures function to diffract or deviate the portion of the
rays impinging thereupon, whereas the remainder of the beam remains
undeviated or undiffracted. The deviated beam is shown by the
dashed lines d, whereas the undeviated beam is shown by the solid
lines u in FIG. 1. As is well known, since the difference in path
length between the microstructures and the surrounding is very
small, the phase shift introduced by the microstructures will equal
one quarter of a wavelength. The diffraction or phase plate 44
functions to introduce an additional quarter wave shift between the
deviated and the undeviated beam. In this manner, when the beams d
and u combine or interfere in the image plane 46 there will now be
a difference in contrast between the specimen microstructures and
the surrounding media, which difference can be seen and detected.
For a more detailed discussion of the phenomena of phase optics or
phase illumination reference should be had to "Phase Microscopy
Principles and Applications" by Bennett, Jupnik, Osterberg and
Richards, John Wiley & Sons, Inc. 1951. The microcapillary
passage 33 is preferably of flattened rectangular cross-section as
illustrated to avoid the necessity of special corrections that
would be required with circular cross-section passages.
The image of the cell passing through the transilluminated
observation chamber portion of passage 33 will be magnified many
times, 1,000 times for example, and projected onto the surface of
the detector 48. In FIG. 3, the size of the photodetector 48 with
respect to the image of the capillary passage 33i is shown greatly
enlarged in the interests of clarity. Actually, the sensitive area
of the detector is quite small compared to the size of the
projected image of the cell, and many typically be on the order of
0.10 by 0.10 microns. In this manner, the individual components of
the cell microstructure will cause a continuously changing value of
light intensity falling upon detector 48 as the cell travels past
the sensitive area thereof.
The variations in the intensity of radiation incident on detector
48 may cause a resistance or other suitable change within the
detector, depending upon the particular choice of detector, which
creates a voltage fluctuation in output line 49. This voltage is
amplified at A, with suitable modulation if necessary from
reference detector 50 via line 51, and delivered as an input signal
in line 49' to signal analyzer 52. A portion of each magnified cell
image as wide as the detector makes only one pass across the
detector and only the microstructure lying in a plane perpendicular
to the optical axis at the focal point are detected; yet, as will
be discussed hereinbelow, the information contained in such portion
of the cell image, properly analyzed is sufficient to identify and
distinguish the type of cell.
It has been found that Fourier or spectral analysis of the raw
signals developed by the detector will yield a distinct power
spectrum for each cell type; this being true regardless of the cell
size, maturity and orientation. In other words, an analysis of a
large number of cells of the same type will yield substantially
identical power spectrums.
The data illustrated by the curves in FIG. 5 is exemplary of the
power spectrum for the five types of white cells, as determined by
a Fourier analysis of a larger number of raw signals of each cell
type. As shown in FIG. 5, the curves are expressed in terms of
relative spectral power levels versus frequency, in cycles per unit
length. It is to be noted that other signal analysis techniques may
yield curves that differ from those shown in FIG. 5. These curves,
however, would also be distinct for each cell type.
Once having determined these distinctive power spectrums, it
becomes a relatively simple matter to choose those relative power
spectrum values within particular frequency bands that distinguish
one cell type from another. In as much as there may be many
distinguishing relative power level/frequency band values as well
as many different combinations thereof that can be incorporated
separately or jointly in a particular design, the examples to be
given hereinbelow are to be taken as only illustrative of one set
of values that perform satisfactory; others can be used which would
perform equally as well.
Thus, in order to identify the white cells, the signals emanating
from the amplifier A and applied to the input of the signal
analyzer at 49' are analyzed thereby to determine the
following:
1. At one cycle per unit length is the relative power greater than
100?
2. At six cycles per unit length is the relative power greater than
or equal to the power at one cycle per unit length?
3. At 10 cycles per unit length is the relative power less than
10?
From an inspection of FIG. 5 it can be seen that; an affirmative
answer to question (1.) coupled with negative answers to questions
(2.) and (3.) will identify a neutrophil; a negative answer to all
three questions will identify a monocyte; a negative answer to
questions (1.) and (3.) coupled with an affirmative answer to
question (2. ) will identify a lymphocyte; an affirmative answer to
questions (1.) and (3.) coupled with a negative answer to question
(2.) will identify an eosinophil; and negative answers to questions
(1.) and (2.) coupled with an affirmative answer to question (3.)
will identify a basophil.
Due to the large hemoglobin content of red cells and because,
unlike the white cell, the red cell does not contain a nucleus or
granules, it has a power spectrum completely different from the
white cells. Although not shown in FIG. 5, it has been found that
the relative power of the red cell is over 100 at 1, 6 and 10
cycles per unit length. Therefore an affirmative answer to question
(1.) and (2.) coupled with a negative answer to question (3.) will
identify a red cell. The platelets, being the only formed element
left, can be identified when there is a signal that does not fall
within any of the values given above.
In accordance with the above described technique for
identification, the narrow band-width filters 520, 522 and 524
function to allow those signals centered about three frequencies in
the ratio of 1, 6 and 10 to be passed respectively to detectors
526, 528 and 530 which function to remove the signal transients in
a known manner. Threshold device 538 will develop a signal in line
56 only if the power of the signal in line 544 is greater than a
predetermined value (greater than 100 in the example given). Thus,
a signal at line 56 would be equivalent to an affirmative answer to
question (1.). Electronic comparator 540 compares the power of the
signal from detector 528 (at six cycles per unit length in the
example given) with that from detector 526 (at one cycle per unit
length in the example given), developing a signal in line 58 only
if the power in line 546 is greater than or equal to that in line
544, which would be equivalent to an affirmative to question (2.),
supra. Threshold unit 542 will develop a signal in line 60 only if
the power of the signal in line 548 is less than a predetermined
value (less than 10 in the example given). Thus, a signal in line
60 would be equivalent to an affirmative answer to question (3.)
supra. It therefore should be apparent that the presence or absence
of signals in lines 56, 58 and 60 in particular predetermined
combinations will be indicative of the five types of white cells
and the red cell. Signals not falling in these combinations, by
process of elimination, will be indicative of the platelets.
The signals from lines 56, 58 and 60 are fed into logic network 54,
which could typically comprise a conventional three-bit binary
decoder which functions in response to three inputs to deliver up
to eight decision outputs. only seven of which are used as
indicative of the identity of each of the five white cell types,
the red cells and the platelets. Thus, a signal in output line 64
could indicate a neutrophil; that in line 66 could indicate a
monocyte; that in line 68 could indicate a lymphocyte; that in line
70 could indicate an eosinophil; that in line 72 could indicate a
basophil; that in line 74 could indicate a platelet; and that in
line 76 could indicate a red cell. These signals are applied to a
conventional counter or the like 62 which tallies the number of
times each cell type is identified in real time as the images
thereof flow past the detector. Signals from the counters via lines
92, 94, 96, 98, 100, 102 and 104 can be delivered to suitable
indicators and/or recorders which could indicate or print out the
following data:
1. Number or red cells per cubic millimeter of whole blood.
2. Number of platelets per cubic millimeter of whole blood.
3. Number of white cells per cubic millimeter of whole blood.
4. Number of each type of white cell.
5. Percent of total of each type of white cell.
Referring again to FIGS. 1, 6 and 7, the output signals indicative
of the cell type from logic network 54 can be employed as control
signals via lines 80, 82, 84, 86, 88 and 90 leading to sorter 78. A
signal in any one of these lines will actuate its corresponding
motive device 794, 796, 798, 800, 802 or 804 which will cause the
same to elongate to thereby move one of the circularly arrayed
tubes 806, 808, 810, 812, 814 or 816 directly under the exit
portion of capillary passage 33 to receive the particular cell type
associated with the control signal. As illustrated by the dashed
lines in FIG. 7, tube 816 has been moved by motive device 804 to a
position directly under the capillary passage 33. The particular
cell type samples are delivered via the tubes to collecting vessels
780, 782, 784, 786, 788 and 790. The pump 820 functions to maintain
a positive flow of inert gas, such as nitrogen, from the mouth of
each tube to its associated collecting vessel. In this manner cells
deposited into the collecting tubes from the microcapillary will be
drawn into the collecting vessels. When there is no signal to
actuate any of the motive devices the effluent stream from
capillary 33 will be received by central tube 818 leading to the
central collecting vessel 792, which can be used to collect the red
blood cells since these have the greatest population. To this end,
line 76 from logic network 54 which is indicative of the red blood
cell need have no control connection to the sorter. Thus, in the
absence of a control signal the sorter will function to normally
collect the red blood cells.
The size of the white cells is generally greater than the size of
microcapillary passage 33 and some elongation of the white cells
will take place upon passage therethrough. Therefore, the detector
48 will always "see" the image of a portion of the white cells,
which, as pointed out above, is sufficient to make an
identification. As regards the red cells and platelete however, it
is possible that a small number of these might not be "seen" by the
detector. The platelets, being smaller than the capillary opening,
could flow outside the area of the detector. The red cells, being
very thin, could avoid the detector when flowing edgewise against
the microcapillary passage walls.
To insure that all of the cells, especially all of the red cells
and platelets, are counted, the FIG. 1 apparatus can be modified as
illustrated in FIGS. 8 and 9, wherein similar numerals are employed
to depict similar parts. A second detector 110, which may be a
strip photosensor, is located to span across the entire
microcapillary and is upstream of the detector 48 as illustrated in
FIG. 9. Since the strip sensor spans the entire microcapillary, the
small platelets and edgewise red cells are still observed. The
strip sensor could be quite narrow, about 0.5 by 10 microns in
sensitive area. The signal from strip photosensor 110 via line 111
is suitable amplified by amplifier A' and delivered via line 111'
to a detector 112. Amplifier A' may be modulated from the reference
illumination detector signal via line 51 as in the previous
embodiment.
Detector 112 might typically comprise an electronic integrator with
threshold circuits which function to directly classify red cells
and platelets on the basis of their distinctly different
characteristics both with respect to each other and with respect to
the white cells. Thus, the signal from a platelet would be a sharp
spike in response to the small 2 micron diameter thereof. On the
other hand, the larger red cell signal would last somewhat longer
and produce a greater voltage change. The detector 112 could
suitably have two thresholds, one for the platelets and one for the
red cell. If neither a platelet nor a red cell is observed, a
signal is delivered via line 114 to a conventional enabling circuit
116 which functions to render operative the detector 48 for the
observation of the white cells. The signals for the platelets and
the red cells are delivered respectively via lines 120 and 122 to a
red cell and platelet counter 124 which may be separate from the
white cell counter 62'. The platelet signal via line 122 is
delivered to the sorter 78 via 126 for the control of the platelet
sample collecting vessel as in the previous embodiment.
Although the foregoing description has described two preferred
embodiments of the invention, other modifications will occur to
those skilled in the art without departing from the spirit thereof.
It is therefore intended that the present invention is to be
limited only by the scope of the appended claims.
* * * * *