US 3577267 A
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K. PRESTON, JR., ET AL 3,577,267 METHOD OF PREPA RING BLOOD SMEARS May 4, 1971' Filed March 19, 1968 5 w m? M T .r w m m r mfl A Z v aw 1 United States Patent 3,577,267 METHOD OF PREPARING BLOOD SMEARS Kendall Preston, Jr., New Haven, Conn., and Philip E. Norgren, Boulder, Colo., assignors to the United States of America as represented by the Secretary of Health, Education, and Welfare Filed Mar. 19, 1968, Ser. No. 714,279 Int. Cl. B44d 1/02 US. Cl. 117-101 2 Claims ABSTRACT OF THE DISCLOSURE A method of producing blood smears which includes depositing a quantity of blood on a surface, preferably glass, and rotating the surface about a central, perpendicular axis at a high speed for a short time. Preferably, the surface is accelerated within less than ten milliseconds to a speed in the range of from 4,000 to 10,000 r.p.m. The surface is maintained at this speed for a very short time, on the order of a few tenths of a second. The result is a uniform monolayer of flattened, undamaged blood cells.
This invention relates to the field of medical diagnosis and particularly relates to the preparation of blood samples for analysis.
The preparation of blood samples for diagnostic purposes is a common procedure. Information as to the differential white cell type count and the cell structure is of vital importance in the diagnosis and treatment of many conditions. The present methods of preparing blood samples, commonly called blood smears, involves depositing a drop of blood on a glass plate and then wiping the drop to spread it over the surface, producing a thin layer or smear of blood. However, despite the simplicity of this procedure, the technique of performing it properly is actually quite difficult if a good quality layer is to be produced. Even when carefully performed by an experienced person, the thickness of the smear and the differential distribution of cells varies greatly from point to point across the smear. Furthermore, although some of the initially spherical cells are flattened to a shape which is more easily viewed in a microscope by this technique, the degree of flattening which occurs cannot be controlled and is often not sufficient. Finally, in the process of wiping the drop, many cells are ruptured and the pieces of these cells which are present in the smear increase the difficulty of performing an accurate differential count. In fact, differential cell type counts made of different portions of a smear prepared with utmost care by an acknowledged expert have shown that there is a wide variation in the differential count, depending on where it is taken in the smear. Clearly, in the usual case, a hospital technician with a somewhat lower degree of expertise forms the smear somewhat less carefully and thus produces a smear from which it is even more difiicult to achieve consistent results. Since the differential cell type count is of critical importance in the diagnosis and treatment of many conditions, it is highly desirable that an improved method of preparing blood smears be found.
Accordingly, it is an object of this invention to provide a new and improved method of preparing blood smears.
Another object of this invention is the provision of a new and simplified method of forming a thin layer of blood for analysis.
It is also an object of this invention to provide a novel method of preparing blood smears which produces substantially improved results.
A further object of this invention is the provision of a novel method of preparing blood smears which produces a uniformed monolayer of flattened undamaged cells.
Briefly, in accord with one embodiment of this invention, the novel method includes depositing a quantity of blood on a surface, preferably glass, and rotating the surface about a central, perpendicular axis at a high speed for a short time. Preferably, the surface is accelerated within less than ten milliseconds to a speed in the range of from 4,000 to 10,000 r.p.m. The surface is maintained at this speed for a very short time, on the order of a few tenths of a second. The result is a uniform monolayer of flattened, undamaged blood cells.
The novel features believed characteristic of the invention are set forth in the appended claims. The invention itself, together with further objects and advantages thereof, may best be understood by reference to the following description taken in connection with the appended drawing, which is a perspective view of apparatus suitable for performing the method of this invention.
The illustrated apparatus comprises a motor 1 connected to a suitable power source 2 and having a hollow output shaft 3. The shaft drives a flywheel 4 and is coupled through clutch 5 to a shaft 6 which drives a chuck 7. The shaft 6 may conveniently be located within shaft 3 and mounted so that it rotates only when clutch 5 is engaged. One end of the shaft 6 is also hollow so that means such as vacuum line 8 and stationary bushing 9 having ports 10 provide a vacuum for retaining a plate 11 on the chuck. A quantity of blood 12 is deposited on the plate 11 and a shield 13 is provided to prevent spattering of spunoff blood. Normally, the plate 11 is a small glass member commonly called a cover slip which, in the conventional method, is used to spread the blood on a microscope slide and then is cemented over it to preserve it.
T 0 use this device, an operator deposits the blood sample on substantially the entire surface of a carefully cleaned cover slip and mounts the slip on the chuck 7. After the motor has brought the flywheel up to the selected speed, the clutch lever 14 is depressed, engaging the clutch. Due to the flywheel, the plate 11 is accelerated to the speed of the motor within a few milliseconds. The clutch is disengaged almost immediately thereafter, since the smear is done within a few tenths of a second.
Preferably, the spinning technique of this invention is performed by accelerating the plate and blood sample to a speed on the order of a few thousand to several thousand r.p.m. within a few milliseconds and maintained at that speed for a time in the range of from one-tenth to a few tenths of a second. Specifically, the preferred speed range is from 4,000 to 10,000 r.p.m., to which the plate is accelerated within less than 10 milliseconds, and at which it is maintained from about 0.1 to about 0.5. of a second. Speeds below this range do not produce the improvements in smear quality achieved above 4,000 r.p.m., while high speeds tend to damage the cells.
After spinning, the cover slip is removed from the chuck, inverted and attached to a microscope slide. The slide is then analyzed in the conventional manner.
The method of this invention, whether performed by the device described above or by other suitable apparatus, is very simple but has been found to be surprisingly effective in that the results obtained are substantially improved in all respects over those obtained with any previous technique. In contrast to previous 'blood smears, the cell distribution over the entire cover slip is substantially constant; even the variation which might be expected between the center and the edges on the basis of force considerations does not occur. This feature is very important if accurate differential cell counts are to be made. Furthermore, the layer is completely monocellular, since all cells above those actually in contact with the plate are spun off.
It has also been determined that the cells in contact with the plate are flattened by the spinning force. This is a highly desirable result since it spreads the nucleus over a broad area so that it can be more easily observed in a microscope. Specifically, the White blood cells before deposition are approximately spherical and have a diameter on the order of microns. By previous techniques, an expert performing the smearing carefully is able to flatten some of the white cells so that these become flat discs about -15 microns in diameter. When the method of the present invention is performed, it has been found that essentially all white blood cells on the plate are flattened into discs which are significantly larger in diameter. Thus, the number and the size of flattened cells are both increased. This increases both the ease and the accuracy of microscopic examination.
Another advantage provided by the method of this invention as compared to prior methods lies in the fact that very few cells are broken by the spinning force. In prior methods, many more cells would rupture due to the pressure of the wiping edge, leaving broken pieces called artifacts in the smear. The loss of such cells causes errors in the differential cell type count. The method of this invention essentially eliminates this cause of error.
As is pointed out by the foregoing discussion, the method of the present invention is based on a simple, easily performed technique which overcomes several difiiculties of conventional methods and produces blood smears which are substantially improved in many significant ways. Since it is of critical importance that the blood smears used for differential cell type counting, structure analysis, etc. permit accurate results to be obtained, the layers prepared must be of the best possible quality. The present method produces blood smears which enable the analyst to study a monolayer of flattened, undamaged cells which is coextensive with the plate and therefore as large as can be viewed in the microscope.
A most important feature is that of uniformity of the layer so that, no matter where the count is taken, it is consistent. As an illustration of the degree of improvement achieved, a recent analysis indicates that the differential cell-type count performed at different locations in conventionally prepared smears varies with location by 25% to 40%. In contrast, counts performed at different locations in smears prepared by the method of this invention have been shown to vary by less than 5% It has been assumed in the above statement of preferred ranges that the plate used is the cover slip conventionally used with microscope slides; this is a very thin (0.1 mm.) glass plate on the order of 2 cm. square. If the plate size is changed substantially from this order of magnitude, some adjustment of the time and r.p.m. may be required 4. if the quality of the smear is to be maintained over the entire plate.
It has also been assumed that conventional silica-based glass is used as the material of the cover slip. Other materials such as transparent plastic may also be used provided that there is a suificient differential between the adherence of the bottom layer to the plate and the adherence of the blood layers to each other to permit the upper layers to be spun off without removing the bottom layer. The conventional glass cover slips are preferred because this condition is particularly well satisfied as well as because of their ready availability.
While we have illustrated and described a particular embodiment of our invention, it will be understood that various modifications may be made therein without departing from the spirit of the invention and the scope of the appended claims.
1. A method of producing a thin layer of blood for use in analysis thereof which comprises the steps of providing a substantially horizontal surface, depositing a quantity of blood on said surface, and spinning said surface about an axis substantially perpendicular thereto to remove substantially all of said blood except that in contact with said surface, said spinning being performed by quickly accelerating said surface to a speed in the range of from 4,000 to 10,000 rpm. and maintaining said speed from about 0.1 to about 0.5 of a second, and said surface being a material, such as silica based glass, having a sufiicient differential between the adherence of the bottom layer of cells of the blood to the plate and the adherence of the layers of blood cells to each other to permit the upper layers to be spun off Without removing the bottom layer.
2. The method claimed in claim 1 wherein said acceleration is performed within less than 10 milliseconds.
References Cited UNITED STATES PATENTS 2,744,034 5/1956 Dalton et a1. 1l7l0lX 3,352,280 11/1967 Hughes et a1. 118-52X 3,394,880 7/1968 Carter 424-1 1X ALFRED L. LEAVITT, Primary Examiner C. K. WEIFFENBACH, Assistant Examiner US. Cl. X.R,