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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to flow cytometric applications to be performed on
blood cells and specifically relates to methods for determining the
competence of leukocyte phagocytic and leukocyte killing ability by
utilizing the methods of flow cytometry.
The monitoring of leukocyte function is becoming of increasing clinical
importance, particularly with regard to the pathology of autoimmune and
arthritic diseases. Other reasons for inquiry into leukocyte abilities
pertain to the healing processes such as those encountered in burn
patients. See for instance Ransjo et al., "Some Aspects of Neutrophil
Granulocyte Function in Burn Patients", Burns, 5:255-259 (1979), printed
in Great Britain and given at the Fifth International Congress on Burn
Injuries, Stockholm, June 1978. Further reference may be made to an
article by Halgren et al., "The Serum Independent Particle Uptake by PMN
From Patients With Rheumatoid, Arthritis and Systemic Lupus
Erythematosis", Arthritis and Rheumatism, 21:107-113 (1978) and to
Hakansson, et al., "Neutrophil Function In Infection Prone Children",
Archives of Disease in Childhood, 55:776-781, (1980).
The particular aspects of leukocyte function monitoring which are becoming
of increasing clinical importance involve the cell's ability to
phagocytose, i.e., the ability to ingest foreign particles, and the
leukocyte's killing ability, i.e., the ability to destroy ingested
organisms through the application of reactive oxygen. For further
information, reference may be made to articles by Stossel et al.,
"Quantitative Studies of Phagocytosis by PMN Leukocytes: Use of Emulsions
to Measure the Initial Rate of Phagocytosis", The Journal of Clinical
Investigations, 51:615-624 (1972); Michell et al., "Measurement of Rates
of Phagocytosis: The Use of Cellular Monolayers", J. Cell Biol.
40:216-224, 1969; and Arnaout et al., "Alternative Complement Pathway
Dependent Ingestion of Fluolite Particles by Human Granulocytes", The J.
of Immunology, 127:278-281 (1981).
Active oxygen is the species of oxygen used by phagocytic cells to kill
foreign organisms generally by way of oxidative cytotoxic related
mechanisms. It has been previously measured by monitoring neutrophil
respiratory events for producing active oxygen and detecting the effects
on autofluorescence. See: "Neutrophil Activation Monitored by Flow
Cytometry: Stimulation by Phorbol Diester is an All-or-None Event",
Science, Vol. 215:673-675 (Feb. 5, 1982). Still other microchemical
techniques for the cellular spectrophotometric measurement of oxygen
uptake have been described by Glick in an article entitled "Microchemical
Analytical Techniques of Potential Clinical Interest", in Clinical
Chemistry, Vol. 23, No. 8:1465-1471 (1977). Hultborn et al., described yet
additional studies on oxygen consumption rate and nitroblue tetrazolium
reduction capacity in the absence and presence of phagocytogenic agents.
That article was reported in the Scandanavian Journal of Clinical
Laboratory Investigations, 23, 297-304 (1973) in an article entitled,
"Studies on Leukocyte Function by Measuring Respiration and Nitroblue
Tetrazolium Reduction by Simplified Methods".
Other cellular activation and membrane effects have been described using
solid monolayer membranes, fluorescent lipid probes and bound fluorescent
antibodies in an article entitled, "Specific Antibody Dependent
Interactions Between Macrophages and Lipid Haptens in Planar Lipid
Monolayers", Proc. Natl. Acad. Sci., Vol. 78, No. 7:4552-4556 (July 1981).
Still other references of general interest include Gordon, "Regulation of
Hematopoiesis", Vol. 2, Chapter 42, 1970, Appleton - Century Crofts New
York; and Halgren et al. "The Serum-Independent Uptake of IgG-Coated
Particles by Polymorphonuclear Leukocytes From Uremic Patients on Regular
Dialysis Treatment", J. Lab. Clin. Med. 94:277-284 (1979).
Despite an intense interest in the various phagocytic and killing ability
characteristics of leukocytes, the conventional methods have failed to
provide suitable methods for the convenient assay of both phagocytic and
killing ability.
It is an object of the present invention to provide such methods in a
convenient assay and to thereby enable one to efficiently measure both the
phagocytic and killing ability of leukocytes. It is another object to
allow such measurements to be made on selected leukocyte subclasses. It is
a further object to provide such assays for use in flow cytometry type
instruments thereby permitting the rapid evaluation of large numbers of
cells in a clinically convenient environment and acceptable format.
It is yet another object of the present invention to provide methods
capable of relating phagocytic ability of leukocytes with the presence or
absence of specified cell surface markers or antigens.
SUMMARY OF THE INVENTION
These and other objectives of the present invention are met by the methods
of the present invention which provide for assays permitting the
determination of phagocytic ability of selected subclasses of leukocytes
and the determinations of the cells respective killing abilities. The
leukocytes to be measured, typically contained within a blood sample, are
contacted with target particles having a first label associated therewith.
In a preferred embodiment, this first label is a fluorescent label such as
tetramethyl rhodamine. These same target particles may be preferably
opsonized by having immunoglobulins coated thereon. The thusly treated
leukocytes are thereafter passed in a substantially single file fashion
pursuant to conventional and well-known hydrodynamic focusing techniques,
through a zone illustrated by a focused light source. The passage of the
cells through this zone results in a pattern of scattering characteristic
of the respective leukocyte subclasses. Specifically, the scattered light
is detected at substantially forward angle and wide angle locations and
leukocytes identified accordingly. The relative wide angle scatter
characteristics of different types of leukocytes are relatively
insensitive to angles of measurement over at least the range 32.degree. to
148.degree.. Theoretical and experimental considerations [Hansen, et al.
"Light Scatter as an Adjunct to Cellular Immunofluorescence in Flow
Cytometric Systems", Journal of Clinical Immunology, 2:325-415 (1982)]
indicate that scattering angles below 2.degree. give primarily size
information, while angles above 4.degree. are dependent on granularity
properties of the cell. Thus, the class of leukocytes (white blood cells)
may be differentiated into monocytes, granulocytes and lymphocytes. These
individual leukocyte subclasses may then be further individually examined
by conventional electronic gating means and the fluorescence of the cells
detected whereby the presence or absence of phagocytized fluorescent
particles may be determined.
Other embodiments provide for the correlation of phagocytosing ability as
determined above with additional cellular characteristics such as the
presence or absence of particular surface antigens detectable by
antibodies specific therefor. These antibodies are labeled with a second
detectable label, preferably different from the first label. Most
preferably, the second label will be a chromophore or other fluorescent
type label having fluorescence different than that of the label employed
in the target particle.
Still additional methods provide for the determination of killing ability
to the extent it is related to the production of reactive oxygen. The
presence of reactive oxygen is detected by the change in the ratio of an
oxidatively sensitive fluorophore against a nonoxidatively sensitive
fluorophore, both of which are associated with a target particle ingested
by the leukocyte.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other principles of the instant invention may be more clearly
understood by reference to the figures wherein:
FIG. 1 depicts a conventional gating analysis on the well-known three part
differential;
FIG. 2 depicts the same analysis in a three dimensional view;
FIG. 3 depicts a fluorescence histogram analysis of granulocyte
phagocytosis of fluorescent particles;
FIG. 4 shows a phagocytosis analysis based on a one to two ratio of cells
to target particles;
FIG. 5 shows a phagocytosis analysis based on a one to four ratio of cells
to target particles;
FIG. 6 depicts graphically a phagocytosis analysis on blood sample No. 183;
FIG. 7 depicts graphically a phagocytosis analysis on blood sample No. 184;
FIG. 8 depicts graphically a phagocytosis analysis on blood sample No. 193;
FIG. 9 depicts graphically a phagocytosis analysis on blood sample No. 194;
FIG. 10 depicts graphically a phagocytosis analysis on blood sample No.
196;
FIG. 11 depicts graphically a phagocytosis analysis on blood sample No.
197;
FIG. 12a shows a histogram of the corrected red fluorescent particles in a
correlation study between phagocytosis and C3b receptors;
FIG. 12b is a histogram of the uncorrected red fluorescent particles in a
correlation study between phagocytosis and C3B receptors;
FIG. 12c is a histogram of the corrected green fluorescence;
FIG. 12d is a histogram of uncorrected green fluorescence; and
FIG. 13 graphically depicts a time related correlation study between
phagocytosis and C3b receptors.
DETAILED DESCRIPTION OF THE DRAWINGS AND PREFERRED EMBODIMENTS
Whole blood samples may be analyzed and the leukocytes or white blood cells
therein separated into a three part differential by means of the cells'
light scatter characteristics. Particularly useful in such an analysis is
the forward light scatter and right angle light scatter measurement such
as those made by the Spectrum III.TM. flow cytometry instrument available
from Ortho Diagnostic Systems Inc., Raritan, N.J. The experimenter
typically lyses the red blood cells in a whole blood sample, stains and
dilutes the sample appropriately, and in a process conventionally known as
hydrodynamic focusing, forms a substantially one cell wide column of cells
surrounded by a sheath fluid. This stream is passed through a zone
illuminated by a focused light source. Such a light source may preferably
be a laser, the spectral characteristics of which may be advantageously
selected in accordance with the fluorescent dyes employed.
As the cells pass through the aforementioned zone, the light is scattered
in a manner characteristic of cellular subclasses. As is now well-known,
comparison of forward angle light scatter against right angle light
scatter, permits the leukocytes to be differentiated into the respective
categories of lymphocytes, monocytes and granulocytes. The resulting
categories generally form clusters when the data is presented in a
histogram format. Such a cluster arrangement is depicted in FIG. 1 and the
respective clusters labeled. As is further shown in FIG. 1 and in a manner
well-known in conventional art, a particular subclass such as the
granulocytes may be gated as indicated by the dotted line region
circumscribing the granulocyte cluster. This gating is typically
accomplished by electronic means and is fully operator adjustable. FIG. 2
indicates an alternative manner of presenting the three part differential,
whereby the Z axis or height is indicative of the numbers of cells
detected.
The gated granulocyte cluster indicated in FIG. 1 may then be further
analyzed with regard to one or two color fluorescence. Fluorescence is
typically detected by utilizing a photomultiplier type detector means and
spectral filters in manners well-known in the art. The leukocytes depicted
in FIG. 1 were incubated with fluorescently labeled target particles and
phagocytosis permitted to occur. The granulocyte cluster was then
arbitrarily chosen for further analysis and a trigger region or gate
constructed to surround these cells. Those cells included within the
trigger region were then analyzed for green fluorescence indicative of the
presence of ingested particles labeled with a green fluorescent
chromophore. Those skilled in the art will readily recognize that
fluorescent labels of other colors may be readily employed in substitution
for the fluorescein used in this example and indeed, other types of labels
such as colloidal gold and the like may be employed providing the labels
have detectable effects preferably with respect to light.
FIG. 3 presents a histogram relating the level of green fluorescence
detected against the count or number of cells exhibiting fluorescence. As
may be clearly determined by analysis of FIG. 3, the granulocytes may be
further differentiated into subpopulations based on the number of
fluorescent particles ingested. Those cells having a lower level of
fluorescence correspond to cells having a single particle ingested while
those cells exhibiting greater levels of fluorescence correspond to cells
having additional particles ingested.
As may be readily appreciated, the number of particles ingested is at least
partly related to the number of particles provided per number of cells
capable of phagocytosing the particles. Thus, particle concentration can
become a rate limiting factor. FIGS. 4 and 5 clearly depict this
relationship and show the increase of percentage of cells phagocytosing
identified when the ratio of cells to particles is altered from one to two
(FIG. 4) to one to four (FIG. 5). The particles employed in both
determinations were particles having a diameter of approximately two
micrometers.
By performing a regression analysis on plotted curves of percents of cells
phagocytosing over time, one may calculate an exponential fit relating the
number of phagocytosing cells to time pursuant to the equation
Y=a+b Ln (X)
where Y equals the percent of cells actively phagocytosing, X is the unit
of time permitted for phagocytosis measured in minutes, a equals the Y
intercept and b equals the slope. The constants and the standard
deviations for FIGS. 4 and 5 have been calculated pursuant to such a
regression analysis and are depicted thereon. Indeed, FIGS. 6 through 11
represent curves and calculated values resulting from like analysis of six
different blood samples and clearly show the close correlation of uptake
as a natural log function of time.
The target particles employed in the above experiments were fluorescent,
monodispersed carboxylated microspheres in the one to two micrometer
diameter range such as those obtainable from Polysciences under Catalog
No. 15702. It should be noted, however, that although such a microsphere
or bead represents the preferred target particle embodiment, other target
particles may be employed such as bacteria and the like.
The Carboxylated Microspheres
Example 1--Method for Determining Phagocytic Ability
Fluorescent monodispersed carboxylated microspheres obtained from
Polysciences, P.A. (No. 15702) diluted in glucose medium comprising:
Sodium Chloride--5.9 grams
Anhydrous Socium Acetate--2.5 grams
Potassium Chloride--0.3 grams
Calcium Chloride--0.44 grams
Magnesium Chloride--0.20 grams
Glucose--1.26 grams
(All quantities to 1 liter)
were opsonized by coating the particles with human IgG. 50 mg of human IgG
and 2.times.10.sup.9 particles were added to 10ml of 0.1M sodium
borate-boric acid buffer, pH 8.2, and incubated for 48 hours at 4.degree.
C. with a constant stirring. The particles were then washed with a glucose
suspension medium and suspended to the desired concentration.
The sample was prepared in the following manner: to 200 .mu.l of blood, 200
.mu.l of particles in the glucose medium were added and the resultant
mixture incubated at 37.degree. C. with gentle shaking. An identical
sample was incubated at 0.degree. C. to serve as a control. 100 .mu.l of
the incubated sample was lysed for five minutes with 2 ml of ammonium
chloride based Ortho lysing reagent (available from Ortho Diagnostic
Systems Inc., Raritan, N.J.) and analyzed on the Spectrum III.TM. flow
cytometer (also available from Ortho Diagnostic Systems Inc.). The
Spectrum III.TM. was configured to produce a forward versus right angle
scatter three part differential with gated fluorescence.
It was surprisingly discovered that by employing multiple fluorescent
colors, one may, in addition to determining phagocytic ability of a
selected class of leukocytes, further determine the "killing ability" of
the same cells. Such killing ability has typically been associated with
the generation of so-called "reactive oxygen". It has been theoretically
proposed that reactive oxygen "kills" by oxidizing the phagocytosed
particle, however, the exact mechanisms are still unknown.
By associating two labels with the target particle to be phagocytosed, one
of which is photogenically sensitive to the presence of reactive oxygen,
one may determine "killing ability" of the cell in question. The nonoxygen
sensitive label, which may be chemically insensitive to active oxygen or
protected from active oxygen, e.g., by being embedded in a plastic matrix,
is utilized to indicate the presence or absence of the target particle
within the cell. By measuring the two labels and determining the ratio
therebetween, analysis of the killing ability may be effected without
necessitating the measurement against controls. An example of the present
invention employs fluorescent labels, the first label being tetramethyl
rhodamine incorporated within the plastic bead while the surface of the
bead or particle is labeled with fluorescein (green fluorescence as
opposed to the rhodamine's red fluorescence), which is sensitive to the
presence of the active oxygen. The combination of reactive oxygen with the
fluorescein effectively causes photodecomposition of the fluorescein
thereby resulting in high red/green fluorescent ratios. Cells exhibiting
low red/green ratios, close to those exhibited by the particles
themselves, indicate the presence of little or no reactive oxygen
production. A preferred embodiment of the present invention would use a
dye, such as a cyanine [di-O-C2-(3)], sensitive to active oxygen but not
to the pH of the cell interior. Fluorescein is sensitive to pH as well as
active oxygen.
The principles of the instant invention provide for still greater
versatility and specifically allow for the correlation of phagocytosis
with cellular surface markers of interest. An example of such a marker is
the C3b receptor, the presence or absence of which is becoming
increasingly important in the clinical environment. A correlation may be
determined by the aforedescribed three part differential in conjunction
with multiple gated fluorescence. For instance, red fluorescent particles
are ingested by cells which are additionally incubated with fluorescein
conjugated antibodies specific for the surface marker of interest such as
the C3b receptor. As described earlier, the leukocyte subclass is
indentified on the basis of its light scatter characteristics, the
subclass of interest is gated and fluorescence measured. In the preferred
embodiment, the particles are labeled with a rhodamine dye whereby the
detection of red fluorescence serves as indication of phagocytic activity.
The cells may be further interrogated for the presence of second
fluorescent label (for instance the presence of fluorescein conjugated
anti-C3b antibody) which presence is indicative of the C3b or other
receptor on the cell surface.
With multiple fluorescence determinations, however, corrections may be
necessary in the event of spectral overlap by fluorescence, for instance,
between green fluorescence of the antibody and red fluorescence of the
particle. Such a correction may be easily accomplished by treating three
sets of samples in parallel wherein set 1 represents cells incubated with
particles and anti-C3b; set 2 with particles and no anti-C3b; and set 3 no
particles and only anti-C3b immunoglobulin. The following formulae were
used:
##EQU1##
(Where c=Corrected, m=Measured, G and R refer to measurement made with
calibrating samples with only the green or red fluorochome respectively).
These corrections may be performed on a cell by cell basis by employing the
Ortho 2150 computer in the list mode. FIGS. 12a through 12d indicate
corrected red, uncorrected red, corrected green, and uncorrected green
fluorescence respectively with the associated histogram counts.
FIG. 13 presents in tabular format an experiment preferred with three such
sets of samples, incubated from 3-40 minutes and the resultant data
therefor.
As may be readily appreciated by those skilled in the art, the above
disclosure and figures demonstrate the principles and preferred embodiment
of the instant invention, however, many alternatives are available, for
instance the coating of a particle may be accomplished by absorption or by
covalent linkage employing surface carboxyl groups on the particle etc. as
well as other substitutions and variations on the described processes
without deviating from the spirit or scope of the present invention.
* * * * *
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