JPS6229735B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and apparatus for preparing blood cell samples for diagnostic analysis. More particularly, the present invention relates to techniques for fixing or preserving cell morphology for automated analysis.

The present invention realizes a technology that fixes cells during precision sample preparation and preserves their morphology for later (automated) analysis. This is an attempt to eliminate deformation, which often occurs when samples are prepared. Although the invention is not limited to particular cells or samples, it will be described below in connection with the preparation of red blood cells for rapid automated analysis. One of the previously known variations in the conventional preparation of microscope slides is that the morphological changes in the red blood cells, especially in some blood cells, the central thinned and concave cell area disappears, resulting in similar This means that cells become spherical cells with similar sphericity in diameter or shape.

The various diagnoses of anemia are improved by more accurate classification of red blood cell size and red blood cell parameters such as mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and Wintrobe index. Currently, these data are 1
It is obtained using a blood cell counter that measures the number of red blood cells per cubic centimeter, hemoglobin concentration, and mean blood cell volume using a fluid flow system. In addition to this data, accurately analyze the types of cells present in the sample according to hematological classifications such as normal blood cells, macrocytes, target blood cells, small red blood cells, acicular red blood cells, hypochromic long blood cells, red blood cells, etc. That is beneficial. For a method and apparatus for classifying individual cells as described above, i.e., dividing them into subpopulations and providing red blood cell parameters for each subpopulation, U.S. Pat.
No. 4097845.

The most traditional method of preparing microscope slides for manual examination under a microscope involves placing a predetermined amount of blood on a glass slide and then placing a second slide on top of this slide. There is a wedge slide method in which a thin layer of blood is created on the surface of the first glass slide by wiping. After this thin layer dries for a short time, the slide is immersed in stain and then viewed under a microscope to visually identify and analyze the red blood cells. Not only is this time consuming, but the physical action of wiping the slide may distort the morphology of many blood cells.
Furthermore, even under the best conditions, only a portion of the slide surface is suitable for analysis.
The wedge method for producing such deformations is unsuitable for automated red blood cell analysis.

Regarding white blood cell measurements, a rotation method has been developed to form a monolayer of blood cells on the slide surface and then automatically analyze the white blood cells. In this method, a slide is rotated and blood cells are placed on top of the slide, so that excess blood cells are thrown away from the slide surface. Surface tension and other forces remain in this single blood layer on the sliding surface. In the case of autologous blood cells,
The blood cells in this single layer were allowed to dry and then stained with Wright's stain (or others), as described in the U.S. patent.
Subject to automatic analysis etc. disclosed in No. 3883852.

Drying white blood cells does not cause much deformation from the point of view of white blood cell analysis, but when drying red blood cells,
It has been found that upon drying, many of the red blood cells develop undesirable deformities such as loss of their central pallor. Although the fundamental cause of these shape changes is still unclear, it is thought to be due to surface tension, electrical charges, and drying effects.

The rotation method is preferred because it allows the red blood cells to form a single layer on the slide surface in a way that makes it difficult for them to interact with each other. If the slide is immersed in a fixative or staining solution before the blood cells are dried, the red blood cells will be washed off the slide surface. If the red blood cells are fixed in the liquid before they are deposited on the slide surface, a single layer of separated blood cells cannot be created because the fixation causes them to clump together. In this way, fixation of blood cells needs to be carried out within a limited time after monolayer formation and before drying.

Therefore, the present invention provides a method that fixes the morphology of blood cells on the slide surface after the formation of a single layer of blood cells but before the blood cells dry on the slide surface, thereby making it possible to identify various characteristics in subsequent blood cell analysis. The purpose is to

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

According to the invention, a predetermined amount of blood sample is placed on a microscope slide and then rotated by a centrifugal rotator. This rotational action of the slide springs all but a single layer of blood from the slide. Before the red blood cells become deformed by drying, a suitable fixative is applied to the monolayer of the sample to preserve the shape of the red blood cells, so that the shape of the central pallidum remains as drying progresses. It remains unchanged and does not cause any adverse changes in blood cell morphology.

Referring to FIG. 3, a centrifugal rotating machine 10 includes a housing 12 in which a plate body 16 rotatably disposed at the end of a rotating shaft 18 is housed in a small chamber 14 that opens upward.
It is shown. The plate 16 receives the slide 20 on its horizontal upper surface and rotates the slide in the horizontal plane. The slide 20 is removably held by suitable means such as a stop 22 spaced apart from the plate surface for receiving the slide. The rotating shaft 18 is driven by a motor (not shown) with a rotational speed and rotation time controlled by conventional means known in the art. Suitable rotating devices that are commercially available are disclosed in US Pat. No. 3,853,092 and US Pat.

According to an embodiment of the invention, the top-opening member of the chamber of the rotating device is made closable by means of a covering hood 24 adapted to rest on the housing 12. In the illustrated embodiment, the hood 24 is connected to the pipe 2.
6 communicates with a source of volatile fixative, such as a canister 28 containing fixative. A valve device 30 installed intermediate the ends of the pipe 26
regulates the flow of steam between the source and the chamber 14 of the rotary machine. Here, the valve 30 comprises a semicircular valve plate 31 centrally pivoted by a pin 33 fixed on the cover hood. This valve plate is pivotally mounted, and when the upright knob pin 35 is swung in an arc with a finger, the outlet of the pipe 26 is opened and closed. In order to force the flow of steam into the chamber, the pressure inside the canister is made higher than the pressure inside the rotating chamber by a fan 32 installed in this canister 28 or an air pump 34 supplying air to the canister. Pressure is maintained.

As mentioned above, in preparing a blood sample for analysis, a predetermined amount of blood sample 36 is placed on the slide 20.
It is then placed on the plate 16 of the rotary machine in the chamber 14. The slide is rotated at a predetermined speed for the time necessary to produce an evenly distributed monolayer of red blood cells on its surface. Usually this rotational speed is kept constant and a speed of 4000 to 10000 rpm actually produces a satisfactory monolayer in a short time, e.g. 1 to 2.5 seconds.

"The Journal of Histochemistry and Cytochemistry" 1974, Vol. 22, No. 7, 506-516
A blood sample is diluted to a plasma viscosity and the sample is then heated to a constant concentration, as shown in a paper entitled "Erythrocyte Morphology and Preparation of Thin Blood Films by Centrifugal Rotation" by James W. Bacus on p. If rotated at a constant speed for the rotation time, a single layer of separated blood cells will be produced and the central pallidum will be beautifully preserved.
For example, a well-mixed whole blood sample is diluted with a serum albumin solution with a relative viscosity of 1.3 to adjust the hematocrit of each sample to 18%. This viscosity is
The relative viscosity of plasma is usually in the range of 1.2-1.8 compared to H ₂ O of 1.0. on the other hand,
Isotonic or 1:1 normal saline solutions are also used as diluents for most blood. If the rotation time is too short, the blood cells tend to form clumps and solidify, making it impossible to differentiate and classify them individually. On the other hand, if the rotation time is too long, the shape of the blood cells will be undesirably deformed.

Rather than using a constant rotation time to control plasma viscosity, it is preferable to adjust the rotation time of the rotation device. For this, US Patent No. 3827805
This method uses a light beam to pass through the rotating slide and sample. It measures the scattering rate of blood cells with respect to the light beam and stops rotating when a predetermined blood cell distribution is reached. Another device for controlling this rotation time is the one in US Pat. No. 3,906,890 that measures the approximate blood cell concentration in the sample and sets the rotation accordingly. The present invention may use any of these methods or may use other methods.

It is desirable that the monolayer of red blood cells on the surface of the slide be fixed and dried to the extent that the slide can be handled mechanically without disruption or dislocation of the blood cells. Typically, a single layer of blood on the slide surface dries with air within 10-15 seconds after stopping rotation;
This time varies depending on conditions such as humidity. Previously, drying a single layer of blood as described above resulted in severe distortion and deformation of the red blood cell shape, making it difficult to identify blood cells in the central pallidum, or leading to encountering blood cells that had never been seen before. There was a problem. For example, the red blood cells 40, 42 are shown as solid lines in FIG. 1 and arrows are shown as being fixed with a fixative in FIG. 2 to prevent loss of the central pallidum as the blood cells dry. Before this drying, blood cells 40.4
If the blood cells 2 are not fixed, the blood cells will become flattened as shown by flattened shapes 40a and 42a as shown by dotted lines in FIG. These central pallor areas,
Flattened blood cells 42a, 40a whose regions have disappeared or have been almost deformed do not have a central pallor as shown in FIG. 1, making it difficult to distinguish them from corpuscles 46. This is especially true with automated blood analyzers.

According to the present invention, the red blood cells are properly dispersed to form a single layer, but before the drying process has progressed to the point where the cell morphology is indistinguishable, the red blood cells are removed during or immediately after the rotation. It was found that fixation allowed for proper drying without causing any change in blood cell morphology. Such fixation fixes the physical shape of the blood cells, thereby allowing single layer drying to be accomplished without the cell deformations that are difficult to avoid in the prior art. In a preferred embodiment, the fixing step may be introduced after the monolayer is complete and the rotational forces have been removed, so that fixation occurs before excessive drying occurs. This is achieved according to the invention by introducing a fixative in the form of vapor into the chamber in which the slide is rotated, thereby avoiding any adverse effects on the blood sample. The fixation step described above effectively fixes the blood cell morphology.

It has been found that fixing before applying rotation can prevent blood cells from dispersing within the desired single layer, but when a rotational force is subsequently applied, blood cells tend to aggregate or collect in streaks. Similarly, it has been found that introducing a liquid fixative onto the slide surface after rotation and before drying destroys blood cell dispersion as early as the time when the blood sample is washed from the rotating slide.

Formaldehyde, which is gaseous at room temperature, is effective as a fixative. In the above-described apparatus, formalin (an aqueous solution of 37 to 50% by weight formaldehyde and about 15% by weight methanol) is stored in the canister 28. This formaldehyde escapes from the solution and passes through pipe 26 to rotating machine 10.
Move to small room 14. Alternatively, the slide 20 carrying the monolayer of blood may be quickly removed from the chamber of the rotating machine and placed in a gaseous formaldehyde atmosphere. In this case, if fixation is to be carried out before the problem of drying occurs, it is somewhat difficult in that it must be carried out within the short time required for transfer, that is, within about 5 seconds. In addition, the "self-contained" system of the embodiments described above is advantageous in regulating gaseous formaldehyde, which has hazardous concentration levels above 5 ppm. Favorable fixation of red blood cells is found by exposing the monolayer to an atmosphere of gaseous formaldehyde for about 5 minutes.

When the blood sample is rotated, most of the sample is thrown off the slide towards the inner wall of the rotator housing 12. This blood contains various harmful organic substances, so depending on the rotating machine, the housing 1
By applying running water to the inner wall surface of the chamber 2, it acts as a safe seal to prevent such organic matter from escaping from the chamber. In this way, the scattered blood is washed away and collected at the bottom of the rotating machine. In the present invention, formalin can also be used as the flushing water. As a result,
Formaldehyde vapor will be generated in the rotating machine at the beginning of rotation. The formation of a monolayer occurs quickly enough with this rotation, i.e. within about 0.5 seconds, that blood cells can be fixed until dispersed in a monolayer even though gaseous formaldehyde is initially present in the chamber. will not be carried out.

After fixation and drying of the blood sample, the slide 20 is removed from the rotator or fixation chamber and analyzed by automated equipment. Since the red blood cells 40 and 42 fixed as described above retain their characteristic central pallor 44 and other physical properties, the samples prepared as described above can be analyzed using an automated analyzer. This enables accurate measurement instructions based on the true morphology of blood cells.

Although formaldehyde is mentioned as a fixing agent, short-chain alcohols such as methyl alcohol and ethyl alcohol may be used instead of formaldehyde, or fixation may be performed using heating using microwave energy or other physical or chemical means. It is possible.

[Brief explanation of the drawing]

Figure 1 is an enlarged schematic plan view of a single layer of red blood cells on the surface of a microscope slide, with and without a central pallor; Figure 2 is a side view of the blood cells on the slide shown in Figure 1; The figure shows a perspective view of the device according to the invention in an open state, and FIG. 4 shows a perspective view of the device in a closed state. 36... Blood sample, 16... Plate member, 14...
...Rotation chamber, 40, 42...Red blood cells, 20...
Support (slide), 10...Rotating machine, 30...
Valve, 24...Hood, 12...Housing, 26
……pipe.

Claims (1)

[Scope of Claims] 1. A method for preparing a blood sample having a monolayer of wetted blood cells dispersed on a support surface for automatic analysis, the method comprising: a brief rotation in a closed chamber to scatter excess blood cells radially outwardly from the surface of the support to produce a well and evenly distributed monolayer of blood cells; applying a fixative to a monolayer of blood cells on the surface of the support within a certain period of time before the wet blood cells dry, thereby fixing the unique morphology of the blood cells before the wet blood cells flatten due to drying; A method for preparing a blood sample, comprising the step of drying the thus fixed blood cells in preparation for automatic analysis of the blood sample. 2. In the method described in claim 1,
generating a gaseous fixative and applying the gaseous fixative to a sample of red blood cells dispersed in a single layer to fix the central pallor of the red blood cells and determine their specific morphology and size; A method for preparing a blood sample, comprising the step of retaining it. 3. In the method described in claim 2,
A method for preparing a blood sample, characterized in that the gaseous fixative is gaseous formaldehyde. 4. In the method described in claim 1,
removing the support bearing the monolayer of blood cells from the chamber after the rotation and placing the support in the fixation chamber for the period of time before the monolayer of blood dries; Characteristic blood sample preparation method. 5 a housing defining a rotation chamber; a rotatable plate member disposed substantially horizontally within the rotation chamber for receiving a support carrying moist blood cells; means for controlling the driving time of said plate member to deposit said plate member as a substantially single layer on said support surface; and an external source of fixative for introducing fixative to the wet blood cells on said support surface; and pipe means for transferring gaseous fixative from said external source.