JP2006071475A - Sample stand for blood observation, and separation quantitative evaluation method of blood cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a quantitative evaluation method of a blood cell separation state for evaluating quantitatively the separation/aggregation state of erythrocyte not only for pathologic diagnosis but also for initial examination of a health condition and a nutrition state, and also to provide a sample stand for blood observation to be used for the quantitative evaluation method. <P>SOLUTION: This sample stand for blood observation is equipped with: a flat glass substrate; a spacer thin film having the uniform thickness disposed on the glass substrate; and a cover glass placed thereon. The spacer thin film is equipped with: a maintenance means having a recessed part for storing a prescribed amount of blood; and an escape connected to the recessed part. The sample stand is also equipped with a maintaining means for maintaining the blood amount dropped into the recessed part to be always constant. The spacer thin film has the thickness of 2-9 μm, preferably the thickness of 5 μm (±10%). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention provides a method for quantitative evaluation of blood cell separation state for quantitatively evaluating the separation / aggregation state of red blood cells not only for pathological diagnosis but also for initial examination of health and nutritional state, and to this quantitative evaluation method. The present invention relates to a sample table for blood observation for use.

  Test methods for diagnosing various pathological conditions from blood cell images observed under a microscope using a blood smear stained specimen have been used in clinical practice from an early stage, and are effective in finding abnormal states and determining disease names. The preparation of blood smears for this examination required experience in pulling glass, etc., and it was inevitable that there were individual differences among the creators. In addition, after the blood was made into a thin layer, it was quickly dried to require a process such as fixation and staining.

  A blood smear-stained specimen used for conventional blood cell microscopic image analysis is produced by dropping blood on a glass substrate, drying it, and staining and fixing it, resulting in variations in blood thickness. In addition, according to the conventional blood smear stained specimen, “dead” blood is observed and various pathological conditions are diagnosed. Furthermore, in blood observation, blood cells are thick and overlapped, which is not suitable for performing observation with high accuracy.

  On the other hand, in recent years, there have been attempts to obtain information from a drop of fresh blood itself from the viewpoint of preventive medicine and health science. That is, it is intended to obtain various information on the nutritional status and health status of the subject from the blood cell image directly observed under an optical microscope without pretreatment such as staining or fixation. However, at present, most of the judgment criteria are qualitative, and the present condition depends on the subjectivity of the examiner. The simplicity of this method as a simple initial check has attracted attention, and methods that enable objective quantitative evaluation with reproducibility have been studied.

  In the blood cell microscopic image analysis of fresh blood, the most characteristic and frequently observed is the state of red blood cell separation / aggregation. Usually, erythrocytes are separated from each other because the erythrocyte surface is negatively charged and repels each other. However, it is said that when a neutrally charged substance with a large axial ratio binds to the erythrocyte surface, both ends of this molecule crosslink to the erythrocyte and the erythrocyte binds. In addition, it is said that saturated fats and abnormal proteins are involved as causative substances that bind red blood cells in fresh blood. From these, it is considered that the separation / aggregation state of red blood cells reflects one end of the physiological state.

  In order to evaluate the separation / aggregation state of erythrocytes, it is straightforward to obtain the ratio of the number of separated erythrocytes and aggregated erythrocytes. However, for analysis, it is difficult to accurately count the complicatedly overlapping agglomerated erythrocytes, and the measurement time becomes long.

  The present invention has been made in view of the conventional drawbacks, and the object of the present invention is to analyze the red blood cells that have been separated from the individually separated red blood cells by dynamically observing the live red blood cells. A method of quantifying the proportion, i.e., counting the separated red blood cells and clumped red blood cells present in the image, or quantitatively evaluating using the ratio of the total area of the separated red blood cells to the total red blood cell area present in the image, and The object is to provide a sample table for blood observation used for evaluation of this quantification method.

(1) A sample stage for blood observation according to the present invention comprises a flat glass substrate, a spacer thin film having a uniform thickness disposed on the glass substrate, and a cover glass placed thereon. The spacer thin film has a recess for storing a predetermined amount of blood and an escape passage connected to the recess, and has a maintaining means for constantly maintaining the amount of blood dripped in the recess. It is characterized by.
(2) In the sample table for blood observation of (1),
The spacer thin film has a thickness of 2 μm to 9 μm.
Further, the thickness is preferably 5 μm (± 10%).

(3) In the blood observation sample stage of (1) or (2),
The spacer thin film is formed by forming a metal thin film on a substrate by a plating technique.

(4) In the blood observation sample stage of (1) or (2),
The spacer thin film is formed by forming a non-metallic thin film on a substrate by a printing technique.

(5) The method for separating and quantitatively evaluating blood cells according to the present invention comprises observing blood using the blood observation sample stage according to any one of (1) to (4), and the total number of red blood cells present in the image. The ratio of the number of separated erythrocytes to is defined as the degree of erythrocyte separation, and using this value, the separation / aggregation state of erythrocytes is quantitatively evaluated using the following formula.
Red blood cell separation rate (%) = 100 × (total number of separated red blood cells) / (total number of separated red blood cells + total number of aggregated red blood cells)

(6) The method for separating and quantitatively evaluating blood cells according to the present invention comprises observing blood using the blood observation sample stage described in any of (1) to (4), and total red blood cells present in the image The ratio of the total area of separated erythrocytes to the area of erythrocytes is defined as the degree of erythrocyte separation, and using this value, the separation / aggregation state of erythrocytes is quantitatively evaluated using the following formula.
Red blood cell separation rate (%) = 100 × (total area of separated red blood cells) / (total area of separated red blood cells + total area of aggregated red blood cells)

  In contrast to dynamic observation of live blood, which has been attracting attention as a new method in recent years, we established a quantification standard for objective evaluation methods based on qualitative criteria based on the subject's subjectivity. Tried to do. By defining the red blood cell separation degree as a quantification standard, the separation / aggregation state of red blood cells could be quantified. In order to measure the degree of red blood cell separation with good reproducibility, a slide glass using a thin film as a spacer was invented to enable the preparation of a blood sample with a constant thickness. As a result, objective evaluation of the degree of red blood cell separation is possible in image processing of a blood cell microscopic image.

  As a result of investigating the possibility of detecting physiological changes using such an objective technique, it was found that the degree of red blood cell separation increases with ingestion of sports drink after fasting and fasting. Moreover, the tendency for the degree of red blood cell separation to decrease with sugar load was observed. Considering together with the results when pure water was ingested, it was determined that the change in red blood cell separation upon ingestion of sports drinks was accompanied by hydration. Thus, confirmation of the reliability and usefulness of fresh blood microscopic analysis for physiological state measurement was obtained.

  In order to accurately and quantitatively evaluate the degree of erythrocyte separation, it is necessary to establish conditions under which reproducible and constant results can be obtained. The principle of the blood observation sample stage prepared for this purpose is shown in FIG. FIG. 1 is a schematic view showing a cross-sectional view of a sample table for blood observation. In FIG. 1, reference numeral 10 indicates a glass substrate, which has a flat plate shape and a predetermined uniform thickness. A spacer thin film 12 having a constant thickness is fixedly disposed on the glass substrate 10, and a cover glass 14 is placed thereon for use. In FIG. 1, the spacer thin film 12 deposited on the glass substrate 10 is made of a metal thin film such as gold, silver, copper, platinum, rhodium, palladium, nickel, tin, or chromium by a printing technique, or a printing technique. Thus, a non-metal such as an epoxy resin or a fluorine resin is deposited in a predetermined pattern and a predetermined thickness.

  Here, the deposited thin film has a function of a spacer for maintaining the cover glass at a predetermined height. The thickness t of this spacer thin film is 2 μm to 9 μm, preferably 5 μm (± 10%). That is, when the thickness t of the spacer thin film is 2 μm or less, the red blood cells are crushed, making it difficult to distinguish the blood cell separation state and the aggregation state, and dynamic observation becomes impossible. Further, when the film thickness is 9 μm or more, blood cells are multilayered and are observed as if they were aggregated although they were originally separated. On the other hand, if the thickness t of the spacer thin film is 2 μm to 9 μm, preferably 5 μm (± 10%), the red blood cells are almost one layer, and the blood cells can move freely without being disturbed by each other. In addition, dynamic observation in which the separated state and the aggregated state are distinguished is possible.

  Further, the spacer thin film has a predetermined pattern shape as shown in FIG. That is, the pattern of the spacer thin film has a shape extracted in a substantially circular shape with a diameter d; about 10 mm at the center of the glass substrate. This portion forms a substantially circular recess 16 having a depth t = 2 μm to 9 μm, preferably 5 μm (± 10%), depending on the thickness of the film. A blood escape path 18 (flow path) is connected to and connected to the recess 16 so that blood dropped into the recess does not overflow on the thin film. In FIG. 2, the blood escape path 18 is provided at four predetermined positions of the recess 16. The four escape paths are formed in the longitudinal direction and the longitudinal direction of the glass substrate, respectively, at an angle of 90 degrees from the center of the circular recess 16. The red blood cell escape path 18 is not limited to the four predetermined positions of the above-mentioned recess, but may have various shapes for discharging blood so that the blood does not overflow on the thin film. Thus, the blood dropped into the recess 16 is always maintained at a constant thickness, and the blood sample for microscopic observation is made constant. Further, a convex portion 21 extending in the vertical direction of the substrate is provided on one end side of the glass substrate in order to prevent blood from flowing into the region 20 held by the hand.

  FIG. 3 shows another example of the spacer thin film pattern. In FIG. 3, two flow paths are provided as a blood escape from the circular recess 16. Each of the two escape paths is formed in the longitudinal direction of the glass substrate at an angle of 180 degrees from the center of the circular recess. Of course, the blood escape path 18 can be formed without being limited to four or two places from the predetermined position of the above-mentioned recess.

  Thus, when a blood sample was prepared with a spacer thin film having a thickness of 9 μm or more or 2 μm or less, erythrocytes overlapped thickly and many states were unsuitable for analysis, and blood did not spread well throughout the circular recess. It was. However, when the thickness of the spacer thin film is 2 μm to 9 μm, preferably 5 μm (± 10%), about 0.25 μl of blood is collected from the fingertip and dropped into the circular recess in the center of the glass substrate. It became possible to observe a stable blood sample in a state suitable for analysis.

  When this method was used to observe a plurality of blood samples collected in the same time zone, the blood thickness did not vary and a blood cell image having the same tendency could be observed with good reproducibility. Thereafter, various analyzes were performed using a spacer thin film having a thickness of 2 μm to 9 μm, preferably 5 μm (± 10%).

  When observing the separation / aggregation state of red blood cells with a microscope using the blood observation sample stage with a constant blood thickness as described above, the measurement area is divided as follows to separate and aggregate the red blood cells. The effect of non-uniformity was averaged. Specifically, as shown in FIG. 4, a 2.0 mm × 2.5 mm range at the center of the blood sample was divided into 0.5 mm increments for a total of 30 areas, and each was observed at a magnification of 1000 times. For one blood sample, the separated red blood cells and the aggregated red blood cells were discriminated by image analysis for these 30 region images. The degree of erythrocyte separation was determined from the number of each blood cell or the area of the erythrocytes, and the average of 30 regions was used as the degree of erythrocyte separation of the blood sample.

  In order to confirm the stability of red blood cell separation obtained in this way, the degree of dispersion of red blood cell separation in 30 regions in each blood sample was examined. As a result of measuring five times for each of 25 subjects, the coefficient of variation (standard deviation / average value) of the red blood cell separation degree was within about 30%, confirming that it was a relatively stable measurement parameter. In the subsequent analysis, the average value of the 30 regions was used as the representative value of the blood sample red blood cell separation degree.

  In order to examine the accuracy of this quantification method, 600 images were quantified by a blood cell number ratio and a hemocyte area ratio, and compared. The result is shown in FIG. As shown in FIG. 5, a high correlation was obtained with a correlation coefficient r = 0.97. From this result, it was confirmed that an equivalent quantitative evaluation was possible by the area ratio without counting the number of red blood cells, which is difficult to process.

  Thus, in the present invention, when the red blood cell count can be measured, the red blood cell separation ratio is used as the blood cell count ratio, and when the measurement is difficult, the red blood cell separation ratio is used as the red blood cell separation ratio.・ Evaluate the aggregation state. FIG. 6 shows an outline of the results of discrimination and area calculation of separated red blood cells and aggregated red blood cells present in the image.

Red blood cell separation rate (%) = 100 × (total number of separated red blood cells) / (total number of separated red blood cells + total number of aggregated red blood cells)
Or
Red blood cell separation rate (%) = 100 × (total area of separated red blood cells) / (total area of separated red blood cells + total area of aggregated red blood cells)

  In order to investigate the possibility of detecting a physiological change by the method according to the present invention, the change in the degree of red blood cell separation at the time of taking a sports drink was experimentally examined. In the experiment, the subject was kept close to dehydration, and the change in red blood cell separation before and after taking a sports drink, which is said to be absorbed quickly into the body, was examined. The subjects were fasted and drunk for about 10 hours, and then started to take blood micrographs. Here, the degree of red blood cell separation was measured before and after taking 500 ml of sports drink. In addition, hematocrit was also measured at the same time using a capillary tube to confirm changes in blood state.

  A representative example of the measurement result is shown in FIG. This shows a measurement result of one individual as a change with time of the average value and standard deviation of the 30 regions described above. Hematocrit is an average of 1 to 3 points for each point. You can read the magnitude of change in red blood cell separation, average value change, and standard deviation. It can also be seen that red blood cell separation and hematocrit show almost symmetrical increases and decreases with the intake of sports drinks. The result of putting together the measured values for 25 subjects using these average values is shown in FIGS. In these figures, similar to the data for one individual in FIG. 7, the degree of red blood cell separation increases significantly 20 minutes after taking a sports drink, and then decreases slightly while maintaining that state. Hematocrit decreases 20 minutes after ingestion and increases slightly while maintaining that state.

  From these results, it was quantitatively clarified that the degree of erythrocyte separation reflects a physiological change accompanying sports drink intake.

  As a cause of the change in the degree of red blood cell separation accompanying sports drink intake, water or sugar supplementation can be considered. To clarify this, we examined the change in red blood cell separation during blood glucose tolerance test and pure water intake. In the blood glucose tolerance test, subjects were fasted and drunk for about 10 hours, and thereafter, blood cell micrographs were started. Here, blood was collected from the fingertips before ingestion and every 30 minutes after ingesting 225 ml of sugar solution (Shimizu Pharmaceutical, Toray Run G75) containing 75 g of glucose, and the degree of red blood cell separation was measured. In addition, the blood glucose level was measured using a commercially available blood glucose level measurement kit (Terumo, Medisafe Mini GR-102), and hematocrit was also measured using a thin tube.

  A representative example of the measurement result is shown in FIG. This shows a measurement result of one individual as a change with time of an average value and a standard deviation of 30 regions. It can be seen that 30 minutes after ingesting the sugar solution, the degree of red blood cell separation is markedly reduced, and then returns to its original state with time. Next, the blood glucose level and hematocrit results measured simultaneously are shown in FIG. It can be seen that the blood glucose level increases and decreases almost symmetrically with the degree of red blood cell separation, and that the hematocrit does not show a particularly constant tendency of change. This experiment was performed on six subjects. A summary of the results is shown in FIGS. Although the difference was reduced due to the variation in the measurement results, the results were almost the same as the above-mentioned representative example. From these results, it was quantitatively shown that the degree of red blood cell separation showed a clear change according to the change in blood glucose level. In addition, about the aggregation of the erythrocyte in the case of a sugar load, there exists a report by another measurement.

  Further, considering that the red blood cell separation degree decreases with the sugar load, it has been found that the cause of the increase in the red blood cell separation degree with the intake of sports drink is not due to the sugar component in the sports drink. Based on this result, in order to confirm the cause of the change in red blood cell separation upon ingestion of sports drinks, the red blood cell separation upon intake of pure water was further investigated. The procedure of the experiment is the same as the experiment of taking a sports drink except that the intake is pure water. In addition, blood glucose levels were measured at the same time. FIG. 14 shows the change in red blood cell separation obtained before and after intake of pure water. It can be seen that the degree of red blood cell separation increases with the intake of pure water. Hematocrit showed a symmetrical change in red blood cell separation and blood glucose levels did not show any beneficial changes.

  Summing up these results, it is considered that the change in the degree of red blood cell separation when taking a sports drink is mainly due to moisture. That is, it has been clarified that the degree of red blood cell separation remarkably reflects physiological changes associated with hydration. Moreover, it was shown that the physiological change accompanying a glucose load is reflected in the red blood cell separation degree measured by this method.

  INDUSTRIAL APPLICABILITY The present invention can be easily applied as an examination for obtaining information on a subject's physiological state, nutritional state, and health state, and as a blood observation sample stage for use in the examination. The nature is extremely large.

It is a sample stand for blood observation concerning one embodiment of the present invention, and is a mimetic diagram showing the section. Similarly, it is a plan view showing an example of a spacer thin film pattern of the sample stage for blood observation according to the present invention. Similarly, it is a top view which shows the other pattern of the spacer thin film of the sample stand for blood observation by this invention. It is a figure which concerns on this invention, and is a conceptual diagram which shows the red blood cell separation degree measurement procedure in the sample center part. Similarly, it is a figure which shows the correlation with the separation degree by a blood cell number ratio, and the separation degree by an area ratio. Similarly, it is a figure which shows the outline of the result of the distinction of the separated erythrocyte and the aggregated erythrocyte which exist in an image, and area calculation. Similarly, it is a figure which shows the change of the red blood cell separation degree in one subject's sports drink intake. Similarly, it is a figure which shows the 25 person average change of the red blood cell separation degree in sports drink intake. Similarly, it is a figure which shows the change of 25 average of hematocrit in sports drink intake. Similarly, it is a figure which shows the change of the red blood cell separation degree in the sugar water intake of one test subject. Similarly, it is a figure which shows the change of the blood glucose level and hematocrit in the sugar water intake of one test subject. Similarly, it is a figure which shows the 6-person average change of the red blood cell separation degree in sugar solution intake. Similarly, it is a figure which shows the 6 person change of the blood glucose level and hematocrit in sugar solution intake. Similarly, it is a figure which shows the change of the average of 3 persons of the red blood cell separation degree in pure water intake.

Explanation of symbols

10 Glass substrate 12 Spacer thin film 14 Cover glass 16 Recess 18 Escape (flow path)
20 Region for gripping 21 Protrusion for preventing outflow

Claims (6)

A flat glass substrate, a spacer thin film having a uniform thickness disposed on the glass substrate, and a cover glass placed thereon,
The spacer thin film has a recess for storing a predetermined amount of blood and an escape passage connected to the recess, and has a maintaining means for constantly maintaining the amount of blood dripped in the recess. A sample stage for blood observation characterized by.   The sample table for blood observation according to claim 1, wherein the spacer thin film has a thickness of 2 μm to 9 μm.   The spacer thin film is formed by forming a metal thin film made of one metal selected from the group of gold, silver, copper, platinum, rhodium, palladium, nickel, tin, and chromium on a substrate by a plating technique. The sample table for blood observation according to claim 1 or 2, characterized in that:   The said spacer thin film forms the nonmetallic thin film which consists of one nonmetal selected from the group which consists of an epoxy resin and a fluororesin by the printing technique on a board | substrate, The Claim 1 or 2 characterized by the above-mentioned. Sample stage for blood observation. Blood is observed using the blood observation sample stage according to any one of claims 1 to 4, and the ratio of the number of separated red blood cells to the total number of red blood cells present in the image is defined as the degree of red blood cell separation, and this value is used. A method for separating and quantitatively evaluating blood cells, characterized by quantitatively evaluating the separation / aggregation state of erythrocytes by the following formula.
Red blood cell separation rate (%) = 100 × (total number of separated red blood cells) / (total number of separated red blood cells + total number of aggregated red blood cells) Blood is observed using the blood observation sample stage according to any one of claims 1 to 4, and the ratio of the total area of separated red blood cells to the area of total red blood cells present in the image is defined as the degree of red blood cell separation, A method for separation and quantitative evaluation of blood cells, characterized in that the separation / aggregation state of red blood cells is quantitatively evaluated by the following formula using this value.
Red blood cell separation rate (%) = 100 × (total area of separated red blood cells) / (total area of separated red blood cells + total area of aggregated red blood cells)

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