CA1087970A - Method of preparing blood smear slides - Google Patents
Method of preparing blood smear slidesInfo
- Publication number
- CA1087970A CA1087970A CA290,401A CA290401A CA1087970A CA 1087970 A CA1087970 A CA 1087970A CA 290401 A CA290401 A CA 290401A CA 1087970 A CA1087970 A CA 1087970A
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- CA
- Canada
- Prior art keywords
- blood
- diluent
- venous blood
- slide
- sample
- Prior art date
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
- G01N2001/2846—Cytocentrifuge method
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A method for preparing blood smear slides with substantially uniform separation of the blood cells from venous blood samples of different hematologic properties wherein the venous blood is diluted by mixing it with a substantially isotonic diluent in a proportion ranging from about one part blood to one part diluent, to four parts blood to one part diluent. A quantity of diluted blood is deposited on a slide which is then placed in a slide spinner operative to spin the slide a preselected acceleration, speed and time to produce a monolayer of blood cells on the slide. The method is adapted for preparing blood smears by automation regardless of the hematologic properties of the venous blood samples and the smears produced are especially suited for examination by automated scanning and analysis techniques. The uniformity and quality of these smears also facilitates fast accurate examination by conventional and manual microscopy.
A method for preparing blood smear slides with substantially uniform separation of the blood cells from venous blood samples of different hematologic properties wherein the venous blood is diluted by mixing it with a substantially isotonic diluent in a proportion ranging from about one part blood to one part diluent, to four parts blood to one part diluent. A quantity of diluted blood is deposited on a slide which is then placed in a slide spinner operative to spin the slide a preselected acceleration, speed and time to produce a monolayer of blood cells on the slide. The method is adapted for preparing blood smears by automation regardless of the hematologic properties of the venous blood samples and the smears produced are especially suited for examination by automated scanning and analysis techniques. The uniformity and quality of these smears also facilitates fast accurate examination by conventional and manual microscopy.
Description
~ q~
' ~
BACKGROUND OF TIIE INVENTION
_ _ _____ 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 ~ .
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, ~`' . '` ~.
.
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 ,. .
~ R.E. Wenk, American Journal of Medical Technology 42, 71-58, 1976.) , '-:
, ~ 7~1 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 ~ o 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 ~ t7~
` 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 ` 5 _ , ' ' ,::
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 ~ .
. .
. .
` :
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.
_7_ r~ ~ r~glt~O
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 ; . .
; hematocrit from a smear.
As previously mentioned, the method described above is . .
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.
;'`
``' :
' ' .
:, 9 . : , . : . , .~.
.. . . .
' ~
BACKGROUND OF TIIE INVENTION
_ _ _____ 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 ~ .
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, ~`' . '` ~.
.
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 ,. .
~ R.E. Wenk, American Journal of Medical Technology 42, 71-58, 1976.) , '-:
, ~ 7~1 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 ~ o 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 ~ t7~
` 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 ` 5 _ , ' ' ,::
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 ~ .
. .
. .
` :
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.
_7_ r~ ~ r~glt~O
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 ; . .
; hematocrit from a smear.
As previously mentioned, the method described above is . .
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.
;'`
``' :
' ' .
:, 9 . : , . : . , .~.
.. . . .
Claims (18)
1. A method for preparing a blood smear slide from a sample of venous blood including the steps of:
diluting the venous blood sample with a substantially isotonic diluent wherein the ratio of diluent to blood is in the range between one part diluent to one part venous blood and one part diluent to four parts blood;
placing a quantity of diluted venous blood on a viewing surface of a slide;
spinning the slide about an axis disposed perpendicularly to the viewing surface to form a monolayer of blood cells on the viewing surface, the slide being accelerated to a fixed rotary speed and spun at that fixed rotary speed for a fixed time, the time and rotary speed being arbitrarily selected without regard to characteristics of the particular sample of venous blood for which a blood smear slide is being prepared.
diluting the venous blood sample with a substantially isotonic diluent wherein the ratio of diluent to blood is in the range between one part diluent to one part venous blood and one part diluent to four parts blood;
placing a quantity of diluted venous blood on a viewing surface of a slide;
spinning the slide about an axis disposed perpendicularly to the viewing surface to form a monolayer of blood cells on the viewing surface, the slide being accelerated to a fixed rotary speed and spun at that fixed rotary speed for a fixed time, the time and rotary speed being arbitrarily selected without regard to characteristics of the particular sample of venous blood for which a blood smear slide is being prepared.
2. The method of claim 1 wherein the volume of diluted venous blood placed on the slide viewing surface is approximately 200 µl.
3. The method of claim 1 wherein the ratio of diluent to venous blood in the diluted venous blood is one part diluent to two parts venous blood.
4. The method of claim 2 wherein the ratio of diluent to venous blood in the diluted venous blood is one part diluent to two parts venous blood.
5. The method of claim 1 wherein the diluent comprises a saline solution in water containing between 0.85 and 0.90 per-cent NaCl by weight.
6. The method of claim 4 wherein the diluent comprises a saline solution in water containing between 0.85 and 0.90 percent NaCl by weight.
7. The method of claim 1 wherein the diluent comprises a buffered saline solution containing approximately 0.85 percent NaCl by weight with a phosphate buffer to adjust the Ph to be within a range of from 6.0 to 7.5.
8. The method of claim 4 wherein the diluent comprises a buffered saline solution containing approximately 0.85 percent NaCl by weight with a phosphate buffer to adjust the pH to be within a range of from 6.0 to 7.5.
9. The method of claim 1 wherein the rate of acceleration of the slide is approximately 45,000 RPM per second, the rotational speed is approximately 3500 RPM and the time the slide is maintained at 3200 RPM for substantially 0.5 seconds.
10. A method for preparing a blood smear slide from a sample of venous blood including the steps of placing a quantity of blood on a viewing surface of a slide, spinning the slide about an axis disposed transversely to the slide viewing surface at a fixed rotary speed for a fixed time to form a monolayer of blood cells on the viewing surface, wherein the improvement comprises:
diluting the venous blood sample with a diluent wherein the ratio of diluent to venous blood is in the range between one part diluent to one part venous blood to one part diluent to four parts venous blood.
diluting the venous blood sample with a diluent wherein the ratio of diluent to venous blood is in the range between one part diluent to one part venous blood to one part diluent to four parts venous blood.
11. The method of claim 10 wherein the diluting step comprises diluting venous blood with a diluent in the ratio of one part diluent to two parts venous blood.
12. The method of claim 10 wherein the diluent comprises a saline solution in water containing 0.85 to 0.90 percent NaCl by weight.
13. The method of claim 10 wherein the diluent is substan-tially isotonic.
14. The method of claim 11 wherein the diluent is substan-tially isotonic.
15. The method of claim 10 wherein the diluent comprises a buffered isotonic saline solution containing approximately 0.85 percent NaCl by weight with a phosphate buffer to adjust the pH to be within a range from 6.0 to 7.5.
16. The method of claim 1 additionally including the step of first diluting the sample of venous blood with an isotonic diluent until the resulting diluted venous blood sample has a hematocrit in the range between 15 and 45 and thereafter using the resulting diluted venous blood sample as the venous blood sample in the diluting step.
17. The method of claim 10 additionally including the steps of first diluting the venous blood if the hematocrit is greater than 45 until the resulting blood has a hematocrit in the range from between 15 and 45 and using the resulting blood as the venous blood sample.
18. A method for making a plurality of blood smear slides from a plurality of venous blood samples including the steps of:
1) analyzing each venous blood sample to determine its hematocrit content;
2) diluting each blood sample having a hematocrit of greater than 50 with an isotonic diluent until the resulting blood sample has a hematocrit in the range of between 15 and 45;
3) further diluting each blood sample with a hematocrit in the range between 15 and 50 with an isotonic diluent wherein the ratio of blood having hematocrit in the range of between 15 and 50 to diluent is in the range between one part diluent to one part blood and one part diluent and four parts blood;
4) placing a quantity of further diluted blood on the viewing surface of a slide; and 5) spinning the slide about an axis disposed perpendicularly to the viewing surface to form a monolayer of blood cells on the surface, the slide being accelerated to a fixed rotary speed and maintained thereat for a constant time duration, the rotary speed and time duration being arbitrarily selected without regard to any characteristic of any particular sample of venous blood for which a blood smear is prepared.
1) analyzing each venous blood sample to determine its hematocrit content;
2) diluting each blood sample having a hematocrit of greater than 50 with an isotonic diluent until the resulting blood sample has a hematocrit in the range of between 15 and 45;
3) further diluting each blood sample with a hematocrit in the range between 15 and 50 with an isotonic diluent wherein the ratio of blood having hematocrit in the range of between 15 and 50 to diluent is in the range between one part diluent to one part blood and one part diluent and four parts blood;
4) placing a quantity of further diluted blood on the viewing surface of a slide; and 5) spinning the slide about an axis disposed perpendicularly to the viewing surface to form a monolayer of blood cells on the surface, the slide being accelerated to a fixed rotary speed and maintained thereat for a constant time duration, the rotary speed and time duration being arbitrarily selected without regard to any characteristic of any particular sample of venous blood for which a blood smear is prepared.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US74139176A | 1976-11-12 | 1976-11-12 | |
| US741,391 | 1985-06-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1087970A true CA1087970A (en) | 1980-10-21 |
Family
ID=24980553
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA290,401A Expired CA1087970A (en) | 1976-11-12 | 1977-11-08 | Method of preparing blood smear slides |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS5387289A (en) |
| CA (1) | CA1087970A (en) |
| DE (1) | DE2750885A1 (en) |
| FR (1) | FR2370970A1 (en) |
| GB (1) | GB1589935A (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3577267A (en) * | 1968-03-19 | 1971-05-04 | Us Health Education & Welfare | Method of preparing blood smears |
-
1977
- 1977-11-08 CA CA290,401A patent/CA1087970A/en not_active Expired
- 1977-11-10 FR FR7734025A patent/FR2370970A1/en active Granted
- 1977-11-11 JP JP13558577A patent/JPS5387289A/en active Pending
- 1977-11-11 GB GB4710477A patent/GB1589935A/en not_active Expired
- 1977-11-14 DE DE19772750885 patent/DE2750885A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| FR2370970A1 (en) | 1978-06-09 |
| DE2750885A1 (en) | 1978-05-18 |
| JPS5387289A (en) | 1978-08-01 |
| FR2370970B1 (en) | 1984-03-16 |
| GB1589935A (en) | 1981-05-20 |
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Effective date: 19971021 |