U.S. patent number 3,883,247 [Application Number 05/410,994] was granted by the patent office on 1975-05-13 for method for fluorescence analysis of white blood cells.
This patent grant is currently assigned to Bio/Physics Systems, Inc.. Invention is credited to Lawrence R. Adams.
United States Patent |
3,883,247 |
Adams |
May 13, 1975 |
Method for fluorescence analysis of white blood cells
Abstract
A dye composition for differential analysis of white blood cells
comprising a hypotonic aqueous solution of a metachromatic
fluorochrome dye, e.g., acridine orange. The solution has a pH
factor within normal physiological ranges for human blood. The
white cell analysis is made by straining the cells with the
fluorescent dye by suspending a sample of fresh blood in the dye
solution, subjecting the suspension before dye uptake equilibrium
is reached to radiation from a light source (e.g., radiation from a
blue laser) having a wave length within the range of absorption of
the dye, distinguishing the white cells from other blood particles
by detecting fluorescenses (e.g., green fluorescences) emitted from
the white cells in response to the radiation, and further
distinguishing the various types of white cells by measuring the
magnitudes of their respective fluorescence emissions (e.g., red
and green fluorescences). Differences in the rates of uptake of
fluorochrome dye of the various kinds of white cells are
accentuated by altering the aqueous dye solution so that it becomes
hypotonic.
Inventors: |
Adams; Lawrence R. (Mahopac,
NY) |
Assignee: |
Bio/Physics Systems, Inc.
(Mahopac, NY)
|
Family
ID: |
23627114 |
Appl.
No.: |
05/410,994 |
Filed: |
October 30, 1973 |
Current U.S.
Class: |
356/39;
250/461.2; 356/36 |
Current CPC
Class: |
G01N
21/6428 (20130101); A61B 5/417 (20130101); G01N
1/30 (20130101); G01N 2021/6421 (20130101); G01N
2021/6441 (20130101) |
Current International
Class: |
A61B
5/145 (20060101); G01N 21/64 (20060101); G01N
15/14 (20060101); G01n 001/30 () |
Field of
Search: |
;250/304,365,458,484
;356/36,39 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Wibert; Ronald L.
Assistant Examiner: Koren; Matthew W.
Attorney, Agent or Firm: Pennie & Edmonds
Claims
We claim:
1. A method for distinguishing specific types of cells among white
blood cells and other particles within a blood sample, said method
comprising the steps of:
a. forming an aqueous diluent solution of a metachromatic
fluorochrome dye, said solution being of hypotonic osmolarity and
of physiologically normal pH, and said dye being capable of
staining the nuclear material of the white cells to provide
fluorescence from the stained nuclear material which is different
from the fluorescence of any other blood particle; said hypotonic
diluent solution being further characterized in that it can alter
the normal rates of dye uptake by the various components of the
white cells;
b. staining the vital white cells of a blood sample by suspending
the blood sample in the diluent dye solution prepared in step
(a);
c. subjecting the suspension formed in step (b) before dye uptake
equilibrium is reached therein to illumination from a light source
having a radiation wavelength absorbable by the stained cellular
material; and
d. distinguishing the various white cells by the magnitudes of
their fluorescence emissions.
2. A method for distinguishing white blood cells from other
particles within a blood sample and for differentiating the white
blood cells as between neutrophils, eosinophils, basophils,
lymphocytes, monocytes, and immature granulocytes, said method
comprising the steps of:
a. combining a blood sample with an aqueous solution of acridine
orange having a physiologically normal pH factor to form a
suspension, said solution having a concentration of acridine orange
sufficient to provide a concentration of acridine orange in the
suspension within the range of between about 4 .times.
10.sup..sup.-4 and about 4 .times. 10.sup..sup.-2 grams per liter
of suspension, and said solution having a salt concentration
corresponding to less than the physiologically normal
osmolarity;
b. subjecting the suspension obtained in step (a) to radiation and
distinguishing the white cells from all other blood particles by
the detection of green fluorescence emitted from individual white
cells in response to the excitation from the radiation; and
c. simultaneously detecting the magnitudes of red and green
fluorescences emitted from individual white cells in response to
excitation from the radiation, and differentially classifying the
white cells on the basis of such detection.
3. A method according to claim 2 wherein the blood sample is fresh
peripheral blood and wherein said salt concentration within the
solution of acridine orange is less than about 7.5 grams per liter
of solution.
4. A method according to claim 2 wherein the suspension obtained in
step (a) is directed in step (b) in a fine stream through the flow
channel of an optical chamber wherein the suspension is subjected
to radiation, the fine stream within the flow channel being so
narrow as to be capable of confining the flow to substantially a
single cell width such that the cells traverse the radiation beam
one at a time in single file.
5. A method according to claim 4 wherein the radiation employed in
step (b) is a blue laser of wavelength in the neighborhood of 488
millimicrons and is directed toward the suspension stream in the
form of a narrow beam substantially transverse to the suspension
stream and having a beam dimension in the direction of the
suspension stream which is on the same order of magnitude as the
maximum dimension of an individual cell.
6. A method according to claim 5 wherein the step of subjecting
said suspension to radiation from a blue laser is carried out by
energizing an argon ion laser and directing the resulting radiation
to said suspension.
7. A method according to claim 4 wherein the step of subjecting
said suspension to radiation is carried out by energizing a
helium-cadmium laser and directing the resulting radiation to said
suspension.
8. A method according to claim 2 wherein the red fluorescence
radiation is discriminated by an optical filter which passes
wavelengths on the order of 6,500 angstrom units and the green
fluorescence radiation is discriminated by an optical filter which
passes wavelengths on the order of 5,300 angstrom units.
9. A method according to claim 2 wherein the differences in the
magnitudes of red fluorescence and the differences in the
magnitudes of green fluorescence emitted from individual cells are
detected by converting the optical signals into electrical signals
and displaying the resultant electrical signals upon the face of a
cathode ray tube.
10. A method for making a differential blood analysis of white
cells, said method comprising the steps of:
a. combining undiluted and untreated fresh blood with an aqueous
solution of acridine orange in the ratio of up to about 1 part of
blood to 5 parts of solution to form a suspension, said solution
having a concentration in the neighborhood of 4 .times.
10.sup..sup.-3 grams of acridine orange per liter of solution, said
solution containing a buffering agent to maintain the pH factor of
the suspension within the normal range for human blood, and said
solution having an osmolarity below the normal physiological range
for human blood plasma;
b. passing the suspension formed in step (a) through a
photoanalysis apparatus and subjecting each white cell in the
suspension to radiation;
c. discriminating the white cells from all blood particles
contained in the suspension by detecting fluorescent radiation
emissions above a predetermined threshold value from each cell in
the green portion of the visible spectrum;
d. detecting the magnitudes of the fluorescent radiation emissions
from each white cell in the red and green portions of the visible
spectrum; and
e. classifying cells based upon the differences in magnitudes of
red and green fluorescences measured from the cells.
11. A method according to claim 10 wherein the osmolarity of the
acridine orange solution is between 0.3 and 0.7 isotonic.
12. A method according to claim 11 wherein the green radiation
signals are employed to derive a total count of the white cells
from a measured volume of the suspension.
13. A method according to claim 11 wherein:
the magnitudes of the fluorescent radiation emissions from each
cell in the red portion of the visible spectrum are detected by
converting the red optical signals to electrical signals and
displaying said electrical signals upon the face of a cathode ray
tube, the magnitude of the red signals being indicated in terms of
the cathode ray tube deflection on one axis; and the magnitudes of
the fluorescent radiation emissions from each cell in the green
portion of the visible spectrum are detected by converting the
green optical signals to electrical signals and displaying said
electrical signals upon the face of a cathode ray tube, the
magnitude of the green signals being indicated in terms of the
cathode ray tube deflection the other axis.
14. A method according to claim 13 wherein a camera is placed over
the face of the cathode ray tube to record the pattern of spots
produced by said method.
15. A method according to claim 10 wherein:
voltage thresholds are applied to select red fluorescence signals
within a narrow field and green fluorescence signals within a
narrow field; and
the signals from the overlapping portions of the narrow red and
green fields are individually counted for a carefully measured
blood sample volume to obtain a quantitative measure of those white
cells having the particular characteristics exemplified by
overlapping portions of said red and green fields.
16. A method according to claim 13 wherein:
the green fluorescence signals above a predetermined threshold are
counted up to a predetermined count value to thereby determine the
passage of the number of white cells corresponding to that count
value;
voltage thresholds are applied to select red fluorescence signals
within a narrow field and green fluorescence signals within a
narrow field; and
the signals from the overlapping portions of the narrow red and
green fields are individually counted during the period of the
green fluorescence count to thereby obtain a count ratio of those
white cells having the particular characteristics examplified by
the field common to the selected narrow red and green fields.
17. A method according to claim 16 wherein the method is repeated
with different portions of the same blood sample and with different
settings of the voltage thresholds to thereby obtain a series of
measurements of cells having different characteristics.
18. A method according to claim 16 wherein the voltage thresholds
are applied by parallel threshold circuits to select green
fluorescence signals within a selected narrow field and red
fluorescence signals within a selected narrow field simultaneously
for each of the lymphocyte, monocyte, neutrophil, basophil,
eosinophil and immature granulocyte classes of white cells.
19. A method according to claim 18 wherein:
for each of the said classes of white cells and the green
fluorescence signals within a selected narrow field and the red
fluorescence signals within a selected narrow field are
respectively converted to a digital representation of the
fluorescence values on a digital register by an analog-digital
converter, each of said registers being connected to the input data
buss of a computer;
the computer monitors, for each of the said classes of white cells,
the green fluorescence values corresponding to the red fluorescence
signals within said selected narrow field; and
the computer produces a count of the number of cells having
fluorescence signals within each selected narrow field of red and
green fluorescence signals.
20. A method for making a count of total white cells in a blood
sample comprising the steps of:
a. combining a fresh blood sample with an aqueous solution of
acridine orange to form a suspension, said solution having a
physiologically normal pH factor and a concentration of acridine
orange in said suspension in the neighborhood of 10.sup..sup.-3
grams per liter of suspension, and said solution having an
osmolarity below the physiologically normal level of human
blood;
b. subjecting the suspension obtained from step (a) to radiation
from a blue laser and counting the cells from a measured volume of
suspension based upon the detection of green fluorescence emitted
from each individual cell in response to excitation from the blue
laser radiation.
21. A method according to claim 20 wherein the detection of green
fluorescence emitted from the cells is discriminated by an optical
filter which passes radiation in the neighborhood of 5,300 angstrom
units wavelength.
22. A method for classifying vital white blood cells amoung the
groups of lymphocytes, monocytes, neutrophils, basophils,
eosinophils and immature forms of granulocytes, said method
comprising the steps of:
a. combining a fresh blood sample with a hypotonic colloidal
solution of a metachromatic fluorochrome dye at a physiologically
normal pH; and
b. distinguishing the groups of white blood cells by measuring the
magnitudes of metachromatic fluorescences from the individual cells
in response to optical radiation.
23. A method for distinguishing specific types of cells among white
blood cells and other particles within a blood sample, said method
comprising the steps of:
a. forming an aqueous diluent solution of a metachromatic
fluorochrome dye, said solution being capable of staining the
nuclear material of the white cells to provide fluorescence from
the stained nuclear material which is different from the
fluorescence of any other blood particle; said solution being
further characterized in that it is capable of altering the
differential rates of dye uptake by the various components of the
white cells;
b. staining the vital white cells of a blood sample by suspending
the blood sample in the diluent dye solution prepared in step
(a);
c. subjecting the suspension formed in step (b) before dye uptake
equilibrium is reached therein to illumination from a light source
having a radiation wavelength absorbable by the stained cellular
material; and
d. distinguishing the various white cells by the magnitudes of
their fluorescence emissions.
Description
BACKGROUND OF THE INVENTION
This invention relates to an improved composition for staining
white blood cells, and to an improved method and apparatus for
utilizing that composition in analyzing blood for white cells.
Blood is a fluid, circulating tissue found in all higher animals
and in many invertebrates. It is a tissue, just as a skin, muscle
and bone are tissues, because it contains living cells and has
specific functions, chief among them being the conveyance of
materials from one part of the body to another. The general
principle on which the chemical life of the body is conducted is
that each living cell carries out within itself all the chemical
processes necessary to its existence. Therefore, all the materials
which each cell requires must be carried to it, and those which it
discards must be removed. Throughout the bodies of higher animals a
highly specialized system of transport--the blood vascular
system--has evolved, affording an efficient route for the blood and
providing the necessary, intimate contact thereof with every living
cell.
The principal materials which a living cell requires are sugar,
amino acids, fats, vitamins, oxygen, salts, hormones and water. The
organs of digestion convert the solid constituents of food into
forms that the blood can absorb and deliver to the cells of the
body. The principal substances which the cell must dispose of are
carbonic acid and simple soluble compounds of nitrogen. These are
conveyed by the blood to the various organs functioning in an
excretory capacity, ridding the body of noxious wastes.
In all the higher animals blood consists of an aqueous fluid part,
the plasma, in which are suspended corpuscles of various kinds: the
red blood cells (erythrocytes), the white blood cells (leukocytes)
and the blood platelets.
The plasma has a faint straw color and is clear unless a meal
containing fat has been eaten recently, in which case it is
somewhat milky because of the minute globules of fat which it
transports. The two materials dissolved in greatest quantity are
albuminous substances (proteins) and common salt.
The general nature of plasma resembles that of raw egg white,
diluted with 0.9% solution of salt. In detail, its composition is
roughly as follows: water, 90%; proteins (fibrinogen, globulins,
albumin), 9%; salts (of Na, K, Ca, Mg, Fe, Cu, etc.), 0.9%; sugar,
urea, uric acid and creatinin, traces. Water is present primarily
in order to dissolve the other substances and to afford the blood a
degree of fluidity sufficient to secure its easy flow through the
minute capillaries of the vascular system.
Sodium chloride in blood plasma serves primarily to effect protein
dissolution. Since most proteins in the blood do not dissolve in
pure water, and since protein can form the basis of living material
only if it is in solution, salt is essential.
The concentration of hydrogen ions in normal plasma equals 0.4
.times. 10.sup.-.sup.7 gram of hydrogen per liter (pH=7.4). Any
considerable increase in hydrogen ions (lowering of pH value, or
acidosis) causes increased and violent breathing; this in turn
enhances the expulsion of carbonic acid from the plasma into the
air, thereby tending to raise the pH value of the blood to its
normal level. On the other hand, should the blood become unduly
alkaline (alkalosis) the kidney will relieve this condition by
secreting an increased quantity of alkali in the urine. This
balance of acid and alkali, the maintenance of which preserves the
reaction of the blood at a pH of 7.4, is called the acid-base
equilibrium of the blood.
The cells or corpuscles of the peripheral blood (i.e., blood
outside the bone marrow) are divided into two groups: the red blood
cells (erythrocytes) whose primary object is to transport oxygen
and the white blood cells (leukocytes) whose functions include the
engulfing and destruction of microorganisms and other foreign
bodies in the blood stream (phagocytosis). In addition to these
cellular elements, the blood contains so-called blood platelets
(thrombocytes).
It is well known that mature white blood cells normally exist in
the blood in different forms which can be classified into major
groups. Each group forms a percentage of the total within the
general limits for normal blood as shown in the following table:
Polymorphonuclear leukocytes Neutrophils: 38 to 70 percent
Eosinophils: 1 to 5 percent Basophils: 0 to 2 percent Mononuclear
leukocytes Lymphocytes: 15 to 45 percent Monocytes: 1 to 8
percent
The polymorphonuclear leukocytes not only have segmented nuclei,
but are also characterized by having granules in the cytoplasm, and
they are therefore sometimes referred to as granulocytes.
Granulocytes develop in the bone marrow from cells called
myeloblasts, which are undifferentiated and will mature into either
neutrophils, eosinophils or basophils. The stages of growth of
immature granulocytes have been classified and the appearance of
them characterized. Although immature granulocytes are present in
low numbers in normal blood, their appearance in significant
quantity is particularly significant for the diagnosis of infection
or leukemia. A more detailed description of the various types of
blood cells sufficient for a further understanding of the
background of the present invention can be found in L. W. Diggs et
al., The Morphology of Human Blood Cells, published in 1970 by
Abbott Laboratories, and in M. M. Wintrobe, Clinical Hematology,
224-60 (6th ed. 1967)
Even though they are far outnumbered by the red blood cells
(erythrocytes) by a ratio of approximately 700 to 1, the leukocytes
are extremely important to the body in fighting disease and
infections. Furthermore, probably because of that function, it has
been observed that abnormal body conditions such as disease or
infection often results in marked changes in the leukocytes in the
blood stream. These changes can include a marked increase
(leukocytosis) or decrease (leukopenia) in the total number of
white cells in proportion to the number of red cells in the blood
stream. Also, marked changes in the proportions of different types
of leukocytes in the blood stream have been found to be
characteristic and unique with respect to particular diseases. A
detailed description of this phenomenon appears in M. M. Wintrobe,
Clinical Hematology, 260-94 (6th ed. 1967). Thus, a total white
cell count (i.e., a count of the total number of white cells within
a specified volume of blood) and a differential blood analysis
(i.e., a count which reveals the relative percentages of the
different types of white cells in the blood) are two extremely
valuable diagnostic and medical research procedures.
The usual medical laboratory procedure for obtaining a quantitative
or total white cell count is to combine a precise volume of a blood
sample with a precise volume of a solution (lysing agent) which
destroys the red cells, and then to place a portion of the sample
within a "counting chamber" microscope slide and to visually count
the number of white cells appearing in several squares of the
counting chamber. The result is usually expressed in terms of the
number of white cells per cubic millimeter. This is a tedious
procedure and involves making a count on such a small sample that
inaccuracies are very likely to occur. Furthermore, the skill of
the technician is extremely critical in achieving accurate
results.
In the conventional medical laboratory procedure for obtaining a
differential analysis of white cells within a blood sample, a smear
of the blood sample is dried upon a clean glass microscope slide
and is then treated with an appropriate reagent which typically
contains a fixative such as methyl alcohol in combination with a
mixture of stains. Alternatively, the preparation procedure may
include treatment with the fixative as a separate step. The treated
blood smear is then examined through a microscope under oil
immersion. Sample counts are taken visually in various different
areas of the blood smear and recorded. An even distribution of the
white cells is difficult to obtain and therefore it is recommended
that counts be taken at the edges of the blood smear as well as at
the central portion, and that a minimum of 200 cells be counted. It
is by this means that a differential analysis of white blood cells
is normally made. Unfortunately, this method involves many
difficulties and limitations which seriously affect the accuracy,
including lack of uniformity in the blood smear sample, the
extremely small size of the sample actually counted, the
tediousness of the task (which encourages the laboratory technician
to cut corners and to make short counts), and the inconsistencies
in the skills of different technicians in recognizing and
accurately recording all of the unique cells which he actually
sees.
Because the aforementioned manual procedures are time consuming
even for a highly skilled technician, the cost of each analysis is
necessarily high, and the speed at which urgent tests can be
obtained is limited.
As a result of the aforementioned problems associated with manual
procedures for counting white blood cells and differentiating the
various types thereof, machine or instrumental methods have been
devised with a view toward accomplishing these tests automatically
and on live blood.
In the case of machines intended for making quantitative white cell
counts, these devices have generally involved the procedure of
providing a blood sample suspension having a lysing agent which is
adjusted so as to destroy the red cells without destroying the
white cells. All of the remaining cells are then counted, and the
assumption is made that they are all white cells. This prior
machine method leaves much to be desired because the destruction of
the red cells is not always complete, and any remaining red cells
are then counted as white cells, thus hurting the accuracy of the
count. Also, the condition which destroys the red cells may damage
the white cells, and such damage often interferes with the
subsequent identification of the white cells.
In the case of machines intended for making differential analyses
of living (vital) white blood cells (see, for example, M. Ingram et
al. Scientific American, November, 1970, page 72-82), it has not
been possible in the past to devise a satisfactory instrument
except as described in U.S. Pat. No. 3,684,377 issued to the same
assignee as the present invention; M. R. Melamed et al., European
J. Cancer, 9, 181-184 (Pergamon Press, 1973); M. R. Melamed et al.,
Am. J. Clinical Pathology, 57(1), 95-102 (January, 1972); M. R.
Melamed et al., Cancer, 29(5), 1361-68 (May, 1972); and L. R. Adams
et al., Acta Cytologica, 15(3), 289-91 (1971). The aforementioned
patent and articles, in particular, disclose for the first time the
machine differentiation of vital white blood cells as between
lymphocytes, monocytes, and the general class of polymorphonuclear
leukocytes (granulocytes). However, it has hiterto beem impossible
to instrumentally differentiate further among the various forms of
granulocytes (i.e., basophils, eosinophils and neutrophils) as well
as among mature and immature forms of white blood cells and
particularly to distinguish, when present, the immature forms of
granulocytes. According to the above-cited Abbott Laboratories and
Wintrobe references, the immature forms of granulocytes include as
a general class the so-called myelocytes.
The ability to quantify living white blood cells and to
differentiate them into six major categories (i.e., lymphocytes,
moncytes, neutrophils, eosinophils, basophils and immature
granulocytes (myelocytes)) is highly desirable for the purpose of
diagnosing many bodily ailments and diseases which manifest
themselves in abnormal proportions of the aforementioned six
categories or leukocytes. Therefore, an automatic machine or
instrumental technique for achieving the foregoing white blood cell
count and differentiation rapidly and accurately would provide the
physician with an important diagnostic and prognostic tool for
management of the ill patient.
Accordingly, it is an object of the present invention to provide an
improved automatic machine and method for accomplishing a
differential analysis of white blood cells which is characterized
by a high degree of accuracy and very low cost.
Another object of the invention is to provide an improved machine
and method for producing a differential count of white blood cells
with rapid speed of analysis (less than 1 minute as compared to 10
minutes or more by previously known procedures).
Another object of the invention is to provide an improved machine
and method for producing a differential count of white blood cells
under conditions in which the cells are "shocked" by exposure to a
non-physiologic medium during staining.
Another object of the invention is to provide an improved machine
and method for producing a differential count of white blood cells
which rely on the use of properties which are dependent on the rate
at which the leukocytes develop elicitable fluorescence when
exposed to a non-physiologic medium.
Another extremely important problem in devising a machine method
for obtaining a white blood cell differential count is that of
providing signals from the cells to the machine to enable it to
recognize and distinguish white cells from all other bodies within
the blood such as red cells or platelets. The machine recognition
and discernment of all white cells is essential in the problem of
classifying them so as to provide the desired differential
analysis.
Accordingly, another object of the present invention is to provide
an improved composition for staining white blood cells, and for
generating a signal therefrom, and an improved machine method for
recognizing thereby and distinguishing all white cells within a
blood sample to obtain a white cell differential analysis.
SUMMARY OF THE INVENTION
The foregoing objects are achieved according to the present
invention by the discovery of an improved blood cell staining
composition and an apparatus and procedure for employing same which
provide a clear basis for rapidly distinguishing the aforementioned
six different categories of vital white blood cells (including
immature granulocytes) in a machine analysis.
The staining composition of the present invention comprises an
aqueous solution of a metachromatic fluorochrome dye (i.e., a dye
which undergoes fluorescence at a multiplicity of wavelengths in
response to excitation by radiation within its range of absorption)
such as acridine orange. This solution is formulated so that the pH
thereof is at the normal physiological level of approximately 7.4.
However, the solution is made hypotonic, the osmolarity or salinity
thereof being generally below that normally obtained in human
blood. In this way, the white cells in a sample of live blood
treated with the aforementioned staining composition experience
discomfort or shock, but nevertheless remain intact and their
constituent materials remain undenatured during the time that the
blood analysis is being conducted. By achieving this state of
affairs, it was discovered that the consequent intensities or
amplitudes of metachromatic fluorescences at two different
pre-selected ranges of wavelengths (red and green in the case of
acridine orange) will, at any given moment for a period of time
lasting from about 10 seconds to several minutes following the
addition of the staining composition to the blood sample, depend on
the nature of the particular white cell undergoing fluorescence,
i.e., depending on whether the cell is a lymphocyte, monocyte,
basophil, neutrophil, eosinophil or immature granulocyte. Without
wishing to be bound by theory, it is believed that this phenomemon
is due to the hypotonic nature of the staining composition which
unexpectedly causes the rates of dye uptake by the white cells to
differ among the cellular organelles of the aforementioned six
types. In other words, the different amplitudes of fluorescences
among the various cell types are determined by the rate-controlled
degree of dye uptake which differs from cell type to cell type.
Generally, the staining composition of the present invention is
hypotonic to the extent that the concentration of NaCl therein is
between about 2.5 and 7.5 grams per liter of solution. We have
preferred to use 4.25 grams per liter of solution. Alternatively,
other well-known equivalents of sodium chloride for this purpose,
e.g., sodium citrate, can be used.
In the case where acridine orange is employed as the dye component
of the staining composition, it is generally used at a
concentration on the order of four times that disclosed in the
aforementioned U.S. Pat. No. 3,684,377. In other words, the
concentration of acridine orange in a freshly prepared staining
composition is sufficient to provide a concentration of the dye in
the blood sample suspension to be tested of between about 4 .times.
10.sup.-.sup.4 and about 4 .times. 10.sup.-.sup.2 grams per liter
of suspension. The use of such unusually high dye concentrations
has been found for some unexplained reason, to enhance the degree
of rate dependence of dye uptake by the various types of white
blood cells.
In a preferred mode of carrying out the method of the present
invention, a fresh blood sample is suspended in the aforementioned
hypotonic aqueous acridine orange solution and the resulting
suspension is preferably allowed to stand for a period of up to
about 30 to 100 seconds, preferably with mild stirring during that
time. The suspension is thereafter subjected to radiation from a
blue laser (488 millimicron wavelength) and the cells are
differentially classified on the basis of the differences in the
magnitudes of red and green fluorescences emitted from individual
cells in response to excitation from the blue laser radiation.
The dye acridine orange or euchrysine, which is an important
constituent of the preferred staining composition of the present
invention, is sometimes referred to in abbreviated form as simply
"AO." Its utility in the method of the present invention stems from
the fact that it possesses the properties of a metachromatic
flurochrome. This material is an organic compound for which the
chemical name is 3,6-bis-(dimethylamino)acridiniumchloride and
whose structural formula is: ##SPC1##
Acridine orange is also identified by color index specification
46,005 from the publication entitled COLOR INDEX, Second
Edition--of 1956 and 1957, published jointly by the Society of
Dyers and Colorists of Great Britain, and by the American
Association of Textile Chemists and Colorists of Lowell, Mass.
Acridine orange is commercially available, e.g., from Eastman Kodak
Company, Rochester, N.Y.
Acridine orange has been recognized for some time as a fluorescent
stain which is capable of staining nucleic acids such as
ribonucleic (RNA) and deoxyribonucleic acid (DNA). For instance,
see Rudolf Rigler's articles entitled "Microfluorometric
Characterization of Intracellular Nucleic Acids and Nucleoproteins
by Acridine Orange" in Volume 67, Supplementum 267 of Acta
Physiologica Scandanavica (Stockholm, 1966) and "Acridine Orange in
Nucleic Acid Analysis" in Volume 157, Article 1 of Annals of the
New York Academy of Sciences of Mar. 31, 1969, pages 211-224. The
application of acridine orange in staining cells is disclosed in
U.S. Pat. No. 3,497,690 to Wheeless et al. However, the novel
method of the present invention in which this material is used in
the unique way described herein as a supravital metachromatic
fluorescent dye for white blood cells has never been recognized.
Since it is regarded primarily as a red fluorescence dye for RNA,
and since the amount of RNA is generally considered negligible in
white blood cells, acridine orange has not been considered as a
likely candidate for providing meaningful information concerning
the aforementioned six classes of leukocytes. However, it has been
discovered that the unexpected utility of acridine orange in this
regard is achievable according to the method of present invention
and thereby permits the automatic machine discernment of white
blood cells from all other blood particles and the differentiation
of leukocytes into lymphocytes, monophils, basophils, neutrophils,
eosinophils and myelocytes (immature granulocytes).
Although the method and apparatus disclosed herein are described
with particular reference to using euchrysine or acridine orange,
it is understood that other suitable fluorochromes (i.e., dye
stains possessing the attributes of a metachromatic fluorochrome)
may be used consistent with the scope of the present invention.
As mentioned above, the preferred staining composition comprises
acridine orange in a hypotonic aqueous solution at a concentration
of between 4 .times. 10.sup.-.sup.4 and 4 .times. 10.sup.-.sup.2
grams of acridine orange per liter of solution. The solution
includes other additives to provide an osmolarity below the normal
physiological ranges for human blood plasma. In the preferred
concentrations of acridine orange solution, the mixture may not be
a true solution, but partly a suspension of aggregates of dye
molecules or, perhaps more properly, a colloidal dispersion, in
which extremely minute undissolved particules are suspended in the
liquid. However, this composition is referred to as a solution
throughout this specification. The mixture of the acridine orange
solution with a blood sample is then referred to as a suspension.
Thus, while the acridine orange "solution" may not be a true
solution, the use of that term serves to distinguish the staining
composition per se from the liquid suspension formed after the
addition of the blood sample.
The normal physiological pH value for human blood is generally
considered to be 7.40, plus or minus 0.05 pH unit, and the pH of
the staining solution is likewise preferably adjusted, within
practical limits, to 7.40, plus or minus 1 pH unit. On the other
hand, the osmolarity, which is a function of the concentration of
salts in the solution, is adjusted to below the physiological level
of approximately 8.5 grams of sodium chloride per liter of
solution, whereby the medium becomes traumatic to the blood
cells.
In the description of Example 1, below, a procedure is followed in
which separate solutions of acridine orange and the saline buffer
combination are produced and then combined. The combined solution
tends to form colloidal particles which apparently include the
acridine orange dye. It has been observed that the combined
solution containing the colloidal particles appears to have a
rather limited shelf life. The colloids appear to precipitate out
upon the walls and bottom of the container. Accordingly, it is
preferred to store the solutions separately, one solution
containing only acridine orange, and the other solution containing
only the saline buffer combination. The two solutions are then
combined in the proper proportions on the day when they are to be
used.
Whether the two solutions are stored separately, or in a combined
solution, it has been observed that there appears to be an aging
process which ocurs such that higher acridine orange concentrations
may be necessary to achieve the desired results with older
solutions as compared to relatively fresh solutions.
Without wishing to be bound by theory, it is believed that the high
degree of effectiveness of the composition and method of the
present invention has to do with the fact that there is competition
for the dye among the three compartments: solution, granules and
nucleus. The rate of dye uptake may also be mediated by the cell
membrane. The stain solution of the present invention promotes
large differences between the various types of leukocytes in the
amounts of dye taken up by the cell nuclei and granules. Additional
factors may involve shielding effects of the granules on the
nucleus and differences in the chemistry of the granules and their
numbers in each cell type.
Furthermore, it is believed that the reason for the distinctive
fluorescence (green) which distinguishes the white cells from other
blood particles such as erythrocytes is that the dye combines with
the DNA (deoxyribonucleic acid) of the cell nuclei in a distinctive
way such that a green fluorescence can be elicited by excitation
with blue laser illumination. The major portion of the uptake of
dye by each cell, which does not combine with the nuclear DNA,
appears to stain the cytoplasm, and is particularly concentrated in
the cytoplasmic organelles known as granules. It is believed that
this is the reason why the granulocytes, the white cells which are
particularly distinguished by the presence of granules in the
cytoplasm, apparently take up more of the acridine orange dye in
the cytoplasm, and thus provide a greater red fluorescence signal.
All of the acridine orange dye taken up by the cytoplasm (as
distinguished from that taken up by the nuclei) appears to cause
red fluorescence in response to the blue laser light. It is a
feature of the present invention that, although the green
fluorescence is due to staining of DNA at equlibrium the cells are
measured before equilibrium is reached and differences in green
fluorescence result from the stain depletion by the granules or
shielding of light by them. The upper and lower populations of
granulocytes merge at longer staining times.
Further objects, advantages, and features of the invention will be
apparent from the following description, claims and the
accompanying drawings wherein:
FIG. 1 is a schematic diagram of an apparatus which can be employed
in carrying out the machine method of the invention.
FIG. 2 is an illustration of a cluster display of six categories of
white blood cells which can be produced according to the method of
the present invention.
DESCRIPTION OF THE DRAWINGS
A preferred apparatus for carrying out the method of the present
invention, particularly with respect to the sample flow and optical
systems is constructed in accordance with the teachings contained
in a prior application Ser. No. 2,750 filed Jan. 14, 1970 by
Mitchell Friedman, Louis A. Kamentsky, and Isaac Klinger for
PHOTOANALYSIS APPARATUS, now U.S. Pat. No. 3,705,771, and assigned
to the same assignee as the present application. Other features of
a preferred apparatus for carrying out the method of the present
invention, and particularly relating to the arrangement of the
counters, cathode ray oscilloscope apparatus and associated
circuits, are carried out in accordance with the teachings of
another prior patent application Ser. No. 25,931 filed Apr. 6,
1970, by Louis A. Kamentsky and Isaac Klinger for a PARTICLE
ANALYSIS METHOD AND APPARATUS, now U.S. Pat. No. 3,662,771, and
assigned to the same assignee as the present application as well as
in the above-mentioned U.S. Pat. No. 3,684,377. The disclosures of
both of these prior patent applications and patent are incorporated
herein by reference. However, for the sake of completeness,
portions of the disclosures of these references are reproduced
here, and specifically related to the process of the present
invention.
Referring particularly to FIG. 1, there is illustrated a schematic
diagram of an apparatus which may be employed in carrying out the
method in accordance with the present invention. The apparatus
includes an optical chamber 10 containing a microcuvette 11 through
which a stream 12 of cells may be passed while entrained in the
suspension and supplied through a sample tube 14 from a reservoir
(not shown). The suspension is preferably surrounded by a sheath of
water 15 to confine the particles (cells) to a very fine stream. As
the stream 12 of particles passes through the chamber 10, it passes
through a narrow beam of light 20 from a light source 22. Light
source 22 is preferably an argon laser, and may include a
cylindrical lens 23 for shaping and directing the light.
Different optical reactions of the individual blood cells to the
light beam 20, in the form of fluorescent radiation from each cell,
are detected by photoelectric pick-up elements 24 and 26, which can
be photomultiplier tubes. The signals detected by the
photoresponsive pick-up elements 24 and 26 are converted by those
elements to electrical signal pulses which are supplied through
connections 30 and 32 to solid state amplifiers 34 and 36,
respectively. The pick-up element 24 is arranged to respond to red
fluorescence signals, and element 26 is arranged to respond to
green fluorescence signals. The transmission of optical
fluorescence signals to the pick-up elements is enhanced by
condensing lens 16. A dichroic mirror 18 is provided which has a
nominal cut-off wavelength for light at about 5,800 angstrom units.
Thus, it reflects light of all wavelengths below that limit,
through a filter 18A, to the pick-up 26. All optical signals above
5,800 angstroms in wavelength are transmitted through the dichroic
mirror 18, and through an optical filter 18B to the pick-up 24. The
pick-up 24 receives the red fluorescence signals and the filter 18B
passes a red band of radiation in the neighborhood of 6,300
angstrom units wavelength. Similarly, the pick-up 26 receives the
green fluorescence signals and filter 18A passes a green band of
radiation in the neighborhood of 5,300 angstrom units wave
length.
The red and green photomultiplier signals, after amplification in
amplifiers 34 and 36, respectively, are sampled and held in
sample-hold amplifier circuits 38 and 40, respectively, to produce
two electrical pulses which are proportioned in amplitude to the
magnitudes of the two fluorescent amplitudes. A threshold circuit
42 is connected to the green photomultiplier signal at 43 and
produces a control signal pulse when the green fluorescence is
above a fixed value indicating a leukocyte, and activates the
sample-hold amplifier circuits 38 and 40 and timing signals for the
analog-digital converters 44 and 45 and computer 46. The two held
fluorescence pulses are each converted to a digital representation
of the fluorescence values by the two analog-digital converters 44
and 45 producing two eight-bit members in two digital registers 48
and 50. Digital registers 48 and 50 are connected in turn to the
input data buss of computer 46. An oscilloscope 52 monitors the
outputs of the sample-hold circuits 38 and 40 to produce a display
on the screen 54 of the oscilloscope. A second oscilloscope (not
shown) can also be used which is controlled by the computer 46 to
monitor computer-processed displays.
The computer 46 processes the signals from analog-digital
converters 44 and 45 as well as input from the operator via a
teletype 56 and produces an output indicating the differential
count.
FIG. 2 depicts a cathode ray tube display of a white cell analysis
characteristically produced in accordance with the methods of the
present invention, and with particular reference to Examples XI and
XII. In this display, the green fluorescence signals have been used
for vertical deflection up from the bottom margin (origin), and the
red fluorescence signals have been used for horizontal deflection
to the right from the origin line on the left margin. Typical
clusters of bright spot signal points for groups of cells are
indicated at 60, 70, 80, 90, 100 and 110 which correspond
respectively to lymphocytes (L), monocytes (M), neutrophils (N),
basophils (B), eosinophils (E) and immature granulocytes (IG).
In order to avoid any possibility of the detection of false
signals, the signal circuit for the detection of the green
fluorescence is preferably operated with a threshold circuit, as
indicated for instance by the horizontal threshold line Y.sub.o, so
that only signals having a sufficient green fluorescence signal
value to exceed the threshold line Y.sub.o are actually registered
and indicated in the visual display. Similarly, an upper threshold
limit may be established, as indicated at Y2. By the employment of
additional threshold limits at positions as indicated by lines
X.sub.0, X1, X2, X3, X4 and X5 individual point clusters may be
picked out and displayed alone, or counted as mentioned below in
Example XII, For instance, cluster 70 may be selected by setting
the upper limits at Y2 and X3 and the lower limits at Y1 and
X1.
In using the apparatus shown schematically in FIG. 1 to obtain the
cathode ray tube display of FIG. 2, the computer 46 first monitors
the values of green fluorescence Y within the range of red
fluorescence X = X3 to X = X5 as cells are being measured
immediately after staining.
As the green fluorescence increases the average neutrophil green
fluorescence is calculated to generate a specific time to produce
data for the cell differentiation program. Thus when <Y>= Ys
for X3 < X < X5 the program switches to the data reduction
mode.
The computer builds in memory a two dimensional histogram, i.e.,
number of cells for each of the two values of fluorescence
possible. The first parameter of this histogram is made
proportional to green fluorescence. The second parameter is made
proportional to either red fluorescence or the ratio of red to
green fluorescence. The two parameter histogram is partitioned as
shown in Table I, below. The values of Xi and Yi are fixed by the
operator. The program counts the number of cells in each area and
prints out these counts.
TABLE I ______________________________________ CELL CLASS X LIMITS
Y LIMITS LOWER UPPER LOWER UPPER
______________________________________ LYMPHOCYTE 0 X1 Y1 Y2
MONOCYTE X1 X3 Y1 Y2 NEUTROPHIL X3 X5 Y1 Y2 BASOPHIL 0 X2 Yo Y1
EOSINOPHIL X2 X4 Yo Y1 IMMATURE GRAN- ULOCYTE X4 X5 Yo Y1
______________________________________
DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE I
A staining composition suitable for use in accordance with the
present invention is produced with the following composition: an
aqueous solution containing 4 .times. 10.sup.-.sup.3 grams of AO
per liter, being adjusted to an osmolarity of approximately
one-half of the normal physiologic (isotonic) level through the
addition of 4.25 grams of sodium chloride per liter of solution and
buffered at a pH value of approximately 7.4. Such buffering can be
obtained with phosphates of sodium at a phosphate molarity level of
0.00125 by incorporating 45 milligrams of NaH.sub.2
PO.sub.4.H.sub.2 O and 250 milligrams of Na.sub.2
HPO.sub.4.7H.sub.2 O into each liter of solution.
This composition can be produced by the following steps. A 1-liter
container is partially filled with distilled water and 4.25 grams
of sodium chloride may be added together with 0.045 grams of
NaH.sub.2 PO.sub.4 H.sub.2 O and 0.250 grams of Na.sub.2 HPO.sub.4
7H.sub.2 O. The mixture is stirred or agitated until the salts go
into solution, and the container is then topped up to a total
volume of 1 liter by adding additional distilled water. In a
separate container, 400 milligrams of acridine orange powder are
combined with sufficient distilled water to produce 100 cubic
centimeters of acridine orange solution. This combination is
stirred or agitated until the acridine orange is dissolved in the
water. This results in a clear solution with a reddish-orange tinge
of color. One cubic centimeter of 100 cubic centimeters of acridine
orange solution is then added to the previously mixed one liter of
saline-buffer solution to produce the acridine orange composition
described above. The combination with a saline buffer appears to
cause some of the acridine orange to form a colloidal
suspension.
Preferably, the combined composition is checked with a pH meter,
and if further adjustment is necessary, a drop or two of one-tenth
normal hydrochloric acid is added to lower the pH, or a drop or two
of one-tenth normal sodium hydroxide is added to raise the pH.
This dye composition was used in accordance with the teachings of
this invention and was found to produce a very satisfactory
result.
EXAMPLE II
Example I is repeated, substituting for the phosphate buffer, a
buffer commonly referred to as HEPES (i.e.,
N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) at a molarity
level of 0.005.
EXAMPLE III
Example I is again repeated, substituting for the phosphate buffer
a buffer commonly referred to as TRIS (i.e.,
2-amino-2-(hydroxy-methyl)-1,3-propanediol) at a molarity level of
0.15.
EXAMPLE IV
Example I is again repeated with the exception that the amount of
acridine orange was quadrupled to provide a concentration of 1.6
.times. 10.sup..sup.-2 grams per liter. At this concentration, the
results are found to be very good in the method of the present
invention.
EXAMPLE V
Example I is again repeated, except that the concentration of
acridine orange is increased to a level of 4 .times. 10.sup..sup.-2
grams of acridine orange per liter of solution, all of the other
conditions being as set forth in Example I. This composition
provides results which are satisfactory, but which are not quite as
effective as the lower concentrations of acridine orange described
in the preceding examples. Accordingly, this is believed to
represent the approximate upper limit of acridine orange
concentration which is effective in accordance with the present
invention. The staining effect appears to be too great to provide
good differentiation between different classes of white cells.
EXAMPLE VI
Example I is again repeated, except that the acridine orange
concentration is reduced to 3 .times. 10.sup..sup.-3 grams of
acridine orange per liter of solution, all other conditions
remaining the same as in Example I.
This composition provides results which are satisfactory for the
purposes of this invention. Thus, it provides a clear distinction
between white cells and other blood particles. However, the results
in terms of distinguishing one white cell group from another appear
to be somewhat less satisfactory than Example I. Accordingly, this
concentration of acridine orange is believed to represent the lower
limit of the preferred range of concentration.
EXAMPLE VII
Example I is repeated, except the acridine orange concentration is
reduced to 4 .times. 10.sup..sup.-4 grams of acridine orange per
liter of solution, all other conditions remaining the same as in
Example I.
This composition provides results which are satisfactory for some
of the purposes of this invention. Thus, it is satisfactory for
providing a clear distinction between white cells and other blood
particles. However, the results in terms of distinguishing one
white cell group from another are seriously impaired. Thus, it
appears that this is the minimum concentration of acridine orange
which is truly useful in the practice of the present invention.
In all of the Examples II through VII, the acridine orange may, at
least to some extent form a colloid when added to the saline-buffer
solution, just as described in connection with Example I.
EXAMPLE VIII
Example I is repeated, except that the staining composition is
produced so as to have an osmolarity which is approximately 0.3
isotonic through the addition of 2.55 grams of sodium chloride per
liter of solution. All other conditions remain the same as in
Example I.
This composition provides results which are unsatisfactory for
purposes of the present invention due to the fact that some of the
white cells are killed before they can be analyzed. Therefore, an
osmolarity level which is 0.3 isotonic represents what is believed
to be the lower limit of hypotonality which is effective in the
present invention.
EXAMPLE IX
Example I is repeated, except that the staining composition is
produced so as to have an osmolarity which is approximately 0.7
isotonic through the addition of 5.95 grams of sodium chloride per
liter of solution. All other conditions remain the same as in
Example I.
This composition provides results which are unsatisfactory for
purposes of the present invention, due to the fact that the
clusters of bright spot fluoroscence signal points are
insufficiently separated. Therefore, an osmolarity level which is
0.7 isotonic represents what is believed to be the upper limit of
hypotonality which is effective in the present invention.
EXAMPLE X
In practicing the method of the present invention, a quantity of
0.10 milliliter of a fresh blood sample is added to 1 milliliter of
the acridine orange diluent staining composition of Example I. The
resulting suspension is mildly agitated from time to time and
allowed to stand for a period varying between 10 seconds and
several minutes to permit the dye to be taken up by the white cells
of the blood sample. The suspension is then introduced into an
apparatus having a flow system which provides for a flow of the
sample liquid in an exceedingly fine stream through an optical
chamber. The stream is so narrow as to be capable of confining the
flow so that the individual cells (both red cells and white cells)
generally traverse the stream one at a time in single file. In the
optical chamber, the cells are caused to pass through a uniform
light field provided from a blue laser beam, preferably an argon
ion laser at a wavelength of 4880 angstrom units (488
millimicrons). The uniform light field from the laser preferably
has a very short dimension in the direction of travel of the cell
sample stream. That dimension is of the same order of magnitude as
the maximum dimension of an individual cell so that the cells are
subjected to radiation one at a time.
Green fluorescent radiation having a range of wavelengths centered
at about 5300 angstrom units resulting from the optical excitation
of the white cells by the argon laser beam is then detected, and
the total number of white cells is counted in terms of optical
green fluorescent pulses. This counting is continued for a
carefully measured volume of the suspension sample to obtain an
exact white cell count per unit of volume.
It has been discovered that at the acridine orange concentrations
noted above there is essentially no take-up of acridine orange dye
by red cells which therefore remain substantially invisible in the
detection method just described. (However, there is an exception to
this statement, as noted below). By contrast, each white cell takes
up a concentration of the acridine orange dye which results in
green fluorescent emission. Thus, a rapid and accurate count of
white cells is produced. Immature red cells, referred to as
reticulocytes, do take up some of the acridine orange dye, but the
dye is taken up in such a way that there is substantially no green
fluorescene emission from the reticulocytes. Thus, the green
fluorescence emission is an accurate basis for distinguishing white
cells from all other blood particles.
EXAMPLE XI
The method as set forth in Example X is repeated, except that, in
addition to detecting the green fluorescence characteristic, the
red fluorescence of each cell is detected, at a range of
wavelengths in the order of 6500 angstrom units. The red and green
signals are optically separated by a dichroic mirror and suitable
filters, and amplified by separate photomultiplier tubes. The green
signals are used to obtain a total white cell count, while the
green and red signals are combined to provide a display pattern
upon a cathode ray tube. Thus, the green signal may be used as the
vertical displacement coordinate and the red signal as the
horizontal displacement coordinate for a single display spot on the
cathode ray tube for each white cell. Various amplitudes of red and
green fluorescences from the different white cells are thus
displayed upon the cathode ray tube. It is believed that the
different green and red fluorescence amplitudes are due to the
variation in characteristics of different types of white cells, the
white cells having the largest number of granules in the cytoplasm
generally displaying the largest red fluorescence signal. However,
the chemical composition of various granules among the granulocytes
are characteristically different and result in differences in red
fluorescence intensity and although the white cells all contain the
same amount of DNA, this green fluorescene intensity of the cell
nucleus may be modulated in characteristic manners by the
differences of avariciousness for the dye in the cell by the
granules of various cells resulting in different green fluorescent
signals. It has been discovered that each blood sample produces a
display pattern having distinct clusters of points indicating the
distribution of different types of white cells. Thus, a
two-dimensional display pattern is produced upon the face of the
cathode ray tube which provides qualitative information about the
distribution of white cells within the sample. By comparing the
patterns produced by blood samples from normal inidviduals with
patterns produced by blood samples from individuals who have
diseases or infections which cause abnormalities in the balance of
white cells, it is quite practical to detect the differences and to
quickly recognize, in a qualitative sense, white cell imbalance
conditions which are characteristic of particular diseases or
infections.
The pattern displayed in connection with the last described example
of the method is preferably photographed to provide a permanent
record which can be analyzed and studied, and which can be compared
with later tests from the same patient.
EXAMPLE XII
Example XI is repeated, and threshold circuits are employed to
select green and red fluorescence signals within individual narrow
fields of clusters, and those signals are individually counted for
a pre-selected total white cell count sample. The method is then
repeated for other settings of the threshold circuits to
successively count separate cluster portions of the display
corresponding to different classes of white cells. By this means,
individual counts of the quantities of white cells of each
different type are obtained. Thus, the ratio of the population of
each type of white cell to the total white cell count may be
obtained. These ratios are preferably presented as percentages.
Multiple threshold circuits may be employed to count several narrow
fields of clusters at once.
Since the apparatus operates very rapidly, white cells counting
rates as high as 1,000 cells per second are achievable. previously
unattainable accuracies can be realized by counting thousands of
white cells of each type from each sample, as contrasted with the
usual manual microscope method of counting a very small total
quantity in the order of 200 individual cells. Furthermore, since
the cells are actually living at the time they are counted, and
since they have been undamaged by the use of any procedure to
destroy the red cells, the measured cell population corresponds
very accurately to the cell population in the living blood stream
of the patient.
It has been discovered that the staining composition and the
methods of the present invention are extremely effective in
detecting and counting white cells, in distinguishing white cells
from red cells on the basis of green fluorescence (essentially
ignoring the presence of the red cells), and most particularly in
distinguishing the different types of white cells. The different
types are distinguishable in the cathode ray tube display by reason
of creation of a discrete cluster of points for each distinct type.
It has been determined thus far that six classes of white cells,
namely, the lymphocytes, monocytes, neutrophils, basophils,
eosinophils and immature forms of granulocytes are distinguishable
on this basis.
EXAMPLE XIII
Example XI is repeated using a helium-cadmium laser instead of an
argon laser. The results are satisfactory, although the
distinctness of some of the oscilloscope clusters are not as well
maintained as with the argon ion laser, probably due to the quality
of the helium-cadmium laser tested.
EXAMPLE XIV
All of the emphasis thus far has been upon the objective of
distinguishing white cells from all other particles in the blood,
and upon deriving useful information about the white cells.
However, the discoveries of the present invention are also useful
for deriving other important information about the blood. For
instance, the present invention can be employed to detect and count
the number of reticulocytes per unit of volume of blood.
Reticulocytes are red blood cells which contain a network of
granules or filaments representing an immature stage in
development. Reticulocytes normally comprise about one percent of
the total red blood cells, but this percentage of reticulocytes can
change dramatically under abnormal conditions, and such a change
may be symptomatic of disease.
It has been observed that the reticulocytes take up acridine orange
dye, under the conditions generally outlined in the above examples,
to a much greater extent than the other red cells. The uptake of
the acridine orange by the other red cells is insignificant, but
the reticulocytes take up enough dye to provide a red fluorescence
signal which is of substantially the same magnitude as the
lymphocytes class of white cells. However, the reticulocytes do not
provide a significant green fluorescence signal. Thus, it is
possible to distinguish reticulocytes from all other blood
particles by staining a blood sample with the acridine orange
composition of the present invention, and then distinguishing the
reticulocytes by excluding from detection all of the green
fluorescing white cells, and detecting those remaining cells which
have a significant red fluorescence. The following example
illustrates this modification of the process.
The process of Example X is repeated, except that the dye
concentration is raised to that of Example V and the optical green
fluorescent radiation from the white cells is employed as a
discrimination signal to exclude any count of the white cells, and
with the further exception that in addition to detecting the green
fluorescence characteristics, the red fluorescence of each cell is
detected at a range of wavelengths in the order of 6,500 angstrom
units to thereby select all of those cells having a significant red
fluorescence signal, and which at the same time do not produce any
green fluorescence signal. As described in connection with Example
XI, the green signal may be used as the vertical displacement
coordinate, and the red signal as the horizontal displacement
coordinate as a basis for setting a low green fluorescence
threshold which excludes all of the white cells, and for setting
red fluorescence thresholds which particularly select the
reticulocytes.
EXAMPLE XV
The process of Example XI is repeated using an apparatus of the
type schematically represented in FIG. 1. The red and green
photomultiplier signals after amplification are sampled and held to
produce two pulses whose amplitudes are proportional to the
magnitudes of the two fluorescent amplitudes. A threshold circuit
is connected to the green photomultiplier tube signal and produces
a control signal pulse when the green fluorescence is above a fixed
value indicating a leukocyte, and activates the sample-hold
amplifiers and timing signals for the converters and computer. The
two held fluorescence pulses are each converted to a digital
representation of the fluorescence values by two analog-digital
converters producing two eight-bit numbers in two digital
registers. The registers are connected in turn to the input data
buss of a computer, such as a Data General. The computer processes
the signals as well as input from the operator via a teletype and
produces an output on the teletype indicating the differential
count.
The computer first monitors the values of green fluorescence Y
within the range of red fluorescence X = X.sub.3 to X = X.sub.5 as
cells are being measured immediately after staining. As the green
fluorescence increases the average neutrophil green fluorescence is
calculated to generate a specific time to produce data for the cell
differentiation program. Thus when <Y> = Ys for X.sub.3
.ltoreq. X .ltoreq. X.sub.5 the program switches to the data
reduction mode.
The computer builds in memory a two dimensional histogram, i.e.,
number of cells for each of the two values of fluorescence
possible. In our system this grid has a dimensionality of 64
.times. 64. The first parameter of this histogram is made
proportional to green fluorescence. The second parameter is made
proportional to either red fluorescence or the ratio of red to
green fluorescence.
In a simple differentiation program, 10,000 cells are read in after
<Y> = Ys, to form the histogram of green versus red
fluorescence or green versus the ratio of red to green
fluorescence. The two parameter histogram is partitioned as shown
in Table I, above. The values of X.sub.i and Y.sub.i are fixed by
the operator. The program counts the number of cells in each area
and prints out these counts. Also, the first three statistical
moments of the data in each area are computed for both parameters
to derive indices which can be correlated with specific
diseases.
In a more complex program, the lines separating populations are not
fixed but are established by the program. To find the proper value
for X.sub.1, for example, the data between Y.sub.1 and Y.sub.2 are
compressed to generate a one dimensional histogram of the values of
red or ratio fluorescence within the green fluorescence range
Y.sub.1 to Y.sub.2. There will be a value of fluorescence between
the lymphocyte and monocyte populations for which there is a
minimum number of cells. This minimum can be determined in several
ways, for example, by regression analysis with a parabolic
function.
The foregoing examples have been presented for the purpose of
illustrating (but not limiting) the compositions, apparatus and
methods of the present invention. It will be understood that
changes and variations therein may be made without departing from
the spirit and scope of the invention as defined in the following
claims.
* * * * *