CN113959912B

CN113959912B - Method and reagent for detecting white blood cells for resisting platelet aggregation interference and application of reagent

Info

Publication number: CN113959912B
Application number: CN202010702062.2A
Authority: CN
Inventors: 张子千; 高飞
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2024-06-21
Anticipated expiration: 2040-07-20
Also published as: CN113959912A

Abstract

The invention discloses a detection method, a reagent and application of platelet aggregation interference resistance. The detection method comprises the following steps: treating a blood sample with a deagglomerating agent to prevent and/or eliminate platelet aggregation in the blood sample, wherein the deagglomerating agent comprises at least one selected from the group consisting of a compound having an amino group and a compound containing an ammonium ion; and detecting the blood sample treated with the depolymerizing agent with a blood analyzer to obtain at least detection information of white blood cells after eliminating the interference of blood cell aggregation. The depolymerizing agent has depolymerization effect on the aggregation of various platelet conditions, can depolymerize the platelets in a short time, does not need additional conditions such as water bath temperature control and reaction time prolongation, can conveniently eliminate the aggregation of the platelets in a sample, and can obtain accurate detection parameters of the blood cells.

Description

Method and reagent for detecting white blood cells for resisting platelet aggregation interference and application of reagent

Technical Field

The present invention relates to blood detection, and more particularly, to a method for detecting white blood cells and a reagent for detecting white blood cells.

Background

In the analysis of blood cells, the false aggregation of platelets can cause erroneous blood cell counts and classification, which in turn can lead to erroneous diagnosis and treatment of patients. We easily confuse platelet false aggregation with certain life threatening diseases such as heparin-induced in vivo platelet aggregation (HIP), disseminated Intravascular Coagulation (DIC), or incorrect treatment decisions such as incorrect dosing, improper platelet infusion to the patient, even splenectomy. The causes of pseudo-platelet aggregation are numerous and complex, common causes including: ethylenediamine tetraacetic acid-dependent pseudothrombocytopenia (EDTA-PTCP), multiple anticoagulant-dependent platelet aggregation, platelet satellite phenomenon, hypercholesterolemia and hypertriglyceridemia, platelet cold aggregation caused by low temperature environment, blood sampling disorder, aggregation caused by the material of anticoagulant tube, and the like. There have been few studies on the mechanism of pseudo-aggregation of platelets, most focusing on EDTA-PTCP. EDTA is a widely used clinical anticoagulant recognized by the International blood standardization Commission (ICSH), and the first report of the false aggregation of platelets caused by EDTA was probably due to the existence of EDTA-dependent anti-platelet antibodies in the blood of EDTA-PTCP patients, which recognize the adhesion receptor glycoprotein IIb-IIIa (GpIIb-IIIa) on the platelet membrane when the blood is mixed with EDTA anticoagulant in vitro, causing the expression of platelet aggregation activating antigens such as granulesten 140 (GMP 140, alias CD62P or P-selectin), type III lysosomal glycoprotein (Gp 55, alias CD 63), thrombin sensitive protein, etc., and thus activate tyrosine kinase, leading to platelet aggregation.

The false aggregation of the blood platelets not only leads to the error of clinical parameters related to the blood platelets, but also can influence the accuracy of the clinical parameters of other blood cells, thereby bringing adverse effects to clinical diagnosis and treatment. Thus, the discovery and elimination of platelet aggregation in blood samples is a continuing problem to be addressed in extracorporeal blood testing.

Currently, several solutions have been clinically adopted to eliminate or reduce the pseudo-aggregation of platelets. For example, a blood sample with pseudo-platelet aggregation is heated to 37 degrees celsius while shaking for a period of time, or additives are added to the blood sample to prevent platelet aggregation. Such as anticoagulants (e.g., sodium citrate, heparin sodium, ACD, CTAD, CPT, caCl ₂ and heparin sodium mixtures, etc.); platelet count dilutions (containing mixtures of sodium azide, calcium azide, sodium fluoride, etc.); antiplatelet agents (added within 10 minutes after blood withdrawal); aminoglycoside antibiotics (such as amikacin and kanamycin) were added within 1 hour after blood collection, but were effective on only a portion of the samples.

These methods and reagents for preventing platelet aggregation in an ex vivo blood sample either result in complex detection steps or the effect of deagglomerating platelets is not ideal or is effective for aggregation for only a portion of the reasons, often requiring re-sampling and formulation of specific deagglomerating agents. In practice, it is also necessary to examine microscopically whether the platelets are disaggregated after the addition of the additive to determine whether the disaggregation effect is sufficient for blood analysis. If necessary, the action time is prolonged in an ice bath or water bath to ensure the depolymerization effect of the platelets.

Thus, there remains a need for a simple, versatile method and reagent for platelet depolymerization in extracorporeal blood tests to obtain accurate white blood cell detection parameters.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for detecting white blood cells, which is capable of eliminating the interference of pseudo-aggregation of platelets, is simple and easy to implement, does not require additional steps, does not change the existing detection steps and detection conditions, and does not have any influence on detection. It is another object of the present invention to provide a reagent for leukocyte detection suitable for use in the method of the present invention, which contains a substance capable of preventing and/or eliminating platelet aggregation, which does not interfere with other reagents used in blood detection, reacts rapidly, and does not require additional reaction conditions.

According to a first aspect of the present invention there is provided a method of detecting white blood cells against platelet aggregation interference, the method comprising:

Treating a blood sample with a deagglomerating agent to prevent and/or eliminate platelet aggregation in the blood sample, wherein the deagglomerating agent comprises at least one selected from the group consisting of a compound having an amino group and a compound containing an ammonium ion; and

And detecting the blood sample treated by the depolymerizing agent by a blood analyzer to obtain at least detection information of the white blood cells.

According to a specific embodiment, the compound having an amino group is selected from the group consisting of compounds of the following formula (I): R1-NH-R2 (I)

Wherein R1 and R2 are the same or different and are each independently a group selected from the group consisting of R1 and R2 are not simultaneously H, the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkylaryl, substituted or unsubstituted C7-14 arylalkyl, -C (O) -Q1 and-C (O) -O-Q2,

Wherein Q1 is H, -NH ₂, substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkylaryl or substituted or unsubstituted C7-14 arylalkyl;

Q2 is H, substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkylaryl or substituted or unsubstituted C7-14 arylalkyl;

Wherein, said substitution is by at least one member selected from the group consisting of-NP 1P2, -SO ₃ H, -OH, halogen, -CN, -C (O) -O-P3, -O-C1-16 alkyl, -O-C6-10 aryl, -O-C7-14 alkylaryl, -O-C7-14 aralkyl, -C (O) -C1-16 alkyl, -C (O) -C6-10 aryl, -C (O) -C7-14 alkylaryl, -C (O) -C7-14 aralkyl and-C (O) -NP1P2, wherein said-O-C1-16 alkyl, -O-C6-10 aryl, -O-C7-14 alkylaryl-O-C7-14 aralkyl, -C (O) -C1-16 alkyl, -C (O) -C6-10 aryl-C (O) -C7-14 alkylaryl and-C (O) -C7-14 arylalkyl are each unsubstituted or further substituted with at least one group selected from the group consisting of-NH ₂、-OH、-SO₃ H, halogen, -CN, -COOH and-C (O) NH ₂,

P1, P2 and P3 are each independently a group selected from the group consisting of: H. c1-16 alkyl, C6-10 aryl, C7-14 alkylaryl, and C7-14 aralkyl, wherein the C1-16 alkyl, C6-10 aryl, C7-14 alkylaryl, and C7-14 aralkyl are each unsubstituted or further substituted with at least one group selected from the group consisting of-NH ₂、-OH、-SO₃ H, halogen, -CN, -COOH, and-C (O) NH ₂.

More specifically, in the formula (I), R1 and R2 are the same or different and are each independently a group selected from the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, substituted or unsubstituted C7-10 aralkyl, -C (O) -Q1 and-C (O) -O-Q2,

Wherein Q1 is H, -NH ₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl or substituted or unsubstituted C7-10 aralkyl;

Q2 is H, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl or substituted or unsubstituted C7-10 arylalkyl;

Wherein said substitution means substitution with at least one group selected from the group consisting of-NP 1P2, -SO ₃ H, -OH, -CN, -C (O) -O-P3, -O-C1-10 alkyl, -O-C6-10 aryl, -O-C7-10 alkylaryl, -O-C7-10 aralkyl, and-C (O) -NP1P2, wherein said-O-C1-10 alkyl, -O-C6-10 aryl, -O-C7-10 alkylaryl, and-O-C7-10 aralkyl are each unsubstituted or further substituted with at least one group selected from the group consisting of-NH ₂、-OH、-SO₃ H, -CN, -COOH, and-C (O) NH ₂;

P1, P2 and P3 are each independently a group selected from the group consisting of: H. c1-10 alkyl, C6-10 aryl, C7-10 alkylaryl and C7-10 aralkyl, wherein said C1-10 alkyl, C6-10 aryl, C7-10 alkylaryl and C7-10 aralkyl are each unsubstituted or further substituted with at least one group selected from the group consisting of-NH ₂、-OH、-SO₃ H, -CN, -COOH and-C (O) NH ₂.

Still further, in the formula (I), R1 and R2 are the same or different and are each independently a group selected from the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, substituted or unsubstituted C7-10 aralkyl, -C (O) -Q1 and-C (O) -O-H,

Wherein said substitution means substitution with at least one group selected from the group consisting of-NP 1P2, -SO ₃ H, -OH, -CN, -C (O) -O-H, and-C (O) -NP1P 2;

P1 and P2 are each independently a group selected from the group consisting of: H. c1-10 alkyl, C6-10 aryl, C7-10 alkylaryl and C7-10 aralkyl, wherein said C1-10 alkyl, C6-10 aryl, C7-10 alkylaryl and C7-10 aralkyl are each unsubstituted or further substituted with at least one group selected from the group consisting of-NH ₂、-OH、-SO₃ H, -CN, -COOH and-C (O) NH ₂.

According to the invention, the compounds of formula (I) have a total number of primary, secondary and/or imine groups of from 1 to 20.

According to the invention, the compounds having an amino group have an amino pKa value of from 1 to 16, preferably from 4 to 14.

Preferably, the amino group pKa value of the amino group-containing compound is equal to or less than the pH value of the depolymerizing agent.

In the present invention, the concentration of the compound having an amino group in the depolymerizing agent is 1 to 50mmol/L, preferably 2 to 20mmol/L.

According to another embodiment, the ammonium ion containing compound contains an anion selected from the group consisting of chloride, bromide, iodide, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, thiocyanate, sulfate, hydrogen sulfate, sulfite, hydrogen sulfite, carbonate, hydrogen carbonate, formate, acetate, oxalate, propionate, malonate, citrate, and combinations thereof.

More specifically, the ammonium ion-containing compound is at least one selected from the group consisting of ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium nitrate, ammonium thiocyanate, ammonium hydrogen sulfite, ammonium oxalate, ammonium hydroxide, ammonium bisulfate, and ammonium bicarbonate.

According to the invention, in the depolymerizing agent, the concentration of the compound containing ammonium ions is 1 to 50mmol/L, preferably 2 to 20mmol/L.

According to one embodiment, the depolymerizing agent comprises at least one compound having an amino group and at least one compound containing an ammonium ion.

In this embodiment, the total concentration of the at least one compound having an amino group and the at least one compound containing an ammonium ion in the depolymerizing agent is 2 to 50mmol/L, preferably 2 to 20mmol/L, more preferably 2 to 10mmol/L.

In this embodiment, the molar ratio of the at least one compound having an amino group and the at least one compound containing an ammonium ion is in the range of 10:1 to 1:10, preferably 4:1 to 1:4.

According to the detection method of the present invention, the pH value at the time of treating the blood sample with the depolymerizing agent is at least 3.0, preferably at least 7.0, more preferably 7.5 to 11.0.

According to one embodiment, the blood analyzer comprises an impedance detector, the blood sample is hemolyzed with a red blood cell lysing agent prior to performing the detection, and the method comprises the steps of:

the blood sample treated with the deagglomerating agent and the erythrocyte lysing agent is detected with an impedance detector in a blood analyzer to obtain an electrical signal of particles in the blood sample and further obtain a white blood cell detection parameter after eliminating the aggregation interference.

According to another embodiment, the blood analyzer comprises an optical detector, the blood sample is stained with a fluorescent dye and hemolyzed with a red blood cell lysing agent, and the method comprises: detecting the blood sample treated by the depolymerizing agent, the fluorescent dye and the erythrocyte lysing agent by an optical detector in a blood analyzer to obtain at least two optical signals of particles in the blood sample so as to obtain detection information of white blood cells;

Preferably, the at least two light signals are selected from the group consisting of fluorescence intensity signals, forward scattered light intensity signals and side scattered light intensity signals.

According to a second aspect of the present invention, there is provided a reagent for detecting leukocytes in an extracorporeal blood test, wherein the reagent comprises at least one selected from the group consisting of a compound having an amino group and a compound containing an ammonium ion, a erythrocyte lysing agent, a buffer and an osmotic pressure regulator, and optionally a fluorescent dye and/or a surfactant, wherein the concentration of the compound having an amino group is 1 to 50mmol/L, and the concentration of the compound containing an ammonium ion is 1 to 50mmol/L.

According to a specific embodiment, the compound having an amino group is selected from the group consisting of compounds of the following formula (I):

R1-NH-R2 (I)

Wherein the compound of formula (I) is as defined above.

In the present invention, the compound of formula (I) may have a total number of primary amino groups, secondary amino groups and/or imino groups of 1 to 20.

In the present invention, the compound having an amino group may have an amino pKa value of 1 to 16, preferably an amino pKa value of 4 to 14.

According to a preferred embodiment, the amino pKa value of the compound with an amino group is equal to or lower than the pH value of the reagent.

In the reagent, the concentration of the compound having an amino group may be 2 to 20mmol/L.

According to a specific embodiment, the ammonium ion containing compound is as defined above.

In the reagent of the present invention, the concentration of the ammonium ion-containing compound may be 2 to 20mmol/L.

According to one embodiment, the reagent comprises at least one compound having an amino group and at least one compound containing an ammonium ion.

In this embodiment, the total concentration of the at least one compound having an amino group and the at least one compound containing an ammonium ion in the reagent is 2 to 50mmol/L, preferably 2 to 20mmol/L, more preferably 2 to 10mmol/L.

The reagent of the invention has a pH of at least 3.0, preferably at least 7.0, more preferably between 7.5 and 11.

According to a third aspect of the present invention there is provided the use of a platelet depolymerising reagent in the detection of leukocytes, wherein the platelet depolymerising reagent is as defined above for the depolymerising reagent.

In such applications, the platelet depolymerization agent has a pH of at least 3.0, preferably at least 7.0, and more preferably between 7.5 and 11.0.

According to a specific embodiment, the platelet depolymerizing agent further comprises a red blood cell lysing agent, a buffer, and an osmolality adjusting agent, and optionally a fluorescent dye and/or a surfactant.

In the blood detection method of the present invention, the depolymerization of the aggregated platelets by the depolymerizing agent can depolymerize the platelets in a short time without additional conditions such as water bath temperature and prolonged reaction time, so that the existing detection steps are not required to be changed, and the method is suitable for any blood analyzer. The blood sample after depolymerization treatment can obtain accurate leukocyte detection parameters. Moreover, the reagent for detecting the white blood cells has no adverse effect on other conventional reagents for detection, can obtain better depolymerization effects under the condition of multiple platelet aggregation, can conveniently eliminate the pseudo aggregation of the platelets in the sample, and can obtain accurate detection parameters of the blood cells.

Drawings

FIG. 1 shows a schematic of the mechanism of depolymerization and repolymerization of aggregated platelets by compounds having amino groups;

FIG. 2 shows a three-dimensional scatter plot of particles in a blood sample obtained in an optical detection device, wherein the optical information features of aggregated and disaggregated platelets, respectively, are shown;

FIG. 3 shows a graph of the deagglomeration effect of a compound having an amino group on aggregated platelets at different ambient pH conditions;

FIG. 4 shows a graph of the deagglomeration effect of compounds with different amino pKa values on aggregated platelets at pH 9.5;

FIG. 5 shows a graph of the induction of platelet aggregation in blood samples over time of ADP at various final concentrations;

FIG. 6 shows a graph of the effect of compounds with amino groups on the depolymerisation of platelet aggregation induced by ADP at different concentrations over time;

FIG. 7 shows a graph of the induction of platelet aggregation in blood samples over time of THR at various final concentrations;

Figure 8 shows a graph of the deagglomeration effect of compounds with amino groups on platelet aggregation induced by THR at different concentrations over time;

FIG. 9 shows a graph of the induction of platelet aggregation in blood samples over time for COL at various final concentrations;

FIG. 10 shows a graph of the depolymerization effect of compounds with amino groups on platelet aggregation induced by COL at different concentrations over time;

FIG. 11 shows a graph of the induction of platelet aggregation in blood samples over time of RIS at various final concentrations;

FIG. 12 shows a graph of the effect of compounds with amino groups on the deagglomeration of RIS-induced platelet aggregation at different concentrations over time;

FIG. 13 shows the depolymerization effect of various ammonium ion containing compounds on ADP-induced aggregated platelets at different concentrations;

FIG. 14 shows the deagglomeration effect of 1-acetylguanidine and ammonium chloride, respectively and in combination, on aggregated platelets at different concentrations;

FIG. 15 shows a three-dimensional scatter plot of a blood sample before and after induced aggregation using a conventional lysate and containing a lysate with amino compounds in an optical detection device to detect leukocytes in the sample;

FIG. 16 shows a three-dimensional scatter plot of a blood sample before and after induced aggregation in an optical detection device using a conventional lysate and a lysate containing ammonium chloride to detect leukocytes in the sample;

FIG. 17 shows a three-dimensional scattergram of a blood sample before and after induced aggregation in which white blood cells in the sample are detected using a conventional lysate and a lysate containing a combination of an amino compound and an ammonium ion-containing compound in an optical detection device;

FIG. 18 is a graph showing the comparison of the deagglomeration effect of conventional dilutions and dilutions containing amino compounds in clinic for samples exhibiting pseudo-aggregation of platelets;

figure 19 shows a graph comparing the deagglomeration effect of conventional dilutions and dilutions containing ammonium chloride in clinic for samples showing pseudo-aggregation of platelets.

Detailed Description

The following description of embodiments of the present invention will be made more apparent and fully by reference to the accompanying drawings and specific examples, in which it is evident that the embodiments described are only some, but not all, embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

It should be noted that, in the present invention, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a method or article of manufacture that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such method or article of manufacture. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other related elements in a method or apparatus that includes the element.

As used herein, "amino" unless specifically indicated otherwise refers to primary and/or secondary amino groups, and does not include tertiary amino groups.

As used herein, unless otherwise indicated, "compounds having an amino group" refers to compounds having at least one amino group (primary and/or secondary amino groups). The compounds may optionally also contain tertiary amino groups and/or imino groups, in particular may also optionally contain imino groups.

The inventors have unexpectedly found that a class of amino-containing compounds has a significant deagglomeration of the pseudo-aggregates of platelets caused by different causes, and that the aggregates of platelets can be eliminated after about 30 seconds without additional heating or cooling, and without long action time, by adding a small amount of this substance to the solution of the blood sample to be tested. Thus, the present invention provides the use of such substances to prevent and/or eliminate platelet aggregation in blood samples. The de-pseudo-aggregated platelets not only can obtain the real number of platelets in the blood sample, thereby providing a reliable reference basis for clinical decision, but also can eliminate the influence of larger particles formed by the aggregation of the platelets on the classification and counting of other blood cells.

Without wishing to be bound by theory, the principle of deagglomeration of pseudo-aggregated platelets by the amino-bearing compounds of the present invention is speculated that the amino/imino groups in the compounds bind in solution to the phosphatidylserine membrane of the platelets and hydrogen bond with the carbonyl ester groups (-O-C (O) -) on the phosphatidylserine membrane (see fig. 1, which illustrates the principle of deagglomeration of amino-bearing compounds). The amino group of the compound thus bound to the phosphatidylserine layer membrane blocks the calcium ion channel, preventing the inflow of calcium ions. Whereas aggregation of platelets is known to require participation of calcium ion influx, such compounds disaggregate aggregated platelets by blocking calcium ion influx by amino groups. As the pH gradually decreases, the amino groups on the compound become protonated. Although the specific cause is not clear, it is speculated that the protonated amino groups on the compound are no longer hydrogen bonded to the carbonyl ester groups on the phosphatidylserine layer membrane, and that calcium ions re-flow into the blood cells, which in turn cause aggregation. In theory, the primary, secondary or imino groups, with the exception of the tertiary amino group, to which at least one hydrogen atom is attached to the N atom, have a certain platelet depolymerization function.

The present inventors have further studied and found that the larger the number of amino groups/imino groups in the compound having amino groups, the better the depolymerization effect. In addition, the depolymerization effect of the primary amino group, the secondary amino group and the imino group alone is sequentially reduced. In general, the depolymerization effect of a compound having strong hydrophilicity is also higher than that of a compound having weak hydrophilicity. Hydrophilic substituents such as hydroxyl, carboxyl, amide, sulfonate and the like have substantially no effect on the depolymerization effect of the amino/imide groups, and in some cases (e.g., hydroxyl, polyhydroxy) even facilitate depolymerization. This may be related to the fact that these groups increase the water solubility of the compound and the affinity with the phosphatidylserine layer membrane. Hydrocarbyl groups, such as alkyl, aryl, etc., have little effect on platelet depolymerization as long as they do not affect certain water solubility of the compound. Hydrophobic groups, such as ester groups (-C (O) -O-), carbonyl groups (-C (O) -) reduce the depolymerization effect to some extent.

Therefore, compounds having a hydroxyl group, a carboxyl group, a sulfonic acid group, such as an alcohol amine, an amino acid, a sulfonic acid, or the like are more preferable. In addition, substances having a plurality of amino groups and/or imino groups are also preferable such as guanidine, short peptide, and the like. Preferably, the amino-bearing compound has at least one primary or secondary amino group, preferably 1 to 20 primary, secondary and/or imino groups. Preferably, the amino compound has at least one primary amine group, more preferably 1 to 4 primary amine groups.

According to the above-presumed mechanism, the amino group in the amino group-bearing compound exists in a solution in a deprotonated form upon depolymerization. The amino/imino groups in different compounds have different ability to bind hydrogen ions in solution. This ability to bind/dissociate hydrogen ions can be expressed in terms of dissociation constant pKa.

The amino compound has the following equilibrium formula in solution:

To obtain a suitable platelet depolymerization effect, the amino groups of the compound should have a suitable pKa value. For compounds having multiple amino/imino groups, the pKa of the amino group referred to herein refers to the first dissociation constant, i.e., the pKa of the amino or imino group of the first dissociated hydrogen ion.

Typically the amino group of the compound should have a pKa value of from 1 to 16, preferably from 1 to 14, more preferably from 4 to 14.

In addition, the depolymerization ability is improved when the pKa value of the amino group in the same compound is smaller than the pH value of the solution. From the above principle of the compound's depolymerization of aggregated platelets, it can be appreciated that as the pH gradually increases, the equilibrium of the amino groups in the compound with hydrogen ions in solution moves in the direction of dissociation, more amino groups are deprotonated, and thus more amino groups are bound to the phosphatidylserine membrane of platelets through hydrogen bonds, blocking the influx of calcium, and acting as depolymerization. Such binding further promotes deprotonation of the amino groups of the more chemical compounds, thereby allowing the depolymerization to be accomplished in a shorter period of time without increasing the temperature.

Generally, in extracorporeal blood tests, the pH of the sample to be tested can be varied over a wide range as required. However, in order to obtain a preferable depolymerization effect, the prevention and/or elimination of pseudo-aggregation of platelets is carried out in a solution having a pH of at least 3.0, preferably at least 7.0, more preferably 7.5 to 11, particularly preferably 9.5 to 11. The pH of the solution may be adjusted according to the type of compound used and the detection requirements to obtain the desired deagglomeration effect.

The effective concentration at which a compound having an amino group can deagglomerate pseudo-aggregated platelets is related to the number and type of amino groups contained in the compound, and also to factors such as the hydrophilicity of the compound. Generally, the concentration of the compound having an amino group for platelet depolymerization is in the range of 1 to 50mmol/L, for example: 1. 2,5, 10, 15, 20, 25, 30, 35, 40, 50 mmol/L. According to a preferred embodiment, said concentration is in the range of 2 to 20mmol/L, more preferably in the range of 5 to 15 mmol/L.

The present invention provides the use of a compound having an amino group as shown in the structural formula (I) and salts thereof for preventing and/or eliminating pseudo-aggregation of platelets in blood samples in an in vitro blood test:

R1-NH-R2 (I)

According to one embodiment, wherein, in the compound of formula (I), R1 and R2 are identical or different and are each independently a group selected from the group consisting of R1 and R2 are not simultaneously H, the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, substituted or unsubstituted C7-10 aralkyl, -C (O) -Q1, and-C (O) -O-Q2,

Preferably, in the compound of formula (I), R1 and R2 are the same or different and are each independently selected from the group consisting of R1 and R2 are not simultaneously H, the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, substituted or unsubstituted C7-10 aralkyl, -C (O) -Q1, and-C (O) -O-H,

Further, in the compound of formula (I), R1 and R2 are the same or different and are each independently a group selected from the group consisting of H, -C (NH) -NH ₂, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted phenethyl, and-C (O) -Q1, provided that R1 and R2 are not simultaneously H, wherein Q1 is defined above.

Further, in the compound of formula (I), Q1 is a substituted or unsubstituted C1-6 alkyl group, a substituted or unsubstituted phenyl group, a substituted or unsubstituted benzyl group or a substituted or unsubstituted phenethyl group;

Wherein said substitution means substitution with at least one group selected from the group consisting of-NP 1P2, -SO ₃ H, -OH, and-C (O) -NP1P2,

P1, P2 and P3 are each independently a group selected from the group consisting of: H. c1-6 alkyl, phenyl, benzyl and phenethyl, wherein said C1-6 alkyl, phenyl, benzyl and phenethyl are unsubstituted or further substituted with at least one group selected from the group consisting of-NH ₂、-OH、-SO₃ H, -COOH and-C (O) NH ₂.

The alkyl group of C1-16 as described herein may be a straight or branched chain alkyl group, preferably a C1-14, C1-12 alkyl group, more preferably a C1-10 alkyl group, and even more preferably a C1-6 alkyl group. Examples of the alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-octyl, dodecyl, and hexadecyl groups.

The C6-10 aryl groups described herein may be phenyl or naphthyl.

The C7-14 alkylaryl groups described herein can be mono-or polyalkyl-substituted aryl groups. C7-10 alkylaryl groups such as tolyl, ethylphenyl, propylphenyl, butylphenyl, xylyl, cresyl, diethylphenyl, and the like are preferable, but are not limited thereto.

The C7-14 aralkyl groups described herein may be phenylalkyl groups. Aralkyl groups of C7-10 such as benzyl, phenethyl, phenylpropyl, phenylbutyl, etc. are preferred, but are not limited thereto.

The term substituted as used herein refers to substitution by at least one of the defined substituents. For the amino substituent-NP 1P2, it may be preferred according to the present invention that the amino substituent is polyamino, up to 20 amino groups. For example, 1 to 18, such as 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, etc. Preferably, the amino group is a primary amino group and/or a secondary amino group, more preferably a primary amino group. For the hydroxyl substituent-OH, a more preferred substituent is one. The number of substitution of hydroxyl groups may be 1 to 6, such as 2, 3, 4, 5, 6. Further, sulfonic acid groups, carboxyl groups, and nitrile groups are also preferable.

Halogen as described herein generally refers to-F, -Cl, -Br, -I, preferably-F, -Cl, -Br.

Preferred amino-bearing compounds according to the invention are guanidines, alcohol amines (in particular polyhydroxy alcohol amines, such as1, 3-diamino-2-propanol), amino acids (preferably lysine, glutamine, glycine), short peptides (e.g. dipeptides) and polyamino-substituted, multi-branched or linear alkanes (e.g. 2- (aminomethyl) propane-1, 3-diamine, 1, 3-propanediamine, etc.), according to the examples below.

Compounds of formula (I) that prevent and/or eliminate pseudo-aggregation of platelets may be cited: sulfonic acids, such as sulfamic acid, taurine, sulfanilic acid, 2, 5-diaminobenzenesulfonic acid; amino acids such as glutamic acid, glutamine, arginine, lysine, alanine, glycine, N-tris (hydroxymethyl) methylglycine, 4-aminobutyric acid; short peptides, such as dipeptides, e.g. polylysine, glycine dimer; alcohols/phenols amines (in particular polyhydroxy-substituted amines), such as ethanolamine, triethanolamine, 3-amino-1-propanol, 4-amino-1-butanol, 4-hydroxybenzylamine, tyramine (4-hydroxyphenylethylamine), 1, 3-diamino-2-propanol, 3-amino-1, 2-propanediol, tris-hydroxymethyl-aminomethane, p-hydroxyaniline, 3, 5-dihydroxyaniline, 4-aminophenol; arylamines such as m-phenylenediamine, m-trimellitic amine; alkylamines, such as ethylamine, n-propylamine, n-butylamine, t-butylamine, 1, 3-propanediamine, 2- (aminomethyl) propane-1, 3-diamine; aminonitriles, such as aminoacetonitrile, triaminopropionitrile; amides such as formamide, acetamide, carboxamide, asparagine methyl ester, 2- (methylamino) butanediamide, 2-aminoacetamide, 2-aminopropionamide, glutamine; guanidine such as guanidine, biguanide, aminoguanidine, methylguanidine, { [ amino (imino) methyl ] amine } -acetic acid, 1-acetylguanidine, etc., but is not limited thereto.

More preferred compounds of formula (I) may be hydroxy-substituted amines, alkylamines, benzenesulfonamines, amino acids and short peptides, and guanidines.

Salts of the compounds having an amino group are also within the scope of the invention. In particular, salts of compounds having acidic sulfonic acid groups, carboxyl groups and the like. The salt may be a salt of an alkali metal (e.g., potassium, sodium), a salt of an alkaline earth metal (e.g., magnesium), a salt of an ammonium cation, or the like; or may be an internal salt. Salts of ammonium salt ions are particularly preferred.

In the method for detecting the exclusion of platelet aggregation interference of the present invention, the blood sample may be treated with a depolymerizing agent comprising at least one of the above-mentioned compound having an amino group and its salt, particularly a compound represented by formula (I) and its salt. The above treatment may of course also be performed using a depolymerizing agent comprising two or more of a compound having an amino group and a salt thereof.

In the depolymerizing agent of the present invention, as described above, the concentration of the compound having an amino group is approximately in the range of 1 to 50mmol/L, preferably in the range of 2 to 20mmol/L, and more preferably in the range of 5 to 15 mmol/L. If two or more of the compounds are used, the total concentration of each compound corresponds to the above range. As described above, the causes of pseudo-platelet aggregation are various, and the degree of aggregation and the degree of deagglomeration vary greatly. Therefore, the compound having an amino group, particularly the compound represented by the above formula (I), is applied in a wide concentration range. Wherein for the case where a compound having a strong depolymerization ability (e.g., a compound having a large number of amino groups and/or a good cell membrane affinity) is applied to a case where depolymerization is easier, a lower concentration may be used, whereas for the case where a compound having a slightly weak depolymerization ability is applied to a case where depolymerization is difficult or the degree of aggregation is high, a higher concentration may be used. The selection of the appropriate concentration can be reasonably determined by one skilled in the art based on the examples of the specific embodiments below in combination with the actual need.

Furthermore, further studies according to the present inventors have found that a good platelet depolymerization effect can be obtained also when the blood sample is treated with an ammonium ion-containing reagent. Moreover, the depolymerization of the aggregated platelets by the ammonium ions can also be accomplished in a short time without additional reaction conditions.

As will be seen from the examples below, each of the ammonium salts has a substantially similar deagglomerating effect on aggregated platelets at a given pH. The ammonium ion-containing compound according to the present invention may contain an anion selected from the group consisting of chloride, bromide, iodide, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, hydrogen sulfide, thiocyanate, sulfate, hydrogen sulfate, sulfite, hydrogen sulfite, oxalate, carbonate, hydrogen carbonate, formate, acetate, oxalate, propionate, malonate, citrate, and combinations thereof.

Examples of the ammonium ion-containing compound include, but are not limited to, ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium nitrate, ammonium thiocyanate, ammonium hydrogen sulfite, ammonium oxalate, ammonium hydroxide, ammonium hydrogen sulfate, ammonium bicarbonate, and the like.

The depolymerizing agent may include one kind of compound containing an ammonium ion, or may be plural. Preferred compounds containing ammonium ions are ammonium chloride, ammonium bromide, ammonium phosphate, ammonium hydrogen phosphate, and the like.

When the ammonium ion-containing compound is further included in the composition, the concentration of the ammonium ion-containing compound may be in the range of 1 to 50mmol/L, for example 1,2, 5, 10, 15, 20, 25, 30, 35, 40, 50mmol/L. The concentration of the compound containing an ammonium ion is preferably in the range of 2 to 20mmol/L, more preferably 5 to 15 mmol/L.

The inventors found that when the platelet depolymerization is performed using an ammonium ion-containing compound, the pH is preferably at least 7.0, more preferably 7.5 to 11, particularly preferably 9.5 to 11.0.

Furthermore, the present inventors have further found that when the compound having an amino group is combined with a compound containing an ammonium ion, a good depolymerization effect can be obtained at a low concentration. That is, when the total concentration of both is comparable to that when either one of them is used alone, a better effect can be obtained than when the compound containing an ammonium ion or the compound having an amino group is used alone. That is, the combined use of the two has a synergistic effect on platelet depolymerization. Thus, when used in combination, the total concentration of the two can be in the range of 2 to 50mmol/L, such as 2, 5, 10, 15, 20, 25, 30, 35, 40, 50mmol/L. Preferably, the depolymerizing agent may include 1 to 20mmol/L of the compound having an amino group and 1 to 20mmol/L of the compound having an ammonium ion, more preferably the depolymerizing agent may include 1 to 10mmol/L of the compound having an amino group and 1 to 10mmol/L of the compound having an ammonium ion, and even include 1 to 5mmol/L of the compound having an amino group and 1 to 5mmol/L of the compound having an ammonium ion. The use ratio of the two is not particularly limited. As an example, the molar ratio of the compound having an amino group and the compound containing an ammonium ion may be in the range of 1:10 to 10:1, preferably in the range of 1:4 to 4:1 and may be 1:4, 1:2, 1:1, 2:1, 4:1, etc. Most preferably, the molar ratio of the two is 1:1.

Without wishing to be bound by theory, the mechanism by which ammonium ions depolymerize platelets is not yet clear. It is presumed that the concentration of ammonium ions in the solution forms hydrated ammonia. However, if ammonia is directly used, the depolymerization performance is inferior to that of the use of an ammonium ion-containing compound, particularly ammonium salts such as ammonium chloride, ammonium phosphate and ammonium halide, although it is also superior. Therefore, it is presumed that some kinds of anions in the solution may also play a role in promoting platelet depolymerization. This is in contrast to the presumed mechanism of depolymerization of compounds having amino groups.

As can be seen from the examples below, when a compound having an amino group and/or a salt thereof is used in combination with a compound containing an ammonium ion, a significantly improved depolymerization effect is obtained.

According to the present invention, the blood analyzer for performing the detection is not particularly limited. And there is no particular limitation on the detector, such as an impedance detector, an optical detector, etc., may be used.

Taking the optical detector as an example, a plurality of aggregated platelets are identified as one particle in the optical channel of the hematology analyzer, and thus such aggregated platelets are larger in volume than individual platelets, stronger in fluorescence intensity than individual platelets, and smaller in number than unagglomerated platelets. As reflected in the detection information, as can be seen from the three-dimensional scattergram measured in the merits blood cell analyzer shown in fig. 2, the aggregated platelet-aggregated sample showed a larger platelet forward scattered light intensity information (FS), reflecting a larger particle volume, and a high fluorescence intensity (FL). The volume of partially aggregated platelets reaches the volume of leukocytes and also affects the count of leukocytes. After deagglomeration, platelets in the optical channel, particle signal recognition volumes decrease, fluorescence intensity decreases, and number increases. Further referring to fig. 2, it can be seen from the three-dimensional scatter diagram that the particle population of the depolymerized platelets reflects a significant decrease in platelet volume corresponding to the forward scattered light (FL), an increase in particle count, and a decrease in fluorescence intensity.

For impedance detectors, similarly, the aggregated platelets will also be treated as a particle, producing an electrical signal. Since the particle volume becomes large after aggregation, the volume of the partially aggregated platelets reaches the volume of the white blood cells, which affects the count of white blood cells and the change of the histogram. This effect is evident in early three-class hematology analyzers.

According to the method of the present invention, by adding a depolymerizing agent comprising at least one of the above-mentioned compound having an amino group and a salt thereof and an ammonium ion-containing compound to a sample, aggregation of platelets can be prevented, and platelets that have already been aggregated can be depolymerized, so that accurate platelet detection information can be obtained.

As used herein, "detection information" includes raw signals (e.g., electrical pulses, light intensities, etc.), processed information such as histograms, scatter plots, etc., and finally reported classification and/or counting information.

The depolymerizing agent of the present invention is effective for platelet aggregation due to various reasons, and thus there is no particular limitation on the blood sample. The sample may be from peripheral blood, such as from peripheral blood or venous blood. The sample may be obtained by, for example, lancing with a fingertip needle or by venous sampling with an anticoagulant tube. Freshly collected blood may use anticoagulation tubes coated with conventional anticoagulants such as EDTA, as the composition of the invention also works well for platelet pseudo-aggregation due to EDTA.

Generally, the sample after blood collection may be subjected to any conventional process to obtain a test sample. The process may be, for example: dilution treatment, dyeing treatment, lysis treatment, and the like. In the methods of the invention, the deagglomerating agent may be added to the sample as a treatment agent alone (e.g., as a platelet deagglomerating agent) or may be added to the sample with the treatment agent at any one of the steps of treating the sample. Thus, the depolymerizing agent of the present invention can be formed as a single treatment agent; the diluent, the stain or the lysing agent having the platelet depolymerization effect can also be formed by including at least one of the compound having an amino group and a salt thereof, and/or the compound containing an ammonium ion in a reagent for dilution, staining or lysing treatment, thereby simultaneously exerting the effect of preventing/eliminating platelet depolymerization in any one treatment without impeding the treatment.

The reaction to depolymerize platelets in the method of the invention does not require additional incubation (e.g., low temperature or heat). The depolymerization agent and the sample are mixed uniformly, and then the depolymerization of the platelets can be completed in about 30 seconds, so that the sample can be detected.

According to the invention, the sample may be from a mammal, preferably a primate, more preferably a human.

More specifically, the detection method of the present invention may include treating a blood sample with a depolymerizing agent comprising at least one compound selected from the group consisting of a compound having an amino group and optionally an ammonium ion-containing compound to prevent and/or eliminate platelet aggregation in the blood sample, and detecting the blood sample treated with the depolymerizing agent with a blood analyzer to obtain at least detection information of white blood cells.

The detection information may be a pulse signal for particles in the sample or an optical signal, depending on the detector used. The blood analyzer may process the detection information to further obtain detection parameters such as histograms, scatter plots, classification and/or counting of blood cells.

According to one embodiment, the blood analyzer comprises an impedance detector, the blood sample may be further hemolyzed with a red blood cell lysing agent in addition to being treated with a depolymerizing agent prior to performing the detection, and the method comprises the steps of: the blood sample treated with the deagglomerating agent and the erythrocyte lysing agent is detected with an impedance detector in a blood analyzer to obtain an electrical signal of particles in the blood sample, and further to obtain a leukocyte detection parameter.

Three classifications and counts of white blood cells were obtained by impedance detectors.

According to yet another embodiment, the blood analyzer comprises an optical detector, the blood sample is further stained with a fluorescent dye and hemolyzed with a red blood cell lysing agent, and the method comprises: detecting the blood sample treated by the depolymerizing agent, the fluorescent dye and the erythrocyte lysing agent by an optical detector in a blood analyzer to obtain at least two optical signals of particles in the blood sample so as to obtain detection information of white blood cells.

Preferably, in this embodiment, the at least two light signals are selected from the group consisting of a fluorescence intensity signal, a forward scattered light intensity signal and a side scattered light intensity signal.

In leukocyte detection, for example, four classifications (i.e., eosinophils, neutrophils, monocytes, and lymphocytes) can be obtained using the DIFF detection channel of the michaeli blood detector, or further detection information of basophils can be obtained using the WNB channel as in the michaeli blood detector. "

In the above detection method, the depolymerizing agent, the fluorescent dye and the erythrocyte lysing agent may be separate reagents or may be mixed reagents in any combination. For example, the depolymerizing agent may be formed as a diluent, a stain, a lysing agent, a stain-lysing agent, or the like having a platelet depolymerizing effect.

To this end, the invention also provides a reagent for detecting leukocytes in an extracorporeal blood test.

Since in extracorporeal blood tests the volume of the reagent used for treating the blood sample is much greater than the volume of the blood sample itself, the treatment conditions of concentration, pH, etc. for the purposes described above are generally equally applicable to the reagent of the invention against platelet aggregation.

According to the present invention, the reagent may include at least one selected from the group consisting of an amino group-having compound having a concentration of 1 to 50mmol/L and a salt thereof and an ammonium ion-containing compound having a concentration of 1 to 50mmol/L, a buffer and an osmotic pressure regulator, and optionally a fluorescent dye and/or a surfactant. The concentration defined in the present invention is the total concentration of two or more compounds having amino groups. Likewise, the concentration is also the total concentration for two or more compounds containing ammonium ions. The at least one compound having an amino group is as defined above and will not be described in detail herein. The concentration is also preferably 2 to 20mmol/L, more preferably 5 to 10mmol/L.

Further at least one compound containing an ammonium ion is also as defined above, while its concentration is preferably 2 to 20mmol/L, more preferably 5 to 10mmol/L. According to a preferred embodiment, the reagent contains both the compound having an amino group and the compound having an ammonium ion, both of which may vary in concentration in the range of 1 to 20mmol/L, 1 to 10mmol/L, and even 1 to 5 mmol/L. The molar ratio of the two may be any ratio, and may vary, for example, in the range of 1:10 to 10:1, preferably 1:4 to 4:1, of the compound having an amino group to the compound containing an ammonium ion.

The pH of the reagent is not particularly limited, and is related to the reagent use requirements thereof. Preferably, the pH of the reagent is at least 3.0, at least 7.0, preferably 7.5 to 11, particularly preferably 9.5 to 11. The pH of the reagent may be adjusted by conventional buffers. The present invention is not particularly limited as to the kind of buffer used for the reagent. For example, a citric acid buffer pair, a phosphoric acid buffer pair, a boric acid buffer pair, a phthalate buffer pair, a 3- (N-morpholino) ethane sulfonic acid buffer pair, a 4-hydroxyethylpiperazine ethane sulfonic acid buffer pair, etc., may be used, but is not limited thereto.

The agent should have a certain osmotic pressure. The osmotic pressure may vary depending on the actual requirements of the agent, for example for diluents, it may be in the range of 180 to 240mOsm/L, preferably 200mOsm/L; as a further example, for a lysing agent, it may be in the range of 70 to 130mOsm/L, preferably 90mOsm/L. The agent may include an osmolality adjusting agent. The agent of the present invention is not particularly limited, and for example, may be an inorganic salt such as sodium chloride, potassium chloride, etc.; sugars such as glucose, mannose, fructose, maltose, etc., but are not limited thereto.

The agent may also include a surfactant. Depending on the purpose of the assay, the reagents may include surfactants of different functions. For example, in the case of leukocyte detection, surfactants that alter the permeability of the cell membrane may be included in the reagent to facilitate the entry of nucleic acid dyes into the cell interior through the cell membrane for binding to nucleic acids. Such surfactants are for example: phenoxyethanol, sodium dioctadecylamine hydrochloride, sodium N-methylamide carboxylate, and the like, but is not limited thereto.

In addition, the agent may include other necessary components such as a preservative, an antibacterial agent, a cell membrane protecting agent, a chelating agent, etc., as needed. Those skilled in the art can choose to add as desired.

According to one embodiment of the invention, the reagent may act as a diluent for the blood sample. For example, the diluent may be used for impedance detection or may be used for optical detection. The formulation of diluents which may be mentioned are, for example:

According to the invention, the reagent may also be a dye. In general, because dyes are more soluble in organic solvents, in platelet detectors, the dyes are stored separately in the form of organic solutions. Dyes may be added to the above diluents as needed to prepare a colorant. The dye in the stain may be a nucleic acid dye. Nucleic acid specific dyes such as asymmetric cyanines, thiazole orange TO, oxazole orange YO, acridine orange AO and the like, specifically, PI, DAPI, hoechst series (e.g., hoechst33258, hoechst 33342) and the like are exemplified, but not limited thereto. A more preferred nucleic acid dye is an asymmetric cyanine dye, such as SYBR Green, for example. The pH at which such dyes dye optimally dye is substantially consistent with the pH at which the compounds of the present invention having amino groups and compounds containing ammonium ions optimally function. The stain may also be a mitochondrial stain (e.g., janus Green B, mitoLite Red, rhodamine 123, mitotracker Green, mitotracker Deep Red, mitotracker Red, etc.) or a membrane stain (e.g., diA, diD, diI, diO, diR, diS, FDA, alexa Fluor 488, super Fluor 488, etc.), depending on the detection needs.

Such a stain formulation may be:

in addition, the reagent of the invention can also be a lysing agent and can be used for detecting white blood cells. The lysing agent used for the reagent may be any conventional erythrocyte lysing agent. As previously mentioned, the amino group-containing compound, particularly the ammonium ion-containing compound, in the reagent of the present invention has no adverse effect on erythrocytes in the defined concentration range, and thus the type and concentration of the lysing agent in the reagent, and the manner of use, are the same as in the prior art. As the erythrocyte lysing agent, there may be mentioned, but not limited to, quaternary ammonium salts (e.g., dimethylbenzyl ammonium chloride, dodecyltrimethyl ammonium chloride, dimethylbenzyl alkyl ammonium chloride, etc.). Such a lysing agent formulation may be:

again, according to the foregoing, the reagent of the present invention comprising the compound having an amino group and the compound having an ammonium ion has a low concentration of an active ingredient in preventing/eliminating pseudo-aggregation of platelets, and does not require additional reaction conditions (such as cooling or heating) nor an increase in reaction time, and can complete depolymerization of platelets for 30 seconds, so that the compound has no adverse effect on the detection of blood, has no effect on the detection step and conditions, and can eliminate interference of platelet aggregation under existing equipment and detection methods.

Examples

Example 1 comparison of structurally similar ability of amino-and amino-free Compounds to depolymerize platelets

This example compares the effect of amino-containing and non-amino-containing compounds of similar structure and substituents on platelet depolymerization. The test compounds are shown in table 1 below:

Table 1: tested compounds having amino groups and structurally similar compounds having no amino groups

The compounds shown in Table 1 were added to dilutions of blood cell analyzers to prepare dilutions of blood samples. Platelet aggregation blood samples were obtained from blood samples treated with platelet aggregation inducer ADP at a final concentration of 0.01 mmol/L. The composition of the diluent is as follows: citric acid (0.5 g/L), surfactant phenoxyethanol (0.1 g/L), bacteriostat (6 g/L), sodium chloride (3 g/L) and EDTA (0.1 g/L). To the diluted solutions, the compounds shown in Table 1 (final concentration: 0.01 mol/L) were added, respectively, and the pH of the diluted solutions was adjusted to 14, and the osmotic pressure was adjusted to 200mOsm/L to obtain treatment solutions.

Venous blood from healthy subjects is collected into a blood anticoagulant tube for use. Two 1ml portions of blood were taken, one of which was added with the above-mentioned diluent, and after mixing, the platelet count was measured in a blood cell analyzer (michaelbc-6000 Plus), and was noted as: plt_o (not polymerized); the other part is added with platelet aggregation inducer ADP (final concentration is 0.01 mmol/L) and mixed evenly. After 5 minutes, the platelets form aggregates, and the platelet count is recorded by detecting the blood containing the aggregated platelets by the blood cell analyzer after the diluent is added: plt_o (not depolymerized). 1ml of blood is taken, platelet aggregation inducer ADP (final concentration is 0.01 mmol/L) is added, the mixture is uniformly mixed, the treatment liquid is added after 5 minutes, the count of platelets in the blood after the treatment is detected by the blood cell analyzer after the uniform mixing, and the count is recorded as follows: plt_o (depolymerization).

In general, plt_o (not depolymerized) < plt_o (not depolymerized).

The term "depolymerization rate" referred to herein is calculated using the following formula:

depolymerized samples: depolymerization rate = plt_o (depolymerization)/plt_o (unpolymerized); or alternatively

Non-depolymerized (control) samples: depolymerization rate = plt_o (not depolymerized)/plt_o (not depolymerized).

The treatment solutions containing the compounds of table 1 were tested one by one for the ADP-induced blood treatment according to the methods described above, and the rate of platelet depolymerization was as shown in table 2 below.

TABLE 2 depolymerization effects of amino-and amino-free structurally similar Compounds on platelets

As can be seen from Table 2 above, the amino-only compound had a depolymerization effect on the already aggregated platelets, while other groups, such as sulfonic acid groups, hydroxyl groups, carboxyl groups, carbonyl groups, etc., had little depolymerization effect on the aggregated platelets (comparable to the depolymerization rate of the blank NaCl). From this example, it was determined that the amino groups in each compound are responsible for the primary depolymerization of platelets.

Example 2 relationship of amino number to the ability of platelet to deagglomerate

This example compares the effect of varying amounts of amino groups on structurally similar compounds on platelet depolymerization. Compounds having different amounts of amino groups were tested separately in the same manner as in example 1 to obtain the platelet depolymerization rate and pKa values of each compound as shown in table 3.

TABLE 3 platelet depolymerization Rate and pKa value for Compounds containing different numbers of amino groups

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As can be seen from Table 3 above, where the structures are similar or identical and the number of amino groups substituted is only different, the depolymerization effect of the compounds on aggregated platelets increases with increasing number of amino groups, with short peptides, polyamino substituted alkylamines, polyamino substituted hydroxylamine and guanidines being more preferred.

Example 3 relationship of amino-pka to the ability to depolymerize platelets in the same pH Environment

This example compares the effect of different amino pKa's of structurally similar compounds on platelet depolymerization under the same pH environment. The same procedure as in example 1 was followed except that the pH was adjusted to a suitable value for each of the different types of compounds, and the compounds having different amounts of amino groups were each tested to obtain the platelet aggregation rate and pKa values of each of the compounds as shown in Table 4.

TABLE 4 platelet depolymerization Rate of Compounds with different amino pKa at certain environmental pH

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As can be seen from Table 4 above, the difference in substituents on the amino groups affects the pKa values of the amino groups.

Example 4 relationship of environmental pH to amino pKa of amino-bearing Compounds and Effect of the Environment pH on depolymerization Capacity

From example 3 it can be seen that there is a relationship between the pH of the environment and the pKa of the amino group of the amino compound. To further investigate the relationship between the environmental pH and the amino pKa of amino compounds and the effect on the ability of platelets to depolymerize, a series of tests were performed using 1-acetylguanidine, a compound having a pKa of 8.33, in analogy to example 1, with only a change in the environmental pH between 7.0 and 10. A graph of pH versus depolymerization rate was obtained as shown in fig. 3.

As can be seen from fig. 3, the pH of the environment has a strong correlation with the pka of the substance in platelet depolymerization. When the pH is raised, the depolymerization ability of the amino group-bearing substance is enhanced, and the depolymerization rate is increased from 35.23% to 97.32%. And when the pH is above 9.5, the depolymerization rate is substantially flat. It can be seen that when the ambient pH is greater than the amino pKa of the compound to some extent, continued pH elevation has little effect on the deagglomeration effect. This is consistent with the above-described putative depolymerization mechanism. That is, at a suitable pH, the amino groups of the compound are present in the solution mostly in deprotonated form, thereby promoting even accelerated depolymerization.

Further, in a similar manner, the depolymerization rate of aggregated platelets by compounds having different amino pKa was detected at ph9.5, and the result showed (fig. 4). Wherein, some compounds with pKa values between 8 and 11 are selected: n- (trimethylol) methylglycine (8.1), arginine (9.0), glutamic acid (9.6), glycine (9.6), sulfanilic acid (10.1) and lysine (10.7). As the amino pka8.1 increased to 10.7, the tendency of the depolymerization effect of each compound was gradually decreasing.

It can be seen from this example that adjusting the pH above the pKa of the amino group helps to increase the depolymerization effect. Therefore, by adjusting the environmental pH, the ability of the amino group-containing compound to disaggregate aggregated platelets can be improved. Conversely, an appropriate compound having an amino group may be selected according to the requirements of the detection environment.

EXAMPLE 5 deagglomerating Effect of amino-bearing Compound Structure on aggregated platelets

In order to examine the effect of the compound structure on the depolymerization of aggregated platelets, compounds having a representative structure were selected and tested under the conditions of ph=9.5 in accordance with the method of example 1 to obtain the depolymerization rates of the respective compounds as shown in table 5 below.

TABLE 5 deagglomeration effect of different compounds on aggregated platelets

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It is apparent from Table 5 above that the interaction of amino groups with cell membranes is affected by the structure of the substance itself having amino groups. For example, dimeric glycine has the same pKa as glycine and one more secondary amino group, but the depolymerization effect is still slightly lower than glycine. This is attributable to one more carbonyl group in the molecule. In addition, glutamic acid and glycine both have only one primary amine group with a pKa of 9.6, but glutamic acid has one more carboxyl group than glycine, and the depolymerization rate is also low.

It can be seen from table 5 that carbonyl and carboxyl groups do not appear to contribute to the platelet depolymerization effect of the boosting compound, whereas alkyl chains do not greatly affect the depolymerization effect.

In addition, the larger the number of amino groups/imino groups, in particular, primary amino groups, the better the depolymerization effect. Arginine has more amino/imino groups, but the depolymerization effect is not very good, possibly related to its ability to form a cyclic lactam, resulting in an amino group that is weakened.

In addition, hydroxyl groups appear to be more beneficial in enhancing the depolymerization effect of amino groups in the compounds. For example, tris (hydroxymethyl) aminomethane contains only one secondary amino group, but the depolymerization rate is still higher than glycine. The tris (hydroxymethyl) aminomethane having a primary amino group has a more excellent effect on the depolymerization of platelets. It is speculated that hydroxyl groups facilitate the approach of the compound to the cell membrane of platelets, while primary amino groups have a better depolymerization effect on platelets than secondary amino groups. Of course, higher depolymerization rates also correlate with lower pKa of the two compounds (8.1).

In addition, the tertiary amino group has little depolymerization effect. It is also understood from the above mechanism that the amino group does not have a hydrogen atom capable of forming a hydrogen bond, and thus it is difficult to exert an action.

EXAMPLE 6 deagglomeration Effect of amino-bearing Compounds on different platelet aggregation models

Aggregation of platelets can be induced by a variety of substances, which in turn lead to blood clotting. In order to examine the depolymerization effect of compounds having amino groups on platelet aggregation induced by different factors, this example selects substances inducing platelet aggregation in blood coagulation test schemes according to existing platelet aggregation pathways, and establishes various models of platelet aggregation including Adenosine Diphosphate (ADP), thrombin (THR), collagen (COL) and Riszein (RIS). It was found by this example that ADP, THR, COL and RIS induce platelets to aggregate to different degrees with concentration and time, and that compounds with amino groups are also able to effectively deagglomerate platelets of different causes and different aggregation degrees over a range of concentrations and times.

Referring to FIG. 5, the extent of platelet aggregation induced by different ADP concentrations (0.01-1 mM) as a function of time is shown. Specifically, 1ml of venous blood was taken and ADP was added to give final concentrations of 0.01mM, 0.25mM, 0.5mM and 1mM, respectively. Blood containing aggregated platelets is detected with a blood cell analyzer. The blood cell analyzer obtains characteristics of various cell particles in blood, including number, size, etc., through the impedance channel and the optical channel. After normal blood collection to the blood anticoagulation tube, the platelet particle count detected by impedance channel obtained by the blood cell analyzer was designated as (plt_i). When platelet aggregation occurs, the volume of aggregated platelets is much greater than that of individual platelets, and therefore cannot be counted correctly by the recognition mechanism to which the platelets correspond, resulting in a decrease in platelet count, denoted plt_i (aggregation). The diluent of the impedance channel is not added with the compound containing amino, so the impedance channel reflects the aggregation degree of the platelets in the aggregation model. The count before platelet aggregation was plt_i (not aggregated), the count after platelet aggregation was plt_i (aggregated), and aggregation rate=plt_i (aggregated)/plt_i (not aggregated). The platelet particle count detected by the optical channel obtained by the blood cell analyzer was designated as (plt_o). When platelet aggregation occurs, the volume of aggregated platelets is much greater than that of individual platelets, and therefore cannot be counted correctly by the platelet-corresponding recognition mechanism, resulting in a decrease in plt_o (not disaggregated). The use of such amino group-containing compounds in dilutions of optical channels allows for the disaggregation of aggregated platelets, resulting in a disaggregated plt_o (not disaggregated). The count before platelet aggregation was plt_o (not aggregated) and the count after platelet aggregation was plt_o (not disaggregated). In general, plt_o (not depolymerized) < plt_o (not depolymerized), depolymerization rate = plt_o (depolymerized)/plt_o (not depolymerized). By the above principle, a model is established to quantitatively detect the depolymerization effect of the amino group-containing compound.

As can be seen from fig. 5, platelet aggregation was substantially maximized within a period of 5 minutes after ADP addition. And the greater the amount of ADP added, the greater the degree of platelet aggregation. At a final ADP concentration above 0.25mM, the effect on the extent of platelet aggregation is substantially the same. In addition, ADP at a concentration of 0.01mM has the least effect on the extent of platelet aggregation, and can aggregate up to about 35% of platelets, but platelet deagglomeration is also relatively slow at this concentration.

With further reference to fig. 6, a graph of the effect of compounds with amino groups on platelet depolymerization over time is shown for blood samples induced with different ADP concentrations. Specifically, each blood sample to which an aggregation inducer was added at different concentrations was subjected to treatment and measurement in a similar manner to example 1, with the addition of a compound having an amino group (1-acetylguanidine) to the dilution, and each sample was subjected to measurement at intervals of 5 minutes after the addition of the treatment liquid, to obtain a change in the depolymerization rate with time. The composition of the diluent is as follows: citric acid (0.5 g/L), surfactant phenoxyethanol (0.1 g/L), bacteriostat (6 g/L), sodium chloride (3 g/L), EDTA (0.1 g/L) and 1-acetyl guanidine (10 mmol/L), pH 9.5, osmotic pressure 200mOsm/L.

As can be seen from fig. 6, the same compound has a remarkable depolymerization effect on different platelet aggregation levels at the same concentration, and the depolymerization effect is highest immediately after the addition of the compound having an amino group and decreases with time, but tends to be stable after substantially 10 minutes. In addition, the lower the concentration of ADP, the more stable the effect. In view of the fact that the blood cell detector can rapidly complete the detection of a sample with only a small amount of sample, the detection can be started within 5 minutes, preferably within 2 minutes, more preferably immediately after 30 seconds after the addition of the treatment liquid containing the compound having an amino group.

Thus, by increasing the concentration of ADP, the degree of aggregation can be increased, thereby increasing the difficulty of deagglomeration.

Similarly, figures 7 and 8 are graphs of tests for platelet aggregation induced by different concentrations of THR. The same procedure as described above was followed except that ADP was replaced with THR. Wherein the concentration of THR is 0.1U, 0.15U, 0.2U, 0.23U, 0.25U, 0.28U, 0.30U, and 0.35U, respectively. It can be seen that the effect of aggregating platelets is weaker at low THR concentrations and that the platelet aggregation rate increases rapidly after 1 minute at high concentrations. This is because, after addition of certain concentrations of THR, soluble fibrinogen is converted to insoluble fibrinogen, which causes blood clotting and platelets are also encapsulated by the insoluble fibrinogen, resulting in a sharp increase in aggregation rate. At the same time, the depolymerization effect of 1-acetylguanidine was immediately highest for each THR concentration, but began to decrease significantly after 1 minute. And also to the failure of the depolymerizing agent to exert its effect at this time. But a better depolymerization rate was obtained in the samples detected immediately after the treatment.

Fig. 9 and 10 are graphs of tests for platelet aggregation induced by COL at different concentrations. The same procedure as described above was followed except that ADP was replaced with COL. Wherein the concentration of COL is 0.05mM, 0.1mM, 0.5mM, 1mM, 5mM, 10mM and 20mM, respectively. It can be seen that COL can rapidly bring platelet aggregation to nearly 100% at different concentrations, where the lower the concentration, the faster the aggregation. Meanwhile, the depolymerization effect of 1-acetylguanidine reaches the highest concentration for each of the other COL concentrations immediately except for the sample with the lowest COL concentration, but starts to decrease significantly after about 4 to 8 minutes. This is also due to the fact that soluble fibrinogen is converted to insoluble fibrinogen upon addition of COL, resulting in blood clotting.

FIGS. 11 and 12 are graphs of RIS-induced platelet aggregation assays with different concentrations. The same procedure as described above was followed except that ADP was replaced with RIS. Wherein the concentration of RIS is 0.15mM, 0.375mM, 0.75mM, 1.5mM and 3mM, respectively. It can be seen that the induction of platelet aggregation by RIS takes a longer time to develop, and after 15-20 minutes, the extent of platelet aggregation begins to reach a maximum. Meanwhile, the depolymerization effect of the 1-acetylguanidine can be immediately highest for each RIS concentration, and is stable within 10 minutes. The rapid decrease in the depolymerization rate after 15 minutes was also due to coagulation of the blood in the sample.

From the above models for inducing platelet aggregation, it can be seen that the aggregation inducers have different effects on the aggregation degree of platelets under different paths, but the compounds with amino groups can immediately act on the aggregation caused by each inducer, but the depolymerization effect of the THR and COL induced platelet aggregation samples after coagulation is not reflected due to the fact that soluble fibrinogen is converted into insoluble fibrinogen to cause coagulation. This has little effect on the platelet classification and counting performed by the blood cell detector.

This demonstrates that the amino-bearing compounds can produce good depolymerization for a variety of platelet aggregates at a range of concentrations and times, and can be rapidly effective for immediate detection without additional reaction conditions (e.g., heating or longer reaction times).

EXAMPLE 7 depolymerization of aggregated platelets by ammonium ion-containing Compounds

This example measures the effect of compounds containing ammonium cations on the disaggregation of aggregated platelets.

The test was conducted in the same manner as in example 1 except that the compound of Table 1 was replaced with an ammonium ion-containing compound (ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium thiocyanate, ammonium bicarbonate, ammonium oxalate, aqueous ammonia) and a compound (NaCl) as a control, and the pH of the treatment solution was adjusted to 9.5 and the osmotic pressure was adjusted to 200mOsm/L. The depolymerization rates of the ammonium ion compounds at various concentrations were measured as shown in fig. 13.

As can be seen from FIG. 13, in the sample in which platelet aggregation was induced by ADP at 0.01mmol/L, each of the ammonium ion-containing compounds produced a certain depolymerization at a concentration of 1mmol/L and reached the best effect at a concentration of 10 mmol/L. Specifically, at 10mmol/L, (NH ₄)₂HPO₄ is 99.25%, NH ₄ CL is 95.70%, (NH ₄)₃PO₄ is 94.87%, NH ₄ Br is 93.48%, NH ₄ I is 92.75%, NH ₄HCO₃ is 88.68%, NH ₄C₂O₄ is 87.67%, NH ₄H₂PO₄ is 86.24%, NH ₄ SCN is 85.78%, and NH ₄ OH is 83.05%,. When the ammonium ion compound is changed to NaCl, the depolymerization effect is reduced to 18.90%. It can be seen that ammonium ions rather than anions are responsible for the depolymerization of platelets, and the different anions have some influence on the depolymerization but not very great influence.

Example 8 influence of environmental pH on the platelet depolymerization Capacity of ammonium ion-containing Compounds

This example investigated the effect of environmental pH on the deagglomeration effect of compounds containing ammonium ions on aggregated platelets. According to a similar manner to example 1, the compounds in Table 1 were replaced with 0.01mol/L of ammonium chloride, and the pH of the treatment solution was adjusted to 4.0, 5.5, 6.5, 7.5, 8.5, 9.5 and 11.0, and the depolymerization rates of the ammonium chloride to depolymerization of platelets in the samples were examined, respectively, as shown in Table 6 below.

Table 6: influence of pH on depolymerization of platelets by ammonium ions in ammonium chloride

PH value of	4.0	5.5	6.5	7.5	8.5	9.5	11.0
								Depolymerization rate	18.62％	31.70％	59.61％	94.05％	94.62％	95.70％	97.72％

As is clear from Table 6, when the environmental pH was 7.5 or more, the depolymerization effect of ammonium ions on aggregated platelets was suddenly increased to 94.05%, and the depolymerization rate was maintained high in the range of pH 7.5 to 11.0. This suggests that the depolymerization of platelets can be promoted in the case where ammonium ions form more hydrated ammonia in the solution.

EXAMPLE 9 Effect of Using Compounds having amino groups in combination with Compounds containing ammonium ions on platelet depolymerization

Based on the fact that the compound having an amino group and the compound containing an ammonium ion may depolymerize aggregated platelets by different depolymerization mechanisms, the present example measured the effect of a combination of the compound having an amino group and the compound containing an ammonium ion on the depolymerization of platelets. In a similar manner to example 1, except that the compounds of Table 1 were replaced with ammonium chloride, 1-acetylguanidine, a combination of ammonium chloride and 1-acetylguanidine, and NaCl as a control at concentrations of 1,2, 5, 10, 20 and 50mmol/L, respectively, wherein the concentrations of ammonium chloride and 1-acetylguanidine were equal in the combination of ammonium chloride and 1-acetylguanidine, and the total concentration met the above concentration requirements. In order to show the effect of the combination of both, in this example, platelet aggregation was induced by using 0.1mmol/L of ADP (i.e., 10 times the concentration of ADP in example 1), and the ability of the compound having an amino group and the compound containing an ammonium ion to depolymerize platelets was investigated with increasing the difficulty of depolymerization. The depolymerization rate measured is shown in FIG. 14.

The results showed (FIG. 14) that the depolymerization effects of NH ₄ Cl and 1-acetylguanidine were gradually increased with increasing concentrations in ADP-induced platelet aggregation at a final concentration of 0.1mmol/L, respectively. For example, the depolymerization effect of NH ₄ Cl alone is 66.27%, the depolymerization effect of 1-acetylguanidine alone is 62.14%, and the depolymerization effect of NH ₄ Cl and 1-acetylguanidine together at a total concentration of 50mmol/L reaches 95.48%, thereby further enhancing the depolymerization effect of platelets. This enhanced effect can also be seen at other concentrations. Suggesting that there is a synergy between the two in platelet depolymerization. Therefore, the two are preferably used in combination at lower concentrations (e.g., as low as 1 to 20mM,1 to 10mM, and even 1 to 5mM, respectively).

The application of the reagent comprising the compound having an amino group according to the present invention to the actual detection of anti-platelet depolymerization interference is further examined as follows.

Test example 1 Compounds with amino groups for leukocyte detection in lysates

The present test example provides a lysate for a blood cell analyzer that can be used for the detection of thrombocyte-disaggregated leukocytes. This test example and the following test examples were each measured using a blood cell analyzer (BC-6000 Plus) from Shenzhen Michael biomedical Co., ltd.) and lysates were prepared according to the following formulation:

Control lysate:

Lysate B:

1-acetylguanidine was added to a final concentration of 0.01mol/L based on the control lysate.

Control lysate and lysate B were prepared with distilled water at 25℃and the pH was adjusted to 9 and the osmotic pressure to 90mOsm/L.

Taking 1ml of human venous blood sample collected in an anticoagulation tube, dividing the sample into two groups, and measuring by using the blood cell analyzer, wherein a control lysate and a lysate B are respectively used, mixing in a proportion of adding 1ml of lysate into 20 μl of sample, adding 20 μl of DNA staining agent (Hoechst 33342), maintaining the temperature at about 42 ℃, measuring fluorescence intensity information (FL) of the blood sample cells after treatment by using side fluorescence with a measuring angle of 90 DEG, measuring side scattering light intensity information (SS) of the blood sample cells after treatment by using side scattering light with a measuring angle of 90 DEG, and obtaining a three-dimensional scatter diagram by using forward scattering light intensity information (FS) of the blood sample cells after treatment by using forward scattering light with a measuring angle of 2 DEG to 5 deg. 1ml of the blood sample was taken, and platelet aggregation inducer ADP (final concentration: 0.01 mmol/L) was added thereto and mixed well. After 5min the platelets form aggregates. At this time, the samples were divided into two groups, and similarly examined by a hemocytometer, and treated with a control lysate and lysate A, respectively, and DNA dye was added, and similarly a three-dimensional scattergram was obtained. As described above, the 4-group scattergrams obtained by measuring the samples are shown in fig. 15.

In the optical channel, a plurality of aggregated platelets are identified as one particle, and the volume of the partially aggregated platelets reaches the volume of the white blood cells, affecting the detection of the count of white blood cells. In this test example, the white blood cell count of the unagglomerated sample was measured to be 7.39X10 ⁹, where neutrophils were counted 4.09X 10 ⁹, lymphocytes 2.35X 10 ⁹, monocytes 0.32X 10 ⁹, Eosinophils 0.52×10 ⁹. the sample of lysate B was used to determine a post-deagglomeration white blood cell count of 7.42X 10 ⁹, a differential count of neutrophils 4.13X 10 ⁹, lymphocytes 2.31X 10 ⁹, monocytes 0.34X 10 ⁹, Eosinophils 0.58×10 ⁹. The aggregated white blood cell count was measured as 8.02X10 ⁹, the differential count neutrophil 4.37X10 ⁹, lymphocyte 2.56X10 ⁹, monocyte 0.41X 10 ⁹, Eosinophils 0.63×10 ⁹. It can be seen that the white blood cell count and classification are not affected by platelet aggregation after using lysate B, and the count is more accurate.

Test example 2: detection of ammonium chloride in lysates for white blood cells

The present test example provides a lysate for a blood cell analyzer that can be used to exclude platelet aggregation from interfering with the detection of leukocytes. The lysate was prepared according to the following formulation:

Control lysate:

Lysate a:

Based on the control lysate, ammonium chloride was added to a final concentration of 0.01mol/L.

Control lysate and lysate A were prepared at 25℃with distilled water, the pH was adjusted to 9 and the osmotic pressure to 90mOsm/L.

According to a procedure similar to that of test example 1, 1ml of human venous blood sample collected in an anticoagulation tube was taken, the samples were divided into two groups, and were measured with the blood cell analyzer, wherein a control lysate and lysate A were used, respectively, and mixed in a ratio of 20. Mu.l of the sample to 1ml of the lysate, and 20. Mu.l of DNA stain (Hoechst 33342) was further added, and the temperature was kept at about 42℃to obtain a three-dimensional scatter diagram by measuring fluorescence intensity information (FL) of the treated blood sample cells with side fluorescence at a measurement angle of 90℃and measuring side scattered light intensity information (SS) of the treated blood sample cells with side scattered light at a measurement angle of 90℃and measuring forward scattered light intensity information (FS) of the treated blood sample cells with forward scattered light at a measurement angle of 2℃to 5 ℃. 1ml of the blood sample was taken, and platelet aggregation inducer ADP (final concentration: 0.01 mmol/L) was added thereto and mixed well. After 5min the platelets form aggregates. At this time, the samples were divided into two groups, and similarly examined by a hemocytometer, and treated with a control lysate and lysate A, respectively, and DNA dye was added, and similarly a three-dimensional scattergram was obtained. As described above, the 4 sets of scatter plots obtained by measuring the samples are shown in fig. 16.

In the optical channel, a plurality of aggregated platelets are identified as one particle, and the volume of partially aggregated platelets reaches the volume of white blood cells, affecting the count of white blood cells. Is detected. In this test example, the white blood cell count in the unagglomerated sample was determined to be 7.49X10 ⁹, where neutrophils 4.7X10 ⁹, lymphocytes 2.16X10 ⁹, monocytes 0.42X 10 ⁹, Eosinophils 0.14X10 ⁹. The white blood cell count after deagglomeration was measured as 8.72X10 ⁹, the differential count neutrophils 5.16X10 ⁹, lymphocytes 2.57X10 ⁹, monocytes 0.61X 10 ⁹ using a sample of control lysate, eosinophils 0.32X10 ⁹. The number of white blood cells after deagglomeration was measured as 7.58X10 ⁹ using the sample of lysing agent A, with neutrophils 4.9X10 ⁹, lymphocytes 2.21X10 ⁹, monocytes 0.39X10 ⁹, Eosinophils 0.15X10 ⁹. It can be seen that the white blood cell count and classification is not affected by platelet aggregation after the use of lysing agent a, and the count is more accurate.

Test example 3: combination of a compound having an amino group and a compound containing an ammonium ion in a lysate for leukocyte detection

This test example was conducted in accordance with the method of test example 5, except that lysate C was prepared in place of lysate B, i.e., 1-acetylguanidine+NH ₄ Cl was added in equimolar amounts to give a final concentration of 0.01mol/L based on the control lysate.

Control lysate and lysate C were prepared with distilled water at 25℃and the pH was adjusted to 9 and the osmotic pressure to 90mOsm/L.

The test was conducted in accordance with the similar procedure to that of test example 5, and the same three-dimensional scattergram was obtained as shown in FIG. 17.

In this test example, the white blood cell count of the unagglomerated sample was measured to be 6.19X10 ⁹, in which neutrophils were counted 3.87X10 ⁹, lymphocytes 1.91X10 ⁹, monocytes 0.22X10 ⁹, eosinophils 0.08X10 ⁹ in a classified manner. The white blood cell count after deagglomeration was measured as 6.13X10 ⁹ using the lysate C sample, and the neutrophils were sorted to 3.91X10 ⁹, lymphocytes 1.88X10 ⁹, monocytes 0.21X 10 ⁹, eosinophils 0.07X 10 ⁹. The white blood cell count after deagglomeration was measured to be 6.97X10 ⁹ using a sample of control lysate, and neutrophils 4.21X10 ⁹, lymphocytes 2.21X10 ⁹, monocytes 0.34X 10 ⁹, eosinophils 0.17X 10 ⁹ were counted in a differential. It can be seen that the white blood cell count and classification is not affected by platelet aggregation after the use of lysing agent C, and the count is more accurate.

Test example 4: deagglomerating effect of Compounds with amino groups in clinical settings on pseudo-platelet aggregation

The present test example is to realize the deagglomeration of pseudo-aggregated platelets in clinic. The same diluent as diluent B of test example 1 was used for the detection. The formula of the diluent is as follows: citric acid (0.5 g/L), surfactant phenoxyethanol (0.1 g/L), bacteriostat (6 g/L), sodium chloride (3 g/L), 1-acetyl guanidine (10 mmol/L). The pH of the dilution was adjusted to 9.5 and the osmotic pressure to 200mOsm/L.

The method comprises the following specific steps: blood of outpatient and inpatient is collected, EDTA.K2 anticoagulation, PLT-I platelet count is obviously reduced, platelet aggregation phenomenon is found by blood smear microscopy, 20 samples meeting EDTA-PTCP diagnostic standards are taken as experimental groups, wherein, 11 men and 9 women are taken as experimental groups, and the average age is 56. Blood from a patient with pseudo-aggregation of platelets was drawn, and immediately after drawing blood without anticoagulant, platelets were detected on a blood cell analyzer within 1 minute (when no aggregation of platelets occurred), and the platelet count value was measured as a true value plt_o (true value) of platelets. Blood from the patient was drawn with EDTA anticoagulation tube, and after 2 hours (platelets from the pseudo-aggregated patient were aggregated), the blood was measured on the same blood cell analyzer with a dilution containing 1-acetylguanidine as a depolymerizing substance, and the platelet count value measured by this method was the platelet deagglomeration value plt_o (depolymerization). At the same time, blood from the patient was drawn with EDTA anticoagulant tube, and after 2 hours (platelets from the pseudo-aggregated patient were aggregated), the platelets were counted as non-deagglomerated plt_o (not deagglomerated) with the same blood cell analyzer without dilution of 1-acetylguanidine as the deagglomerating substance. The depolymerization effect is calculated by the formula:

Depolymerization rate (depolymerization) =plt_o (depolymerization)/plt_o (true value); or alternatively

Depolymerization rate (control) =plt_o (not depolymerized)/plt_o (true value).

The detection results of each sample are shown in fig. 18. As shown in FIG. 18, pseudo-aggregated platelets were all able to disaggregate to varying degrees, and the disaggregation effect was still up to 70% in 14 out of 20 samples after 2 hours of patient blood placement. In the control group, the depolymerization effect was 20% or less, and the depolymerization effect of the depolymerization dilution containing 10mM of 1-acetylguanidine was remarkable.

For a sample which is difficult to depolymerize, for example, a sample having a depolymerization rate of 70% or less, a higher concentration, or a compound having a better depolymerization effect, or a combination of a compound having an amino group and a compound containing an ammonium ion may be used to obtain a desired depolymerization effect.

Test example 5: depolymerization effect of Compounds containing ammonium ions on pseudo-platelet aggregation in clinical applications

The present test example is to realize the deagglomeration of pseudo-aggregated platelets in clinic. The same diluent as diluent A of test example 2 was used for the detection. The formula of the diluent is as follows: citric acid (0.5 g/L), surfactant phenoxyethanol (0.1 g/L), bacteriostat (6 g/L), sodium chloride (3 g/L), NH ₄ Cl (0.01 mol/L). The pH of the dilution was adjusted to 9.5 and the osmotic pressure to 200mOsm/L.

The method comprises the following specific steps: blood of outpatient and inpatient is collected, EDTA.K2 anticoagulation, PLT-I platelet count is obviously reduced, platelet aggregation phenomenon is found by blood smear microscopy, 20 samples meeting EDTA-PTCP diagnostic standards are taken as experimental groups, wherein 9 men and 8 women are taken as experimental groups, and the average age is 51 years. Blood from a patient with pseudo-aggregation of platelets was drawn, and immediately after drawing blood without anticoagulant, platelets were detected on a blood cell analyzer within 1 minute (when no aggregation of platelets occurred), and the platelet count value was measured as a true value plt_o (true value) of platelets. Blood from the patient was drawn with EDTA anticoagulation tube, and after 2 hours (platelets from the pseudo-aggregated patient were aggregated), the blood was measured on the same blood cell analyzer with a dilution containing ammonium chloride as a depolymerizing substance, and the platelet count value measured by this method was the platelet deagglomeration value plt_o (depolymerization). At the same time, the patient's blood was drawn with EDTA anticoagulant tube, and after 2 hours (platelets from the pseudo-aggregated patient were aggregated), the platelet count value measured by this method was the non-deagglomerated value plt_o (non-deagglomerated) of platelets, as measured by the same blood cell analyzer without the deagglomerated substance ammonium chloride diluent. The depolymerization effect is calculated by the formula:

Depolymerization rate (control) =plt_o (not depolymerized)/plt_o (true value).

The detection results of each sample are shown in fig. 19. As shown in the figure, the pseudo-aggregated platelets can be disaggregated to different degrees, and the disaggregation effect still reaches 13 cases of 70% in 20 samples after the blood of a patient is left for 2 hours. In the control group, the depolymerization effect was 20% or less, and the depolymerization effect of the depolymerization dilution containing 10mM ammonium chloride was remarkable.

The foregoing description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the invention, but rather, the equivalent structural changes made by the description of the present invention and the accompanying drawings or the direct/indirect application in other related technical fields are included in the scope of the invention.

Claims

1. A method of detecting white blood cells against platelet aggregation interference, the method comprising:

Treating a blood sample with a deagglomerating agent to prevent and/or eliminate platelet aggregation in the blood sample, wherein the deagglomerating agent comprises at least one selected from the group consisting of a compound having an amino group and a compound containing an ammonium ion;

detecting the blood sample treated with the depolymerizing agent with a blood analyzer, wherein,

The blood analyzer includes an impedance detector, the blood sample is hemolyzed with a red blood cell lysing agent prior to performing the detection, and the method includes the steps of: the blood sample treated with the deagglomerating agent and the erythrocyte lysing agent is detected by an impedance detector in a blood analyzer to obtain an electrical signal of particles in the blood sample and further obtain a white blood cell detection parameter after eliminating the aggregation interference, or

The blood analyzer includes an optical detector, the blood sample is stained with a fluorescent dye and hemolyzed with a red blood cell lysing agent prior to performing the detection, and the method includes: detecting the blood sample treated with the deagglomerating agent, fluorescent dye and red blood cell lysing agent with an optical detector in a blood analyzer to obtain at least two optical signals of particles in the blood sample to obtain detection parameters of white blood cells,

Wherein the compound having an amino group is selected from the group consisting of compounds represented by the following formula (I):

R1-NH-R2 （I）

P1, P2 and P3 are each independently a group selected from the group consisting of: H. c1-16 alkyl, C6-10 aryl, C7-14 alkylaryl and C7-14 arylalkyl, wherein the C1-16 alkyl, C6-10 aryl, C7-14 alkylaryl and C7-14 arylalkyl are each unsubstituted or further substituted with at least one group selected from the group consisting of-NH ₂、-OH、-SO₃ H, halogen, -CN, -COOH and-C (O) NH ₂,

Wherein the ammonium ion containing compound contains an anion selected from the group consisting of chloride, bromide, iodide, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, hydrogen sulfide, thiocyanate, hydrogen sulfate, sulfite, hydrogen carbonate, oxalate, propionate, malonate, citrate, and combinations thereof.

2. The detection method according to claim 1, wherein R1 and R2 are the same or different and are each independently a group selected from the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, substituted or unsubstituted C7-10 aralkyl, -C (O) -Q1 and-C (O) -O-Q2,

3. The detection method according to claim 1, wherein R1 and R2 are the same or different and are each independently a group selected from the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, substituted or unsubstituted C7-10 aralkyl, -C (O) -Q1 and-C (O) -O-H,

4. The detection method according to claim 1, wherein the compound of formula (I) has a total of 1 to 20 primary amino groups, secondary amino groups and/or imino groups.

5. The detection method according to claim 1, wherein the compound having an amino group has an amino pKa value of 1 to 16.

6. The detection method according to claim 5, wherein the compound having an amino group has an amino pKa value of 4 to 14.

7. The detection method according to claim 6, wherein the amino group of the compound having an amino group has a pKa value of the amino group of not more than a pH value of the depolymerizing agent.

8. The detection method according to claim 1, wherein a concentration of the compound having an amino group in the depolymerizing agent is 1 to 50 mmol/L.

9. The detection method according to claim 8, wherein a concentration of the compound having an amino group in the depolymerizing agent is 2 to 20 mmol/L.

10. The detection method according to claim 1, wherein the compound containing an ammonium ion is at least one selected from the group consisting of ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium nitrate, ammonium hydrogen sulfite, ammonium thiocyanate, ammonium oxalate, ammonium hydroxide, ammonium hydrogen sulfate, and ammonium bicarbonate.

11. The detection method according to claim 1, wherein the concentration of the compound containing ammonium ions in the depolymerizing agent is 1 to 50 mmol/L.

12. The detection method according to claim 11, wherein the concentration of the ammonium ion-containing compound in the depolymerizing agent is 2 to 20 mmol/L.

13. The detection method according to claim 1, wherein the depolymerizing agent comprises at least one compound having an amino group and at least one compound containing an ammonium ion.

14. The detection method according to claim 12, wherein a total concentration of the at least one compound having an amino group and the at least one compound containing an ammonium ion in the depolymerizing agent is 2 to 50mmol/L.

15. The detection method according to claim 14, wherein a total concentration of the at least one compound having an amino group and the at least one compound containing an ammonium ion in the depolymerizing agent is 2 to 20mmol/L.

16. The detection method according to claim 15, wherein a total concentration of the at least one compound having an amino group and the at least one compound containing an ammonium ion in the depolymerizing agent is 2 to 10mmol/L.

17. The detection method according to claim 13, wherein a molar ratio of the at least one compound having an amino group and the at least one compound containing an ammonium ion is in a range of 10:1 to 1:10.

18. The detection method according to claim 17, wherein a molar ratio of the at least one compound having an amino group and the at least one compound containing an ammonium ion is in a range of 4:1 to 1:4.

19. The assay of claim 1, wherein the pH of the blood sample treated with the deagglomerating agent is at least 3.0.

20. The assay of claim 19, wherein the pH of the blood sample treated with the deagglomerating agent is at least 7.0.

21. The method according to claim 20, wherein the depolymerization agent is used to treat the blood sample at a pH of 7.5 to 11.0.

22. The detection method according to claim 1, wherein the at least two light signals are selected from a fluorescence intensity signal, a forward scattered light intensity signal, and a side scattered light intensity signal.

23. A reagent for detecting white blood cells in an extracorporeal blood test, wherein the reagent comprises at least one selected from the group consisting of an amino group-containing compound and an ammonium ion-containing compound, a red blood cell lysing agent, a buffer and an osmotic pressure regulator, and optionally a fluorescent dye and/or a surfactant, wherein the concentration of the amino group-containing compound is 1 to 50mmol/L and the concentration of the ammonium ion-containing compound is 1 to 50mmol/L,

R1-NH-R2 （I）

24. The reagent of claim 23 wherein

In the formula (I), R1 and R2 are the same or different and are each independently a group selected from the following group, provided that R1 and R2 are not H at the same time, the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, substituted or unsubstituted C7-10 aralkyl, -C (O) -Q1, and-C (O) -O-Q2,

25. The reagent of claim 23, wherein R1 and R2 are the same or different and are each independently selected from the group consisting of H, -SO ₃H、-NH₂、-C(NH)-NH₂, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, substituted or unsubstituted C7-10 aralkyl, -C (O) -Q1, and-C (O) -O-H,

26. The reagent according to claim 23, wherein the compound of formula (I) has a total of 1 to 20 primary amino groups, secondary amino groups and/or imino groups.

27. The reagent of claim 23, wherein the compound having an amino group has an amino pKa value of 1 to 16.

28. The reagent of claim 27, wherein the compound having an amino group has an amino pKa value of 4 to 14.

29. The reagent of claim 28 in which the amino pKa value of the amino-bearing compound is less than or equal to the pH value of the reagent.

30. The reagent according to claim 23, wherein the concentration of the compound having an amino group is 2 to 20mmol/L.

31. The reagent of claim 23, wherein the ammonium ion containing compound is at least one selected from the group consisting of ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium nitrate, ammonium hydrogen sulfite, ammonium thiocyanate, ammonium oxalate, ammonium hydroxide, ammonium bisulfate, and ammonium bicarbonate.

32. The reagent according to claim 23, wherein the concentration of the compound containing ammonium ion is 2 to 20mmol/L.

33. The reagent of claim 23, wherein the reagent comprises at least one compound having an amino group and at least one compound containing an ammonium ion.

34. The reagent according to claim 33, wherein the total concentration of the at least one compound having an amino group and the at least one compound containing an ammonium ion in the reagent is 2 to 50mmol/L.

35. The reagent according to claim 34, wherein the total concentration of the at least one compound having an amino group and the at least one compound containing an ammonium ion in the reagent is 2 to 20mmol/L.

36. The reagent according to claim 34, wherein the total concentration of the at least one compound having an amino group and the at least one compound containing an ammonium ion in the reagent is 2 to 10mmol/L.

37. The reagent of claim 33, wherein the molar ratio of the at least one compound having an amino group and the at least one compound containing an ammonium ion is in the range of 10:1 to 1:10.

38. The reagent of claim 37, wherein the molar ratio of the at least one compound having an amino group and the at least one compound containing an ammonium ion is in the range of 4:1 to 1:4.

39. The reagent of claim 23 wherein the reagent has a pH of at least 3.0.

40. The reagent of claim 39 in which the reagent has a pH of at least 7.0.

41. The reagent of claim 39, wherein the reagent has a pH of 7.5-11.

42. Use of a platelet deagglomeration reagent in the preparation of a reagent for use in the detection of leukocytes, wherein the platelet deagglomeration reagent comprises a deagglomeration agent as defined in any one of claims 1 to 18.

43. The use according to claim 42, wherein the pH of the platelet deagglomeration agent is at least 3.0.

44. The use according to claim 43, wherein the pH of the platelet deagglomeration agent is at least 7.0.

45. The method according to claim 43, wherein the pH of the platelet depolymerization reagent is 7.5 to 11.0.

46. The use according to claim 42, wherein the platelet disaggregation reagent further comprises a red blood cell lysing agent, a buffer, and an osmolarity adjusting agent, and optionally a fluorescent dye and/or a surfactant.