CN107132088B

CN107132088B - Reagent for immediate coagulation of body fluid specimen sediment

Info

Publication number: CN107132088B
Application number: CN201710295759.0A
Authority: CN
Inventors: 邱承敏; 杨道华
Original assignee: SHANGHAI SONGJIANG DISTRICT CENTRAL HOSPITAL
Current assignee: SHANGHAI SONGJIANG DISTRICT CENTRAL HOSPITAL
Priority date: 2017-04-28
Filing date: 2017-04-28
Publication date: 2020-07-17
Anticipated expiration: 2037-04-28
Also published as: CN107132088A

Abstract

The invention discloses a reagent for instantly solidifying precipitates of a body fluid specimen, wherein each 100 volumes of the reagent contains 5-20 volumes of trichloromethane, 0.5-8 volumes of glacial acetic acid, 1-10 volumes of formaldehyde solution and the balance of ethanol solution; the volume concentration of the ethanol solution is 10-100%, and the volume concentration of the formaldehyde solution is 37-40%. The reagent of the invention can be used for instantly coagulating the body fluid specimen precipitate, and has the advantages of simple preparation, low cost and convenient and fast use.

Description

Reagent for immediate coagulation of body fluid specimen sediment

Technical Field

The invention belongs to the technical field of body fluid detection, relates to a body fluid pretreatment technology, and particularly relates to a reagent for instantly solidifying precipitates of a body fluid specimen.

Background

In clinic, the detection of body fluid or excrement can understand the change rule of body fluid under physiological and pathological conditions, and has important significance for diagnosis, prevention and treatment. Because the body fluid composition is relatively complex, the body fluid is correspondingly pretreated before detection aiming at different detection items.

In some items of testing of body fluid specimens, it is necessary to subject the body fluid specimen sediment to coagulation treatment. At present, the method generally adopted by each hospital adopts either a method of adding agar after centrifugation or a method of buying and importing complete sets of reagents and special consumables. The agar adding method after centrifugation has complex operation process, higher requirements on operator operation skills, high cost of using imported reagents and special consumables and complicated method steps.

Disclosure of Invention

The invention aims to solve the technical problem of providing a reagent for instantly solidifying the body fluid specimen sediment in one step, which has low cost and can quickly and conveniently instantly solidify the body fluid specimen sediment, so as to overcome the defects in the prior art.

In order to solve the technical problems, the invention adopts the following technical scheme:

a reagent for instantly coagulating body fluid specimen precipitate is characterized in that every 100 volumes of the reagent contains 5-20 volumes of chloroform, 0.5-8 volumes of glacial acetic acid, 1-10 volumes of formaldehyde solution and the balance of ethanol solution; the volume concentration of the ethanol solution is 10-100%, and the volume concentration of the formaldehyde solution is 37-40%.

In a preferred embodiment of the invention, the ethanol solution has a concentration of 40 to 80% by volume.

The protein solution is a stable hydrophilic sol solution, and the stability factors are two: firstly, the charges on the protein colloidal particles cause the protein colloidal particles to repel each other and are not easy to agglomerate; the second is the hydrated film on the surface of the colloidal particles, which ensures that the colloidal particles are consistent with water and also plays a role in isolation among the colloidal particles. If the two factors that stabilize the protein solution are destroyed, the protein will precipitate out of solution.

The factors that promote protein precipitation are many and can be roughly divided into two categories:

the first type is a reversible precipitation reaction. The spatial conformation of the protein is not greatly changed, and the protein can be redissolved after removing the precipitation factor, such as salting out and low temperature ethanol precipitation.

The second type is irreversible precipitation reaction, after heavy metal salts or alkaloid reagents precipitate proteins, the proteins are no longer soluble in water due to major changes in protein structure. Irreversible protein precipitation is often an indication that the protein has been denatured. When denatured proteins are heated near the isoelectric point, the protein molecules intertwine and become a firm clot.

The spatial structure of the native protein is maintained by secondary bonds such as hydrogen bonds, and after denaturation, the secondary bonds are broken, and the protein molecules change from the original ordered coiled compact structure to the disordered loose stretched structure (but the primary structure is not changed). Therefore, a large amount of hydrophobic groups originally in the molecule are exposed on the surface of the molecule, and the distribution of the hydrophilic groups on the surface is relatively reduced, so that the protein particles cannot be dissolved in water and lose the water film, and the molecules are easy to collide with each other to aggregate and precipitate. When the pH of the solution is less than the isoelectric point of the protein, the positively charged protein binds to the anion of the alkaloid reagent to form a salt, which precipitates.

In the invention, formaldehyde is used as a cell fixing agent, and glacial acetic acid is used as a cell nucleus fixing enhancer; chloroform is used as precipitant for protein extraction. On one hand, the ethanol is used as a dehydrating agent and can compete for a hydration membrane with protein in the body fluid precipitate; the addition of ethanol also reduces the dielectric constant of water, protein dissociation degree and charge amount. So that the addition of ethanol can destroy the colloidal properties of the protein and precipitate the protein; on the other hand, ethanol also serves as a stabilizer and can react with toxic phosgene generated by trichloromethane to generate nontoxic carbonic acid diacetic acid, so that the damage to a human respiratory system and eyes in the preparation process is reduced.

Therefore, the reagent of the invention can be used for instantly coagulating the sediments of the body fluid specimen, and has the following advantages:

1. the reagent raw materials are low in price, convenient to prepare and low in cost;

2. can quickly finish the solidification of liquid specimen sediments during centrifugation, maintain the cell morphology and have high diagnosis accuracy;

3. the used equipment is a test tube for a common laboratory and a common centrifuge, and the operation method is convenient.

Detailed Description

The invention relates to a reagent for instantly solidifying liquid sample sediment, in particular to a reagent for instantly solidifying liquid sample sediment, wherein each 100 volumes of the reagent contains 5-20 volumes of trichloromethane, 0.5-8 volumes of glacial acetic acid, 1-10 volumes of formaldehyde solution and the balance of ethanol solution; the volume concentration of the ethanol solution is 10-100%, and the volume concentration of the formaldehyde solution is 37-40%.

Preparing a reagent:

adding chloroform, formaldehyde solution and glacial acetic acid into the ethanol solution, mixing, and stirring uniformly to prepare the reagent.

Specific preparation examples are as follows: if the preparation is carried out by using 40% ethanol solution, 5 ml of trichloromethane, 0.5 ml of glacial acetic acid and 1 ml of formaldehyde solution are added into 50 ml of 40% ethanol solution, and then the volume of the solution is made up to 100ml by using 40% ethanol solution.

Test method

The code of the ethanol solution is A, the code of the trichloromethane is B, the code of the glacial acetic acid is C, and the code of the formaldehyde solution is D.

The volume concentration of the ethanol solution A is divided into 6 gradients from low to high, which are sequentially represented by a, b, c, d, e and f. The chloroform B, the glacial acetic acid C and the formaldehyde solution D are added in a gradient of 3 parts from low to high, and each part of the prepared reagent is shown in the following table 1.

TABLE 1

In this test, 18 groups of 18 examples were prepared with reagents of different ratios, each having a volume concentration of a, b, c, d, e, and f6 and 3 volumes of a, b, and c added to B, C, D, respectively, and the reagent volume of each example was 100ml, as shown in table 2 below.

TABLE 2

The 18 different ratios of the example reagents were tested together using the same patient sample. In the test, 10 ml of body fluid is used as a volume for centrifugal precipitation, 1 ml of the reagent is added, and the coagulation condition, the sliced cells and the background condition are observed, and the observation results are shown in the following table 3.

TABLE 3

In the above table 3, in which,

and (3) solidification: macroscopic cases of cell coagulation into clumps, indicated as + - +++ with more "+" indicating better coagulation status;

background: the background condition such as blood in the cell mass is shown as +++ - +, and the less "+" shows the better background condition;

cell: cell morphology, staining clarity, indicated as + - +++ the more "+" the better the staining clarity.

In the horizontal direction from Table 3, A^aGroup example A^aB^aC^aD^a、A^aB^bC^bD^b、A^cB^cC^cD^cThe amount of the coagulated tissues is small and scattered; a. the^bGroup example A^bB^aC^aD^a、A^bB^bC^bD^bSlightly more tissue coagulated, example A^bB^cC^cD^cThe increase is obvious; a. the^cGroup example A^cB^aC^aD^a、A^cB^bC^bD^bThe ratio of the amount of coagulated tissue to the amount of coagulated tissue A^bA significant increase, example A^cB^cC^cD^cAnd A^bB^cC^cD^cThere was no significant difference.

In the vertical column direction from Table 3, in example A^aB^aC^aD^aTo A^fB^aC^aD^aTissue coagulation from example A^cB^aC^aD^aStart to basic>0.2 cm: in example A^aB^bC^bD^bTo A^fB^bC^bD^bCoagulation of cell tissue from example A^cB^bC^bD^bStart to basic>0.4 cm; in example A^aB^cC^cD^cTo A^fB^cC^cD^cCoagulation of cell tissue from A^bB^cC^cD^cStart to basic>0.4cm。

Coagulation by looking at with eyes>0.4cm of A^bB^cC^cD^c，A^cB^bC^bD^bThe solidification state is the best.

Under a microscope: a. the^cB^cC^cD^c，A^dB^aC^aD^a，A^dB^bC^bD^b，A^eB^aC^aD^aIs excellent in preservation of cell morphology. Wherein, example A^cB^cC^cD^cThe coagulation state, the cell shape and the staining are excellent, and the diagnosis value is high; example A^dB^bC^bD^bAlthough the background is slightly more, the coagulation state, cell morphology and staining are better, and the kit has diagnostic value. Example A^eB^aC^aD^aCoagulation status, cell morphology, staining are good, the background is too much cell debris, influence the microscope observation. Example A^dB^aC^aD^aIn the state of better cell morphology, better staining, slightly more background, but coagulationIs not ideal.

Claims

1. A reagent for instantly solidifying and blocking body fluid specimen sediments contains ethanol solution, and is characterized in that each 100 volume of the reagent contains 10-20 volume of trichloromethane, 3-8 volume of glacial acetic acid, 5-10 volume of formaldehyde solution, and the balance of the ethanol solution; the volume concentration of the formaldehyde solution is 37-40%, and the volume concentration of the ethanol is 40-60%.