JP2002350429A - Method for increasing plasma separation efficiency of plasma separation membrane - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for acquiring plasma stably and efficiently by improving decline of the plasma separation rate caused by individual difference of the protein existence quantity in the plasma to thereby minimize the individual difference. SOLUTION: Concerning this plasma separation membrane using a polyester- based polymer or a composite raw material applying the plasma separation membrane, a phosphate, citric acid, mannitol, glycine, fibrinogen, γ-globulin or the like are added independently or compositely to the plasma separation membrane.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reaction material for blood component analysis and a blood (plasma) transport medium (container) in the field of clinical examination.
Separation of blood into blood plasma and blood clots (blood cells) in a plasma separation membrane containing a polyester-based polymer as a main material or a composite material using the same, which is widely used as a base material. The present invention relates to a method for alleviating the inhibition of plasma separation by proteins and protein-binding substances and efficiently obtaining plasma.

[0002]

2. Description of the Related Art
As a method for easily obtaining a plasma component based on chromatography in the medical field, particularly in the clinical test field, a plasma separation membrane using a polyester-based polymer has been developed and commercially available.

[0003] However, the present inventors have found that in a plasma separation membrane containing these polyester-based polymers as a main material, the separation rate of plasma from blood clots (blood cells) differs due to the difference in the amount of plasma components in the blood sample. In particular, it was discovered that the plasma separation rate was significantly reduced in high albumin plasma and high lipoprotein (= high neutral fat) plasma. 1 to 4 show albumin and ultra-low-density lipoprotein in plasma in a plasma separation membrane Hemasep L and a composite material Hemasep V (manufactured by Gelman Sciences Inc., New York, USA) containing polyester-based polymer as a main material. FIG. 4 shows how fat inhibits plasma separation.

[0004] In this experiment, a peparin blood collection tube (Phenoject II) was used.
(VP-H052: manufactured by Terumo Corporation), length 60 mm, width
0.25m each for Hemasep L and V cut to 5mm
l, 0.10 ml of heparin blood is developed, and the development length of plasma and blood clot (blood cell) is measured, and the plasma separation rate = (plasma / (plasma +
(Blood clot) × 100) / (100-hematocrit value). In the case of albumin, the plasma separation rate decreases with increasing abundance with a high correlation coefficient of 0.812 for Hemasep L, 0.785 for Hemasep V, and 0.654 and 0.583 for triglycerides, respectively.

[0005] Incidentally, when the plasma separation rate is lowered, when used as an analytical reaction material, there is a possibility that the reliability of the analytical value is reduced due to a failure to obtain a sufficient plasma volume. In addition, there is also a problem that when an extraction operation is performed using the medium as a plasma transfer medium, an extraction error is increased, and when used directly for measurement, the number of measurement items is reduced.

The present invention has been made in view of the above-mentioned problems, and is intended to improve a reduction in the plasma separation rate due to individual differences in the amount of protein present in plasma and to minimize the individual differences to achieve a stable operation. It is another object of the present invention to provide a method for efficiently obtaining plasma.

[0007]

[Means for Solving the Problems] (Prerequisite consideration) In the case of plasma separation membranes based on chromatography, if the difference between the materials and the state of molecular modification are not discussed, the diffusing power between blood cells and plasma is generally considered. Use the difference. When dealing with the diffusion of blood, it is thought that it can be approximated to Fick's diffusion law that deals with the diffusion in a solution containing colloid particles, and is governed by the cross-sectional area of the interface, the concentration gradient, and the inherent diffusion coefficient of the colloid molecules, It is believed that molecular diffusion occurs.

That is, assuming that the blood is a solution containing red blood cells which are giant colloids, and if this solution is a viscous flow flowing through a space having a relatively small cross-sectional area, red blood cells, which are solutes having a small diffusion coefficient, greatly diffuse. Without this, the concentration gradually decreases in the separation membrane. Then, it is assumed that plasma, which is a solvent, is diffused in the membrane by the concentration gradient of the component contained therein in a state where the resistance of the blood cell is lost. When blood is actually dropped on a plasma separation membrane and spread on a plane, plasma-only extension is observed after the extension of blood cell components is stopped (Barlow's Physics & Chemistry 3rd edition (lower), P712. GMBarrow, Tokyo Chemical Co., Ltd.) Doujin, 1976).

[0009] However, plasma is not a uniform solvent as dealt with in the theory of physical chemistry, and the diffusion flow of blood cells and plasma fluctuates due to individual differences, and the above-mentioned differences in the presence of albumin and lipoprotein ( = Differences in plasma properties) as a result. There are two possible causes of the difference in the separation rate: the separation rate is reduced due to insufficient plasma diffusing power, and the separation rate is reduced as the properties of the whole blood (interaction between plasma and blood cells). Research by the inventor has revealed the latter.

That is, as shown in FIGS. 5 and 6, even if the plasma is directly applied to the separation membrane, the fluctuation (decrease) in the plasma flow width due to the difference in the amount of albumin and lipoprotein (neutral fat) is not observed. However, it is understood that the property of the viscous flow of the whole blood is a phenomenon caused by the interaction between the plasma component and the blood cell. The interaction between plasma proteins and blood cells, particularly red blood cells, has been studied for a long time, and is said to be greatly involved in the blood agglutination reaction and erythrocyte sedimentation reaction in the field of clinical examination. In particular, albumin has a function of delaying the erythrocyte sedimentation rate, and it is theoretically said that this delay is caused by electrical repulsion because both the surface charges of albumin and erythrocyte are negative.

[0011] The delay of sedimentation due to the electrical repulsion means the red blood cell diffusion, and apparently an increase in the diffusion coefficient, considering that there is a negative correlation between the sedimentation and the diffusion in physical chemistry. In other words, the increase in the diffusion coefficient of blood cells in the plasma separation membrane with a focus on the small diffusion coefficient of blood cells,
It means a decrease in resolution.

[0012] In addition, when blood cells are spread around a plasma separation membrane, the diffusion of blood cells may act as an increase in resistance (friction) to the plasma flow in a space having a relatively fine cross-sectional area. It is believed that plasma reduces the force to expand in the membrane.

[0013] To support this consideration, the present inventor:
Using Hemasep L, the extension distance of “plasma: blood cells” was compared based on the difference in plasma albumin concentration. Specimen, neutral fat and normal plasma albumin 3.8g / dl or less, 3.9-4.2g / dl,
4.3 g / dl or more were divided into 3 groups, and 10 cases were measured in millimeter units. The extension distance of “plasma: blood cells” in each group was 5.8: 19.0, 4.0: 19.3, 3.0: 20.7. The smaller the amount of albumin, the more the expansion of blood cells was suppressed (blood cell diffusion), and the total flow distance (blood cell resistance) ) Was also extended.

According to the phenomena confirmed by the present inventors, in many cases, blood containing high albumin and high lipoprotein plasma shows an image in which the separation boundary between blood cells and plasma on the plasma separation membrane is unclear. The fact satisfies the above two considerations.

Similar results were obtained for lipoproteins, and it is presumed from the fractionation position by electrophoresis that the lipoproteins have a weaker charge than albumin but have the same action as albumin. As described above, in a commercially available plasma separation membrane mainly composed of polyester, in order to minimize individual differences, it is necessary to eliminate the interaction between components such as albumin in blood plasma and blood cells by some method.

In order to solve this problem, as is apparent from Fick's law, a method of dividing the interaction between the plasma component and the blood cell is taken, or two methods for giving a concentration gradient that is not inferior to the frictional resistance of the blood cell. Although a method is conceivable, the present inventor has found a method of impregnating a plasma separation membrane with various salts, saccharides and amino acids to suppress individual differences in the amount of plasma and increase the amount of plasma to be separated.

That is, the present invention provides a plasma separation membrane using a polyester polymer or a composite material to which the plasma separation membrane is applied, wherein a salt, a saccharide, an amino acid, or a protein is added to the plasma separation membrane alone or in combination. This is a method for increasing the efficiency of separating plasma. In the present invention, addition is a concept including impregnation and coating.

The salt of the present invention is an inorganic acid salt, an organic acid salt or a chloride. As the inorganic acid salt, a phosphate, a nitrate or the like is preferable, and as the organic acid salt, a citrate or an oxalic acid is used. Salts and the like are preferred. Of these, phosphates and citrates which are tribasic salts are particularly preferred. Tribasic acid salt 0.25
PH of about 0.05 mol / l, preferably about 0.2 to 0.1 mol / l.
It is preferable to adjust the pH to 8 (the optimum range is about pH 7 to 8) and add and impregnate. As chlorides, sodium chloride,
Potassium chloride and the like are preferred, of which sodium chloride is particularly preferred. The chloride concentration is 0.25 ~
About 0.05 mol / l is preferable.

As the saccharide of the present invention, a monosaccharide or a disaccharide is preferable, and as the monosaccharide, mannitol, glucose and the like are preferable, and as the disaccharide, sucrose and trehalose are preferable. Of these, mannitol and sucrose are particularly preferred. The concentration of the saccharide is 0.25 to 0.05 mol / l
The degree is preferred.

As the amino acid of the present invention, glycine, alanine and the like are preferable, and glycine is particularly preferable.
Suitable amino acid concentrations are on the order of 0.25 to 0.05 mol / l. As the protein of the present invention, plasma proteins such as fibrinogen and γ-globulin are preferable. A preferred final concentration of the protein is on the order of 40-100 mg / dl.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022]

Embodiment 1 Improvement of plasma expansion rate with Hemasep L (FIG. 7) As an example, plasma separation membrane Hemasep L (manufactured by Germanic Science, Inc., New York, USA) using a polyester-based polymer was cut into a length of 60 mm and a width of 5 mm. The plasma separation ability was compared using a modified membrane and an unmodified membrane impregnated with 0.1 mol / l aqueous solution of potassium phosphate, sodium phosphate, sodium chloride, potassium chloride, and glycine each.

An albumin concentration of 4.8 g / dl · collected using a blood collection tube of Peparin (Genoject IIVP-H052: manufactured by Terumo Corporation).
Neutral fat 103mg / dl (high albumin sample), albumin 4.
1g / dl-neutral fat 432mg / dl (high neutral fat sample), albumin 4.0g / dl-neutral fat 110mg / dl (normal sample) Each 0.25ml is dropped on each modified, unmodified separation membrane, The ratio of plasma / (clot + plasma) was measured.

As shown in FIG. 7, when the unmodified membrane is set to 100, the membrane modified with each phosphate, chloride and amino acid shows plasma expansion more than twice in both high albumin samples and high neutral fat samples. Was. The plasma expansion was observed even in the normal sample, indicating that the action of albumin was elicited even in the normal range.

A similar experiment was performed on Hemasep V (a product of New York, Germany; German Man Science Co., Ltd.) which is a composite material obtained by bonding cellulose fibers and the like to a polyester fiber film, and similar results were obtained.

[0026]

[Embodiment 2] 2. Effect of Fibrinogen (FIG. 8) As in Example 1, the plasma spreading effect of fibrinogen, which is a plasma protein, was measured using Hemasep L. Specifically, blood was collected using a blood collection tube (made in-house) containing potassium ethylenediaminetetraacetate, and fibrinogen dissolved in physiological saline was added to final concentrations of 0, 50, and 100 mg / d.
l, and an addition experiment was performed on 5 samples. FIG.
As shown in Fig. 7, the plasma expansion rate of each sample increased with the increase in the amount of fibrinogen added, with a minimum of 20% and a maximum of 1%.
A 10% plasma spread was observed.

[0027]

[Embodiment 3] 3. Improvement of plasma expansion rate with Hemasep V (Figs. 9 and 10) Hemasep V cut into length 60mm, width 5mm, potassium phosphate, sodium chloride, mannitol, sucrose,
After immersing in a 0.1 mol / l aqueous solution of sodium citrate and drying sufficiently, albumin 4.0 g / dl / neutral fat 526 collected using a blood collection tube (Phenoject IIVP-H052: manufactured by Terumo Corporation).
mg / dl, albumin 4.0 g / dl, and neutral fat 107 mg / dl were dropped 0.1 ml each on each separation membrane, and the ratio of plasma / (clot + plasma) was measured and the total The amount of protein was measured. The same operation was performed on the unmodified membrane dried by impregnation with pure water as a target and compared.

The total plasma protein content was determined by extracting the obtained plasma portion with 0.4 ml of phosphate buffer, and extracting the obtained extract with
It was measured by the burette method modified in consideration of the dilution equivalent (correlation with the original method was confirmed).

[0029] The results were calculated by setting the average of the pure water impregnated separation membrane to 100
9 and 10. In each salt and saccharide, the plasma extension and the protein amount are increased as compared with the unmodified membrane, and the effect of phosphate and citrate is particularly large. 40% by protein
The above remarkable improvements were observed.

In the case of Hemasep V, blood cells easily infiltrate the plasma part in a high lipid sample, and the image of the separation boundary between the blood clot and the plasma is often unclear, and the plasma yield further decreases. Also in this experiment, in the present experiment, the image of the boundary between the blood clot and the plasma separation was improved by the phosphate in the sample of high neutral fat level compared with the case of the impregnation with pure water, and the blood cell infiltration interval was improved from 3 mm to about 1 mm.

[0031]

[Embodiment 4] 4. Salt pH effect at Hemasep V (Figure 1
1, FIG. 12) In the same manner as in Example 2, the pH of the phosphate was adjusted to 5.2.
The blood pressure was changed in five steps from ７ to 7.8, and the changes in the plasma expansion ratio and total protein amount were measured. As a result, the higher the pH, the greater the plasma extension and the amount of separated proteins. However, in addition to the above, a similar experiment was performed for pH 8.5,
Plasma expansion rate and plasma protein content are also lower than pH 7.2,
The clot and plasma boundaries also increased ambiguity. From this result, it became clear that pH 7 to 8 was desirable.

[0032]

Fifth Embodiment Effect of salt concentration on Hemasep V (Figure 1
3, FIG. 14) As in Example 3, the concentration of phosphate adjusted to pH 7.2 was changed in the range of 0.025 to 0.2 mol / l, and the plasma expansion rate and the plasma protein content were measured. As a result, the plasma expansion rate and the plasma protein content increase with the salt concentration. Also, as the salt concentration increased, the reproducibility of both the plasma separation rate and the plasma protein amount improved. However, almost equilibrium was reached at 0.2 mol / l, and 0.3 mol / l
If the concentration is more than / l, a phenomenon that blood cells are destroyed may occur in some cases. Therefore, the salt concentration is optimally 0.1 to 0.2 mol / l.

In the case of the phosphate concentration of 0.2 mol / l in this experiment, the plasma separation of the neutral fat of about 500 mg / dl and the specimen of about 100 mg / dl were performed in almost the same manner, and it was found that Hemasep V was used as it was. Injury and poor reproducibility caused by neutral fats were improved. That is, since the preferred salt modification and the preferred pH of Hemasep V were satisfied, a substantially constant amount of plasma was obtained regardless of the amount of neutral fat.

[0034]

Example 6 Correction of Individual Differences Due to Differences in Plasma Albumin Content and Lipoprotein (FIGS. 15 to 18) Correction of individual differences due to differences in plasma albumin content was observed by membrane modification using potassium phosphate. Potassium phosphate 0.1mol / l, pH 5.5, cut into Hemasep L cut to length 50mm and width 6mm
7.2 After impregnating with an aqueous solution and sufficiently drying, 0.25 ml of heparin blood having a concentration of various albumins and neutral fats collected using a peparin blood collection tube (Henoject IIVP-H052: manufactured by Terumo) was developed, and potassium phosphate was added. A comparison was made between the separation membrane not modified by the above and the plasma separation rate = (plasma / (plasma + clot) × 100) / (100-hematocrit value). As shown in the figure, the individual difference is almost eliminated by modifying the separation membrane with potassium phosphate.

[0035]

Example 7 Example of Actual Measurement of Plasma Component (FIGS. 19 to 22) According to the present invention, various biological components present in each of the obtained plasma and clot can be measured. The measured values of components extracted from the plasma part of Hemasep V modified with l and the measured values of serum were compared.

As a measurement example, a correlation diagram between glutamate pyruvate transaminase (GPT) and triglycerides (FIG. 1)
9, FIG. 21). The correlation between unmodified and serum was also illustrated for comparison (FIGS. 20 and 22). As a method, Hemasep V was cut into a length of 60 mm and a width of 5 mm, and 30 modified blood samples were collected using a Peparin blood collection tube (Henoject IIVP-H052: manufactured by Terumo Co.) for 0.1% on each separation membrane. All the plasma portions obtained by dropping in ml each day and drying all day and night were extracted with 0.4 ml of a phosphate buffer solution containing a surfactant and subjected to automatic analysis and measurement.

For the measurement of the extracted components, AU6 manufactured by Olympus Corporation was used.
00 automatic analyzer, serum is AU5242 manufactured by Olympus
An automatic analyzer was used. GPT was measured using a JSCC-compliant method, and neutral fat was measured using a free glycerol elimination / enzymatic colorimetric method. The extract from the plasma separation membrane was measured by a method modified (correlation with the original method was confirmed) in consideration of the dilution equivalent.

In the neutral fat measurement example, the correlation between the plasma part and serum of potassium phosphate-modified hemasep V was 0.979.
And was good. When the degree of improvement is compared with the unmodified one, the variation expressing the individual difference = the correlation coefficient is improved,
It is understood that the slope of the regression equation representing the obtained plasma volume is also improved. The same results were obtained in the measurement of GPT, and it was confirmed that the plasma separation membrane modification with potassium phosphate resulted in individual differences and an increase in the amount of recovered plasma.

[Discussion on the theoretical background] In the problem to be solved by the present inventors, the blood flowing through the plasma separation membrane has a physical property of the blood flow due to a difference in properties of the blood itself (difference in abundance of albumin and the like). Chemical change = decreased blood cell diffusion coefficient and increased blood cell frictional resistance, differing significantly from the plasma separation rate. He also stated that this phenomenon could be solved by adding various salts, amino acids, saccharides, and proteins to the membrane. The theoretical background of the separation rate improvement by these additions is
Although there are still unknowns, the following predictions hold.

One is an increase in the plasma concentration gradient. In the present invention, when a tribasic acid such as phosphoric acid or citric acid is compared with a simple salt such as sodium chloride, Example 1. Or, as shown in Example 3, tribasic acid salt had the effect of increasing the amount of separated plasma. This fact is considered to be due to the difference in ionic strength that worked effectively in this system.

The other is the disruption of the interaction between plasma components and blood cells. There are two approaches to this, one is the denaturation of interacting proteins and the other is the provision of a positive charge against a negative charge.

1) Modification of properties of albumin and the like due to change in liquid properties It is known that the surface charge of albumin is negative from observations such as electrophoresis. However, it is also known that the appearance of a bi-charged property possessed by a protein and an increase in hydrophobic properties are caused by a change in liquid properties. In a study by Raft et al., In an adsorption experiment with a polymer surface, albumin was adsorbed on a polystyrene surface, and if the ionic strength became high, the adsorption amount increased due to denaturation of albumin itself at a pH higher than the isoelectric point, that is, an increase in hydrophobicity. (Yoshihito Raft: Fundamentals and Applications of Polymer Surfaces (above), p247-
270, Doujin Kagaku, 1986).

By modifying the plasma separation membrane with a tribasic acid salt such as potassium phosphate, the ionic strength added to the blood becomes slightly more than three times,
There is also a possibility that the surface properties of albumin and the like have changed. In any case, it is considered that the interaction between blood cells and plasma proteins is interrupted by the increase in ionic strength.

However, a dark increase in ionic strength causes damage to red blood cells and hinders measurement of plasma components.
Up to 2 mol / l is considered optimal. 2) Addition of positive charge In blood sedimentation reaction, it is said that immunoglobulin and fibrinogen act to promote sedimentation, contrary to albumin. This is said to be due to the positive charge or hydrophobicity of both proteins. This fact indicates that, as shown in Example 2, the plasma separation rate can be increased by the positively charged fibrinogen. Globulin also has the effect of accelerating plasma expansion, and as shown in FIG. 23, has a weak effect with a correlation coefficient of about 0.3, but increases the plasma separation rate.

[0045] In practical terms, the application of these proteins may not be able to measure the total protein content of multi-components, especially plasma, and may have an effect on the immune response. The measurement of the mineral content becomes possible.

[0046]

As described above, according to the present invention,
The plasma separation rate can be stably and efficiently obtained by improving the reduction of the plasma separation rate due to individual differences in the amount of protein present in the plasma and minimizing the individual differences.

[Brief description of the drawings]

FIG. 1 illustrates that the separation performance decreases according to the amount of albumin.

FIG. 2 illustrates that the separation performance decreases according to the amount of neutral fat.

FIG. 3 illustrates that the separation performance decreases with the amount of albumin.

FIG. 4 illustrates that the separation performance decreases depending on the amount of neutral fat.

FIG. 5 illustrates the relationship between serum extension and TG.

FIG. 6 illustrates the relationship between serum extension and albumin.

FIG. 7 shows the results of Example 1 and illustrates the performance of improving the plasma separation rate with salts and amino acids.

FIG. 8 shows the results of Example 2 and illustrates the effect of fibrinogen on plasma spreading.

FIG. 9 shows the results of Example 3, illustrating the performance of improving the plasma separation rate by salts and saccharides.

FIG. 10 shows the results of Example 3, illustrating the performance of improving the plasma separation rate by salts and saccharides.

FIG. 11 shows the results of Example 4, in which p
3 illustrates the effect of H.

FIG. 12 shows the results of Example 4, in which p
3 illustrates the effect of H.

FIG. 13 shows the results of Example 5 and illustrates the effect of salt concentration on plasma expansion.

FIG. 14 shows the results of Example 5 and illustrates the effect of plasma concentration on the salt concentration.

FIG. 15 illustrates that the separation performance decreases with the amount of albumin.

FIG. 16 shows the results of Example 6, showing that the separation performance was improved as compared with FIG.

FIG. 17 illustrates that the separation performance decreases depending on the amount of neutral fat.

FIG. 18 shows the results of Example 6, showing that the separation performance was improved as compared with FIG.

FIG. 19 illustrates the results of Example 7.

FIG. 20 illustrates the results of Example 7.

FIG. 21 illustrates the results of Example 7.

FIG. 22 illustrates the results of Example 7.

FIG. 23 illustrates the relationship between GLB and plasma recovery.

Continued on the front page F term (reference) 2G045 BA13 BB04 BB52 BB60 CA25 2G052 AA30 AD26 EA02 FD02 FD06 JA07 JA08 JA11 JA16 4C077 AA12 BB02 EE01 KK13 KK15 LL21 LL23 4D006 GA07 MB19 MC48 MC72 MC90 PA01 PB09

Claims (8)

[Claims] 1. A plasma separation membrane using a polyester-based polymer or a composite material to which a plasma separation membrane is applied, wherein a salt, a saccharide, an amino acid, or a protein is added to the plasma separation membrane alone or in combination. A method for increasing the plasma separation efficiency of lipase 2. The method according to claim 1, wherein the salt is an inorganic acid salt, an organic acid salt or a chloride. 3. The method according to claim 1, wherein said salt is a tribasic acid salt. 4. The method according to claim 1, wherein the saccharide is a monosaccharide or a disaccharide. 5. The method according to claim 1, wherein said amino acid is glycine. 6. The protein according to claim 1, wherein the protein is fibrinogen or γ-protein.
2. The method according to claim 1, which is a globulin. 7. The pH of the tribasic acid salt of 0.25 to 0.05 mol / l is adjusted to pH
The method according to claim 3, wherein the composition is adjusted to 6 to 8 and added and impregnated. 8. The method according to claim 3, wherein the tribasic salt is a phosphate or a citrate.