JP2000329762A - Method for preparing white blood cell measuring reagent and white blood cell measuring sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve fractional performance of a white blood cell or particularly to improve a separation of a lymphocyte and a monocyte and to more accurately classify the cell by using a solution containing a red blood cell hemolyzing agent made of a hypotonic solution containing a buffer for holding a pH in an acid range and a solution containing a buffer for neutralizing an acid in the buffer and an osmotic compensator having a final osmotic pressure of a specific value for improving the fractional performance of the cell. SOLUTION: A red blood cell hemolyzing agent (first liquid) made of a hypotonic solution containing a buffer for holding a pH in an acid range is usable with any buffer if pka is 3.5±1.5, and is, for example, a maleic acid or the like. A concentration of the buffer is desired to be lowered at its osmotic pressure of the first liquid. The lower the osmotic pressure of the agent is, the more rapid hemolyzing of the red blood cell is done. The solution (second liquid) containing a buffer for neutralizing an acid in the agent and an osmotic compensator having a final osmotic pressure of about 400 to 500 mOsm/kg for improving a fractional performance of the white blood cell is usable even with any of the buffer if pka is 1.0 to 9.5, and, e.g. a phosphoric acid is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for preparing a leukocyte measurement sample from which red blood cells have been removed and a method for preparing a leukocyte measurement sample in the field of clinical examination.

[0002]

2. Description of the Related Art In a blood test in a clinical test field,
Counting leukocytes in blood by type is generally well performed, and it is known that classification of leukocytes can contribute to diagnosis of diseases. A method for classifying leukocytes includes a light source, a flow cell in which cells in a sample flow through a narrow flow path, a photometric unit that detects light emitted from cells, and scattered light depending on the intensity of the detection signal. In order to classify leukocytes according to the intensity of the detection signal. In this method, blood cells are stained, the stained blood cells are illuminated with light, and the fluorescence and possibly scattered light emitted from the blood cells are detected together, and the blood cells are classified according to the intensity of the detection signal. is there.

A first method belonging to this method is Japanese Patent Publication No. 7-23890, which is a red blood cell hemolyzing agent comprising a hypotonic solution containing a buffer for keeping the pH in an acidic range;
A sample for measuring leukocytes from which a red blood cell has been removed is prepared from a solution comprising a buffer for neutralizing the acid in the buffer and an osmotic pressure compensating agent for adjusting the solution to an osmotic pressure that maintains the form of leukocytes. And a method for preparing a leukocyte measurement sample. According to this method, erythrocytes are ghosted to the point where simultaneous passage of erythrocytes and leukocytes can be neglected by the acid hypotonic treatment, and the leukocyte fractionation performance is improved.

As a second method, Japanese Patent Publication No. Hei 7-2695
5, an acidic lysis reagent effectively distinguishes a whole blood sample from a non-erythrocyte fraction and a erythrocyte fraction, and furthermore, the lytic activity is significantly reduced by an inhibitor, and simultaneously neutralized, and a specific indicator-labeled binding substance is used. Is a method of further dividing the non-erythrocyte fraction into subpopulations by immunochemical interaction.
According to this method, rapid and complete hemolysis of the red blood cell fraction can be achieved while maintaining the non-red blood cell fraction in a natural physiological state.

[0005] However, in the above two methods,
There is no disclosure of improving leukocyte fractionation performance by adjusting osmotic pressure with an osmotic pressure compensator (suppressor). In particular, there is no mention of improving the performance of fractionating lymphocytes and monocytes.

[0006] In addition, CDM, a lymphocyte subset, is used to monitor the onset of AIDS in HIV-infected patients, confirm symptoms of HIV-infected patients, and support treatment (delay the onset).
It is known that monitoring of 4 counts and the ratio of CD4 / CD8 is effective. However, it has been found that when red blood cell ghost components and monocytes are mixed in lymphocytes, the CD4 count and the ratio of CD4 / CD8 cannot be accurately obtained. Therefore, it is desired to improve the accuracy of lymphocyte and monocyte fractionation in addition to the erythrocyte ghost component.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above circumstances, and it is an object of the present invention to improve leukocyte fractionation performance by adjusting osmotic pressure with an osmotic pressure compensating agent, in particular, lymphocyte and monocyte fractionation performance. It is an object of the present invention to provide a reagent for preparing a leukocyte measurement sample and a method for preparing a leukocyte measurement sample for the purpose of improving blood pressure.

[0008]

In order to solve the above-mentioned problems, the present inventor has set forth the above-mentioned buffer after the hemolysis treatment with an erythrocyte hemolytic agent comprising a hypotonic solution containing a buffer for keeping the pH in an acidic range. Various studies were made on the osmotic pressure and the leukocyte fractionation performance of a solution consisting of a buffer for neutralizing the acid in the agent and an osmotic pressure compensating agent for adjusting the osmotic pressure, and the following findings were obtained. As a result, the present invention has been completed. That is, the final osmotic pressure of the osmotic pressure compensating agent is about 400 to about 500 mOsm /
When it is in the range of kg, the collecting ability of the leukocyte population increases,
It has been found that the fractionation performance of leukocytes (lymphocytes, monocytes, granulocytes) is improved, and the separation of lymphocytes and monocytes is particularly improved. That is, the present invention provides a reagent for measuring leukocyte,
(A) an erythrocyte hemolytic agent comprising a hypotonic solution containing a buffer for keeping the pH in an acidic range; and (b) a buffer for neutralizing the acid in the buffer, and A final osmotic pressure of about 400 to about 50 to improve fractionation performance
It is a solution comprising an osmotic pressure compensating agent of 0 mOsm / kg.

Further, the method for preparing a sample for leukocyte measurement of the present invention uses the above reagent, and (a) an erythrocyte hemolytic agent comprising a hypotonic solution containing a buffer for keeping the pH in an acidic range. Adding fresh blood subjected to anticoagulation treatment to lyse red blood cells; and (b) a buffer for neutralizing the acid in the buffer and a solution for improving the performance of fractionating white blood cells. Final osmotic pressure of about 400 to about 50
Adding a blood sample that has been treated with a solution comprising an osmotic compensation agent at 0 mOsm / kg.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION An erythrocyte hemolytic agent (first solution) consisting of a hypotonic solution containing a buffer for keeping the pH in an acidic range is as follows:
The one disclosed in Japanese Patent Publication No. Hei 7-23890 can be used.
Any buffer having a pKa of 3.5 ± 1.5 can be used, for example, maleic acid, malonic acid, phthalic acid, diglycolic acid, salicylic acid, fumaric acid, tartaric acid, citric acid, malic acid and the like. It is desirable that the concentration of the buffer reduce the osmotic pressure of the first liquid. For the purposes of the present invention, 50
mM or less, more preferably 5 to 30 mM.
Is desirable.

Further, the lower the osmotic pressure of the erythrocyte hemolytic agent, the faster the erythrocyte hemolysis is performed. For the purposes of the present invention,
In the range of 100 mOsm / kg, especially 0-50 mOsm
/ Kg is preferred.

A buffer for neutralizing the acid in the erythrocyte hemolytic agent and an osmotic pressure compensating agent having a final osmotic pressure of about 400 to about 500 mOsm / kg to improve the leukocyte fractionation performance of the solution. Solution (second solution) has a pKa of 7.0.
Any buffer can be used as long as it is a buffer of ~ 9.5. For example, phosphoric acid, Tris, Tricine, Bicine, 2-amino-methyl-1,3-propanediol,
Taurine, boric acid, serine and the like.

The final osmotic pressure affects leukocyte fractionation performance, and is preferably in the range of about 400 to about 500 mOsm / kg, and more preferably about 400 mOsm / kg. Salts can be used as the osmotic pressure compensating agent. For example, salts such as NaCl and KCl can be used. Here, the final osmotic pressure is the osmotic pressure when the second liquid is added after mixing the first liquid and the blood.

To measure the prepared sample, for example, FIG.
Flow cytometer (for example, FA)
CScan, Becton Dickinson)
There is no particular need for improvement. The light source can be appropriately selected according to the fluorescent dye wavelength to be used.

[0015] The present invention will be described by the following examples, but the scope of the present invention is not limited thereto.

[0016]

EXAMPLE In a combination of 1st liquid: 1 type and 2nd liquid: 8 types, blood collected from a healthy person was mixed with the first liquid, then the second liquid was added, and the final osmotic pressure was added. Influence on the leukocyte fractionation. Specifically, 500 μL of the first solution was added to 50 μL of blood, and 30
After leaving at room temperature for 2 seconds, 1000 μL of the second liquid was added and mixed, and the mixture was left at room temperature for 5 minutes. The finally prepared sample was measured with a generally used flow cytometer to obtain a scattergram (two-dimensional distribution diagram) based on forward scattering intensity and side scattering intensity. The scattergrams thus obtained are shown in FIGS. In the figure, Lymp
Indicates lymphocytes, Mono indicates monocytes, and Gran indicates granulocytes.

The compositions of the first liquid and the second liquid are as follows. First liquid 10 mM citric acid-disodium phosphate buffer pH 3.0, osmotic pressure 62 mOsm / kg Second liquid 50 mM sodium phosphate buffer pH 7.7 Sodium chloride (osmotic pressure compensator) was added to the above buffer, Final osmotic pressure of 100, 200, 300, 400, 5
Eight kinds of solutions to be 00, 600, 700, 800 mOsm / kg

As shown in the scattergrams shown in FIGS. 2 to 5, the final osmotic pressure of the conventional example (200 mOsm / k
g) and the final osmotic pressure of the comparative example (800 mOsm / kg), when the final osmotic pressure is 400 or 500 mOsm / kg, the collecting ability of the leukocyte population is increased, and the leukocyte (lymphocyte, lymphocyte,
It can be seen that the fractionation performance of monocytes and granulocytes) was improved, and the separation of lymphocytes and monocytes was particularly improved.

Next, a histogram of the forward scattered light intensity and the side scattered light intensity of each of the lymphocyte and monocyte populations (the area surrounded by the gate) is used as a measure for measuring the aggregation ability of lymphocytes and monocytes. The standard deviation was determined and shown in the graphs of FIGS. As shown in FIGS.
At 00 mOsm / kg, the standard deviation of the histograms of the side scattered light and the forward scattered light of the lymphocytes and monocytes was small, indicating that the ability to collect lymphocytes and monocytes was increased.

[0020]

The present invention has the following effects. The present invention neutralizes the acid in the buffer after the hemolytic treatment with an erythrocyte hemolytic agent comprising a hypotonic solution containing a buffer for keeping the pH of the blood subjected to the anticoagulant treatment in an acidic range. Solution consisting of a buffering agent and an osmotic pressure compensating agent for adjusting the osmotic pressure is added to make the final osmotic pressure of about 400 to about 500
By maintaining the sm / kg, the leukocyte fractionation performance was improved, especially the separation of lymphocytes and monocytes was improved, and more accurate leukocyte classification was made possible.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a flow cytometer that can be used in the present invention.

FIG. 2 Conventional example (final osmotic pressure: 200 mOsm / kg)
FIG. 4 is a diagram showing a scattergram of FIG.

FIG. 3 The present invention (final osmotic pressure: 400 mOsm / kg)
FIG. 4 is a diagram showing a scattergram of FIG.

FIG. 4 The present invention (final osmotic pressure: 500 mOsm / kg)
FIG. 4 is a diagram shown by a scattergram.

FIG. 5 Comparative example (final osmotic pressure: 700 mOsm / kg)
FIG. 3 is a diagram shown by a scattergram.

FIG. 6 is a diagram showing a standard deviation of a forward scattered intensity histogram of lymphocytes.

FIG. 7 is a diagram showing a standard deviation of a side scatter intensity histogram of lymphocytes.

FIG. 8 is a diagram showing a standard deviation of a forward scattered intensity histogram of a monocyte.

FIG. 9 is a diagram showing a standard deviation of a side scatter intensity histogram of a monocyte.

Claims (2)

[Claims] 1. A leukocyte measurement reagent comprising the following (a) and (b): (A) an erythrocyte hemolytic agent comprising a hypotonic solution containing a buffer for keeping the pH in an acidic range; (b) a buffer for neutralizing the acid in the buffer and a solution for leukocytes A solution comprising a final osmotic pressure of about 400 to about 500 mOsm / kg for improving image performance. 2. A method for preparing a leukocyte measurement sample, which comprises the following steps (a) and (b). (A) a step of adding fresh blood subjected to anticoagulant treatment to an erythrocyte hemolytic agent comprising a hypotonic solution containing a buffer for keeping the pH in an acidic region to lyse erythrocytes; Treating the solution with a solution consisting of a buffer to neutralize the acid in the buffer and an osmotic pressure compensator with a final osmotic pressure of about 400 to about 500 mOsm / kg to improve the leukocyte fractionation performance Adding the obtained blood sample.