Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF TIIE INVENTION
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The present invention relates -to the preparation of blood
smear slides and particularly to a method for producing blood
smear slides tha~ are, despite variationS in the Rheological-
hematological properties of the venous samples of blood from
which slides are to be prepared, of uniformly good quality for
effective examination. Since blood smear slides of uniform quality
` are produced, the method is particularly suitable for automated
analysis of the blood smears by automated pattern recognition
equipment.
he most conventional method of preparing a venous blood
smear slide consists in placing a drop of venous blood on a
slide blank and smearing it across the blank by scraping it with
, the edge of another slide or cover plate. This method is some-
;~ what time consuming and does not give rise to producing slides
of uniform quality.
In recent years, there has been a widespread effort to
automate the prepar~tion of venous blood smear slides as a
means to provide smear slides that are of uniform and good
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quality which are requisite for automated analysis of blood
smears by pattern recognition techniques. Efforts to find
a suitable automatic slide preparation technique have concen-
trated, for the most part, on the use of centrifugal spinners,
which in general consist of a rotating chuck carrying a slide
holder. A slide is placed on the holder and a blood sample is
deposited thereon; the chuck is then rotated at a velocity, and
or a time, sufficient for the blood to spread across the slide
into a monolayer of hlood cells.
The characteristics of a blood smear required for effective
and accurate analysis, particularly by automated pattern recog-
nition techniques, are:
a. good separation of the cell elements (i.e. erythrocytes,
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leukocytes, and thrombocytes) on the slide,
b. uniform distribution of ~he cell elements,
c. preservation of the cell structure and shape, and
d. maximum coverage of the slide sur~ace by the separated,
uniformly distributed, undistorted cells. These characteristics
are almost impossible to achieve with the previously described
manual method or by the spinning method using undiluted bloQd,
Experiments show that a good monolayer of blood cells can
be prepared by spinning if the slide is spun at the right velocity
for the right time, but the right velocity and time differ from
blood sample to blood sample. The most significant differenti-
ating factor appears to be the blood viscosity. tsee "ERY-
THROCYTE MORPHOLOGY AND CENTRIFUGAL 'SPINNER' BLOOD FILM PREPARA~IONS"by James W. sacus, the Journal of Histochemistry and Cytochemistry,
; Vol. 22, No. 7, pp. 506-516, 1974.) It has been found that the
viscosity of whole blood varies linearly, but only slightly, with
J~ hematocrit within a range of hematocrit 15-45, (See W.J. Williams,
et al, Hematology, p. 204, McGraw-Hill Book Co. 1972), which is
; the hematocrit range within which the majority of both normal
and diseased blood samples falls. When the hematocrit is above
~5, the viscosity increases rapidly and non-linearly with
increasing hematocrit.
~- One automated spinning approach for providing uniform
blood smears has been to select the particular spinning velocity
and duration in relation to the hematocrit value. It would be
simpler, however, to spin at the same velocity for the same
time in each case. It has, therefore, been proposed to spin at
a uniform velocity and terminate the spinning when an optical
detection system detects suitable cell separation. However,
these automated spinning techni~ues for obtaining uniform
quality blood smear slides have not proved satisfactoryO ~See
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~ R.E. Wenk, American Journal of Medical Technology 42, 71-58, 1976.)
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When the spinning enerqy is enough to provide good separa~ion
of all the cells, including particularly the erythrocytes,
many erythrocytes as well as the leukocytes, may be distorted,
particularly in high hematocrit samples.
In addition to the above approaches, numerous researchers
have investigated the problems associated with preparation of
blood smear slides. M. Ingram and F.M. Minter, in their article
"Semiautomatic Preparation of Coverglass Blood Smears Using a
` Centrifugal ~evice" in Vol. 51 No. 2 pp. 214-221 of The American
Journal of Clinical Pathology, reported on several variables
and their effect on blood smears. Dilution of the blood sample
is but one approach discussed but the article reaches no
conclusions as to whether dilution is always desirable in pre-
paring blood smears for automated analysis. The article only
mentions that dilution of greater than one part diluent to
one part blood appears to be disadvantageous due to red cell
distortion. No minimun or optimun dilution is suggested for
the purpose of preparing blood smears for automated blood
` sample analysis.
James M. Bacus, in his article "Eryth~ocyte Morphology
and Centrifugal 'Spinner' Blood Film Preparations" in Vol. 22
No. 7 pp. 506-576 of The Journal o Histochemistry and Cyto-
chemistry, discusses various parameters affecting blood smear
slides made with spinners. The author mentions the possibility
of improvements to spun smears by diluting all samples such
that their adjusted hematocrit values would be identical. How-
ever, no experimental investigation of the dilution method is
cited and the proposed dilution approach was not adopted. Indeed,
the ultimate conclusion of the article is that spinning time
must be adjusted to account for variation in viscosity of blood
so that uniformly good blood smears can be obtained by the
spinner technique. As such, each blood smear is prepared by
adjusting at least one parameter of the blood smear
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preparation method described.
A principal object of the present invention is to provide
a method for preparing venous blood smear sliaes that is essen-
tially the same for all samples of normal or diseased blood
whose hematocrit is within the range 15-45.
It is another object of the:iinvention to provide a method
- for preparing uni~orm blood smears with good separation and
uniform distribution of all the cells including erythrocytes
and leukocytes without distorting the structure and shape of
any of the cell elements.
BRIEF SUMM~RY OF THE INVENTION
~- The majority of samples of normal and ~iseased blood analysed ~-
by clinical laboratories have a hematocrit value in the range
of 15 to 50. In accordance with the present invention, it has
been discovered that when a sample of blood, whose hematocrit
value is within this usual range of 15-45, is diluted by mixing
with a substantially isotonic solution, such as a saline solution,
in a proportion of from 1 to about 4 parts diluent to 4 parts
venous blood, by volume, the viscosity of the samples are
brought into a narrow, low range. Moreover, red cell separation
is increased by dilution as is the tendency for cell aggregation.
With the viscosity reduced to within this narrow, low range,
smear slides of good, uniform quality are produced by spinning
the sl~des at a uniform speed for a uniform time.
Blood smear slides produced by spinning diluted blood
samples are c~aracterized by good coverage of the slide surface,
good separation and uniform distribution of the cells. The
structure and shape of all the cells are suitably preserved for
effective examination and identification utilizing conventional
manual methods or by automated optical scanning, data processing
and pattern recognition techni~ues. Erythrocytes are particu-
larly susceptible to distortion by spinning, but the spinning
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` energy utilized in the present method is su~iciently low as
- to significantly decrease the incidence and severity of ery-
throcyte distor~ion in comparison with the typical result of
the known conventional manual spreading or spinning techniques
- of preparing blood smear slides.
Since the new method utilizes the same proportion of
dilution and the same spinning time and duration, i.e., the
same spinning energy, Eor all blood samples, this method makes
possible a fully automated system for preparing blood smear
slides with requisite uniformity and quality to permit analysis
by automated techniques.
In general, the method of the present invention consists
in diluting a sample of whole blood by mixing it with a suitable
diluent in a fixed proportion to reduce the viscosity to a l~vel
within a narrow range and increases cell separation. The
resulting diluted sample is spun a-t a predetermined, relatively
low velocity for a predetermined time thereby producing a
monolayer of cells with good separation and distribution with-
out distortion of cell structure. For this purpose, an adequate
volume of diluted blood is placed on the flat surface of a slide
~'~ which is then spun in the plane of its flat sur~ace at the
predetermined angular velocity for a predetermined time which
results in a smear having the forementioned requisite character-
istics.
The method of this invention may be adapted ~or preparing
~lood smear slides of similar quality from the relatively un-
common samples Oe whole blood whose hematocrit value is in
excess of 45. In this instance, it has been found that simpl~
~` by increasing the proportion of diluent mixed with the blood,
the viscosity of the blood is brought into the above-described
narrow low range of viscosities.
` Thereafter, the preparation of the slide is the same as
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in the basic method just described, that is, the increasingly-
diluted blood sample is deposited on a slide which is spun At
the same velocity and for the same length of time which is
effective in the basic method.
DESCRIPTION OF THE DRAWING
The method of this invention is described below in more
detail with reference to the accompanying drawing which is a
graph showing the relative change in the viscosity o~ a number
of whole blood samples, each of which has a different hematocrit
as a result of mixing with diluent in various proportions. -
DETAILED DESCRIPTION
The accompanying graph illustrates that when samples of
whole blood having a hematocrit value in the range of 20 to 50
are diluted by mixing with an isotonic di~uent, preferably a
; salinei(NaCl) solution, in a proportion ranging from about 1
to 4 diluent to our parts whole venous blood, by volume, the
; viscosity is reduced to within a narrow xange from about 2.5
to about 4.5 minutes relative to water as measured~with an
Ostwald viscometer at room temperature (i.e~, a temperature in
a range of from about 65 to 75F). It has been found that
an adequate range of resultant viscosity is provided by dilution
in the proportion of two parts diluent to four parts whole
venous blood (i.e., 1 to 2) thereby reducing the blood sample
, viscosity to within a range of from about 3.0 to 5Ø
The particular diluent used is not critical; any substan-
tially isotonic diluent suitable for whole blood would be
satisactory. Examples of suitable diluents are: (1) saline
solutions in water containing from about 0.85 to 0.90 percent
by weight NaCl, which is isotonic with blood; ~2) saline
solution containing about 0.80 percent by weight NaCl, which is
slightly hypotonic with respect to blood; and (3) buffered
'~ isotonic, saline solution (eg. 0.85~ NaCl) containing a phosphate
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buf~er to adjust the pH to within a ranye of ~rom 6.0 to 7.5,
however, the preferred pH is 7.4. Any of these diluents may
also contain small quantities of other substances such as
sodium azide, sodium benzoate, and glycerin which are added to
stabilize or preserve the diluting solution without adversely
affecting the distribution or morphologies of blood cells in a
blood sample diluted therewith or in a smear made from a
diluted blood sample.
In accordance with the method o~ this invention, a
quantity of the blood sample, diluted as described above, is
placed on a suitable transparent support, normally a conven-
; tional microscope slide, and is spun by placing it in the chuck
of a centrifugal type blood spinner such as a Coleman Model 90
Uni-Smear spinner or the spinner described in U.S. Patent No.
~,016,828 filed on March 22, 1976 and entitled "Method and
Apparatus for slood Film Preparation". The slide blank is
spun in the plane of its sample bearin~ surface by accelerating
the chuck at a preselected rate to a preselected maximum
spinning velocity which is maintained for a given period time
and the slide is thereafter decelerated to a stop. The accel-
eration, spinning velocity and duration are selected for achieving
; good cell separation an~d substantially uniform cell distribution
while preserving cell structure and shape. Optimum acceler-
ation for blood samples diluted to one part diluent to two
parts whole venous blood is within the range of from 30,000 to
60,000 RP~l per second to a maximum velocity within the range
of from 3,200 to 3,800 RPM which is maintained for a period
in the range from 0.4 to 0.6 seconds after which the power is
~: turned off and the chuck is decelerated to a stop either by
allowing it to coast to or by braking to a stop. Deceleration
is not particularly critical, however, it should be done
quickly enough to avoid drying of the blood smear.
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Optimum acceleration, speed and duration for o-ther blood sample
dilutions are somewhat different than those described above
for 1:2 diluent to blood samples.
The acceleration, velocity and duration of the spinning
may be set to a preset value as a means to optimize particular
aspects of the blood smear. For example, it is desirable to
enhance the separation of the cells without distorting cell
shape and structure. It is also desirable to produce a blood
smear with a monolayer of cells. In general, optimum smears
suitable for analysis by automated pattern recognition techniques
utilizing optical scanning and d~ta processing are provided
by dispensing approximately 200,ul of venous blood, which has
been diluted by 1 part buffered isotonic saline to 2 parts
whole blood, onto a slide and spinning the slide with an
- acceleration of about 45,000 RPM per second, to a maximum velocity ;~
of about 3,500 RPM which is maintained for about 0.5 seconds.
Thereafter, the spinner is quickly stopped.
In addition to being particularly suited for automated
analysis, blood smear slides prepared by the foregoing method
may also be analy2ed by conventional manual microscopic exam-
~nation.
Additionally, it will ~e appreciated that since the method
of this invention utilizes a dilution proportion that preferably
is the same for each sample, the dilution step, as well as
the deposition of diluted sample blood on the slide blank, are
also particularly suited to being carried out by automated
mechanisms.
It has been found, as well, that the method of the invention
enhances the capability to do ~uantitative estimates of platelet
^ 30 sufficiency, red blood cell and white blood cell count, and
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; hematocrit from a smear.
As previously mentioned, the method described above is
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particularly suited for preparing blood smear slides of good
quality from samples of blood having a hematocrit value in a
range of from 15 to 45. If a particular blood sample has a
hematocrit value in excess of 45, the method is simply
modified to the extent of merely increasing the proportion of
diluent until the hematocrit is about 45; then the steps of
the method as described above are repeated and the smear slide
produced will be a like ~uality, suitable for automated
analysis.
10 From the above description, it is clear that the objects
of the invention have been achieved. Those skilled in the art,
however, will realize the above description relates to a
preferred method exemplary of the invention and that modifi-
cations can be made thereto without departing fromsthe spirit
and scope of the invention as defined by the claims.
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