Platelet disaggregation uses of a composition, disaggregation reagents and disaggregation methods

Abstract

The use of a composition for preventing and / or eliminating platelet aggregation in a blood sample in vitro blood testing, wherein the composition comprises at least one ammonium-containing compound. A reagent for preventing anti-platelet aggregation interference in vitro blood testing comprising the compound, and a method for preventing and / or eliminating platelet aggregation in a sample in vitro blood testing. The compound can disaggregate platelets in various aggregation states, and can disaggregate platelets in a short time without additional conditions such as water bath temperature control and prolonged reaction time, thereby conveniently eliminating platelet aggregation in the sample and obtaining accurate blood cell testing parameters.

Description

Technical Field

[0001] This invention relates to in vitro blood testing, and particularly to the use of compounds that can eliminate platelet aggregation in platelet depolymerization, reagents including such compounds, and depolymerization methods. Background Technology

[0002] In blood cell analysis, platelet pseudoaggregation can cause incorrect blood cell counts and classifications, leading to misdiagnosis and mistreatment of patients. It is easily confused with certain life-threatening diseases, such as heparin-induced platelet aggregation (HIP) and disseminated intravascular coagulation (DIC), or it can lead to incorrect treatment decisions, such as incorrect medication, inappropriate platelet transfusions, or even splenectomy. The causes of platelet pseudoaggregation are numerous and complex, with common causes including EDTA-dependent pseudothrombocytopenic purpura (EDTA-PTCP), multi-anticoagulant dependent platelet aggregation, platelet satellite phenomenon, hypercholesterolemia and hypertriglyceridemia, cold aggregation of platelets caused by hypothermia, and aggregation caused by improper blood collection and the material of the anticoagulant tubing. Research on the mechanisms of platelet pseudoaggregation is limited, with most studies focusing on EDTA-PTCP. EDTA is a widely used anticoagulant recognized by the International Committee for Standardization in Hematology (ICSH). EDTA-induced platelet pseudoaggregation was first reported in 1969. The main reason may be the presence of EDTA-dependent antiplatelet antibodies in the blood of EDTA-PTCP patients. When blood is mixed with EDTA anticoagulant in vitro, these antibodies can recognize adhesion receptor glycoprotein IIb-IIIa (GpIIb-IIIa) on the platelet membrane, causing the expression of platelet aggregation activation antigens, such as granule membrane protein 140 (GMP140, also known as CD62P or P-selectin), type III lysosomal glycoprotein (Gp55, also known as CD63), and thrombin-sensitive protein, thereby activating tyrosine kinase and leading to platelet aggregation.

[0003] Platelet pseudoaggregation not only leads to errors in platelet-related clinical parameters but also affects the accuracy of clinical parameters for other blood cells, particularly white blood cells, resulting in adverse consequences for clinical diagnosis and treatment. Therefore, detecting and eliminating platelet aggregation in blood samples has been a long-standing and desired problem in in vitro blood testing.

[0004] Currently, several solutions have been adopted in clinical practice to eliminate or reduce platelet pseudoaggregation. For example, blood samples with pseudoaggregated platelets are heated to 37 degrees Celsius and agitated for a period of time, or additives are added to the blood samples to inhibit platelet aggregation. These additives include anticoagulants (such as mixtures of sodium citrate, sodium heparin, ACD, CTAD, CPT, CaCl2, and sodium heparin); platelet counting diluents (containing mixtures of sodium azide, calcium azide, and sodium fluoride); antiplatelet drugs (added within 10 minutes of blood collection); and aminoglycoside antibiotics (such as amikacin and kanamycin, added within 1 hour of blood collection, but only effective for some samples).

[0005] These methods and reagents for preventing platelet aggregation in isolated blood samples either complicate the testing process, are not sufficiently effective at deaggregating platelets, or are only effective against aggregation caused by certain factors. Often, repeat blood collection and the preparation of specific deaggregating agents are required. In practice, after adding additives, microscopic examination is necessary to observe whether platelets have deaggregated, to determine if the deaggregation effect is suitable for blood analysis. If necessary, the reaction time in an ice bath or water bath needs to be extended to ensure the deaggregation effect.

[0006] Therefore, simple and universally applicable platelet disaggregation methods and reagents are still needed in in vitro blood testing. Summary of the Invention

[0007] To address the problem of false platelet aggregation in in vitro blood testing, the inventors unexpectedly discovered that adding a solution containing ammonium cations to blood samples yielded an unexpected effect in preventing platelet aggregation and effectively de-aggregating samples where platelet aggregation had already occurred. Therefore, this invention aims to provide the use of such substances in preventing and / or eliminating platelet aggregation in blood samples during in vitro blood testing, and a simple, convenient reagent for this purpose that is applicable to different detection methods and equipment to combat platelet aggregation interference. The substances and reagents of this invention for platelet de-aggregation can eliminate most platelet aggregation, with stable and reliable effects, requiring no repeated confirmation, and have no adverse effects on normal blood testing.

[0008] Therefore, a first aspect of the present invention provides the use of a composition in an in vitro blood test to prevent and / or eliminate platelet aggregation in a blood sample, wherein the composition comprises at least one compound containing an ammonium ion.

[0009] According to specific embodiments, the ammonium-containing compound contains anions selected from the group consisting of chloride, bromide, iodide, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, hydrosulfide, thiocyanate, sulfate, bisulfite, sulfite, carbonate, bicarbonate, formate, acetate, oxalate, propionate, malonate, and citrate, and combinations thereof.

[0010] More specifically, the ammonium salt is selected from at least one of 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.

[0011] In this invention, the prevention and / or elimination of platelet aggregation in blood samples is carried out in a solution with a pH of at least 7.0. Preferably, the pH is 7.5–11, more preferably 9.5–11.

[0012] According to the present invention, in the solution for preventing and / or eliminating platelet aggregation in blood samples, the concentration of the ammonium ion-containing compound in the composition is 1 to 50 mmol / L, preferably 2 to 20 mmol / L.

[0013] According to one embodiment, the composition further comprises at least one amino-containing compound, said amino-containing compound containing at least one primary or secondary amino group. Preferably, said amino-containing compound contains 1 to 20 groups selected from primary, secondary, and imine groups.

[0014] According to one specific embodiment, the compound having an amino group is selected from the group consisting of compounds of formula (I) and their salts:

[0015] R1-NH-R2(I)

[0016] Wherein, R1 and R2 may be the same or different, and each is independently a group selected from the group consisting of H, -SO3H, -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 aralkyl, -C(O)-Q1, and -C(O)-O-Q2.

[0017] Wherein, Q1 is H, -NH2, 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.

[0018] 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;

[0019] Wherein, the substitution refers to being replaced by at least one group selected from the group consisting of -NP1P2, -SO3H, -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. The substitutions, wherein the -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 aralkyl are either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, halogen, -CN, -COOH, and -C(O)NH2.

[0020] 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 either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, halogen, -CN, -COOH, and -C(O)NH2.

[0021] More specifically, in the compounds of formula (I), R1 and R2 may be the same or different, each independently being a group selected from the group consisting of H, -SO3H, -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.

[0022] Wherein, Q1 is H, -NH2, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, or substituted or unsubstituted C7-10 aryl.

[0023] 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 aryl;

[0024] Wherein, the substitution refers to being substituted by at least one group selected from the group consisting of -NP1P2, -SO3H, -OH, -CN, -C(O)-O-P3, -O-C1-10 alkyl, -O-C6-10 aryl, -O-C7-10 alkylaryl, -O-C7-10 aryl, and -C(O)-NP1P2, wherein -O-C1-10 alkyl, -O-C6-10 aryl, -O-C7-10 alkylaryl, and -O-C7-10 aryl are either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, -CN, -COOH, and -C(O)NH2;

[0025] 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 the C1-10 alkyl, C6-10 aryl, C7-10 alkylaryl, and C7-10 aralkyl are either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, -CN, -COOH, and -C(O)NH2.

[0026] Furthermore, in the compounds of formula (I), R1 and R2 may be the same or different, each independently being a group selected from the group consisting of H, -SO3H, -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)-OH.

[0027] Wherein, Q1 is H, -NH2, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, or substituted or unsubstituted C7-10 aryl.

[0028] Wherein, the substitution refers to being replaced by at least one group selected from the group consisting of -NP1P2, -SO3H, -OH, -CN, -C(O)-OH and -C(O)-NP1P2;

[0029] 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 the C1-10 alkyl, C6-10 aryl, C7-10 alkylaryl, and C7-10 aralkyl are either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, -CN, -COOH, and -C(O)NH2.

[0030] According to the present invention, the compound having an amino group has an amino group with a pKa in the range of 1 to 16, preferably 4 to 14.

[0031] According to a preferred embodiment, the pKa value of the amino group of the amino compound is less than the pH value of the solution used to prevent and / or eliminate platelet aggregation in the blood sample.

[0032] According to the present invention, in the solution used to prevent and / or eliminate platelet aggregation in the blood sample, the concentration of the amino compound is 1 to 50 mmol / L, preferably 2 to 20 mmol / L.

[0033] In the application of this invention, the in vitro blood test includes at least one test selected from the following: platelet count, red blood cell count, and white blood cell count. The red blood cell count can further distinguish reticulocytes. The white blood cell count can also perform three-part, four-part, or five-part differential analysis. More specifically, the red blood cells are at least one of mature red blood cells, reticulocytes, and nucleated red blood cells. The white blood cells are at least one of neutrophils, eosinophils, basophils, lymphocytes, and monocytes.

[0034] The detection device may include an impedance detector and / or an optical detector.

[0035] The blood sample is from peripheral or venous blood of a mammal.

[0036] According to a second aspect of the invention, a reagent for inhibiting platelet aggregation interference in in vitro blood testing is provided, wherein the reagent comprises at least one ammonium ion-containing compound at a concentration of 1 to 50 mmol / L, preferably 2 to 20 mmol / L.

[0037] According to one specific embodiment, the ammonium-containing compound contains anion selected from the group consisting of chloride, bromide, iodide, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, hydrosulfide, thiocyanate, sulfate, bisulfite, sulfite, carbonate, bicarbonate, formate, acetate, oxalate, propionate, malonate, citrate, and combinations thereof.

[0038] More specifically, the ammonium-containing compound is selected from at least one of 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.

[0039] According to the present invention, the pH value of the reagent is at least 7.0, preferably 7.5 to 11, and more preferably 9.5 to 11.

[0040] According to one embodiment, the platelet depolymerization reagent further comprises at least one amino compound at a concentration of 1–50 mmol / L, preferably 2–20 mmol / L, said compound containing at least one primary or secondary amino group. Preferably, the amino compound contains 1–20 groups selected from primary, secondary, and imine groups.

[0041] According to one specific embodiment, the compound having an amino group is selected from the group consisting of compounds of formula (I) and their salts:

[0042] R1-NH-R2(I)

[0043] Compound of formula (I) is as defined above.

[0044] According to the present invention, the compound having an amino group has an amino group with a pKa in the range of 1 to 16, preferably 4 to 14.

[0045] According to a preferred embodiment, the pKa value of the amino group of the amino compound is less than the pH value of the reagent.

[0046] According to one specific embodiment, the reagent further includes a buffer, an osmotic pressure regulator, and optionally at least one selected from surfactants, fluorescent dyes, and erythrocyte lysis agents.

[0047] According to a third aspect of the invention, a method for preventing and / or eliminating platelet aggregation in an in vitro blood test is also provided, the method comprising the step of treating the blood sample with the composition or reagent defined above. Optionally, the method further comprises the step of testing the treated blood sample with a blood analyzer.

[0048] In this invention, the reagent containing the aforementioned compound, its uses, and its deagglomeration effect on aggregated platelets in in vitro blood testing methods can deagglomerate platelets in a short time without the need for additional conditions such as water bath temperature control or extended reaction time. Therefore, it does not require changes to existing detection procedures and is applicable to any blood analyzer. Moreover, the reagent has no adverse effects on platelets and other detection reagents, and can achieve good deagglomeration effects in various platelet aggregation situations, conveniently eliminating false platelet aggregation in samples and obtaining accurate blood cell detection parameters. Attached Figure Description

[0049] Figure 1 A schematic diagram illustrating the mechanism by which compounds with amino groups depolymerize and repolymerize aggregated platelets is shown.

[0050] Figure 2 A three-dimensional scatter plot of particles in a blood sample obtained in an optical detection device is shown, in which the optical information features of aggregated platelets and deaggregated platelets are shown respectively.

[0051] Figure 3 The effects of various ammonium ion-containing compounds at different concentrations on the depolymerization of ADP-induced platelet aggregation were shown.

[0052] Figure 4 The graphs show the depolymerization effect of an amino compound on aggregated platelets under different environmental pH conditions.

[0053] Figure 5 The graphs show the depolymerization effects of compounds with different amino pKa values ​​on aggregated platelets at pH 9.5.

[0054] Figure 6 The graph shows the effect of ADP on platelet aggregation in blood samples at different final concentrations over time.

[0055] Figure 7 The graph shows the effect of amino compounds on the depolymerization of platelet aggregation induced by different concentrations of ADP over time.

[0056] Figure 8 The graphs showing the effect of THR on platelet aggregation in blood samples at different final concentrations over time are shown.

[0057] Figure 9 The graph shows the effect of amino compounds on the depolymerization of platelet aggregation induced by different concentrations of THR over time.

[0058] Figure 10The graph shows the effect of COL on platelet aggregation in blood samples at different final concentrations over time.

[0059] Figure 11 The graph shows the effect of amino compounds on the depolymerization of platelet aggregation induced by different concentrations of COL over time.

[0060] Figure 12 The graphs showing the effect of RIS on platelet aggregation in blood samples at different final concentrations over time are shown.

[0061] Figure 13 The graphs show the effect of amino compounds on the depolymerization of platelet aggregation induced by different concentrations of RIS over time.

[0062] Figure 14 The effects of 1-acetylammonium and ammonium chloride, individually and in combination, on the deagglomeration of aggregated platelets at different concentrations were shown.

[0063] Figure 15 The image shows three-dimensional scatter plots of platelets and erythrocytes in blood samples before and after induced aggregation, detected using conventional staining solutions and staining solutions containing ammonium chloride in an optical detection device.

[0064] Figure 16 The image shows three-dimensional scatter plots of platelets and red blood cells in blood samples before and after induced aggregation, detected using a conventional diluent and a diluent containing ammonium chloride in an optical detection device.

[0065] Figure 17 The diagram shows histograms of platelets in blood samples before and after induced aggregation, obtained using a conventional diluent and a diluent containing ammonium chloride, detected on an impedance detection device.

[0066] Figure 18 The image shows three-dimensional scatter plots of platelets and leukocytes in blood samples before and after induced aggregation, detected using a conventional lysis buffer and an ammonium chloride-containing lysis buffer in an optical detection device.

[0067] Figure 19 The image shows a three-dimensional scatter plot of platelets and erythrocytes in blood samples before and after induced aggregation, detected using a conventional lysis buffer and a diluent containing a combination of amino compounds and ammonium ion-containing compounds in an optical detection device.

[0068] Figure 20 The image shows a three-dimensional scatter plot of platelets and erythrocytes in blood samples before and after induced aggregation, detected using a conventional staining solution and a staining solution containing a combination of amino compounds and ammonium ion-containing compounds in an optical detection device.

[0069] Figure 21 The image shows a three-dimensional scatter plot of platelets and red blood cells in blood samples before and after induced aggregation, detected using a conventional diluent and a diluent containing a combination of amino compounds and ammonium ion compounds in an optical detection device.

[0070] Figure 22 The diagram shows a comparison of the deagglomeration effects of conventional diluent and diluent containing ammonium chloride on samples exhibiting platelet pseudoagglomeration in clinical settings. Detailed Implementation

[0071] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0072] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, 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 pertains. In the event of any conflict, this specification shall prevail.

[0073] It should be noted that, in this invention, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a method or product comprising a list of elements includes not only the elements expressly stated, but also other elements not expressly listed, or elements inherent to implementing the method or product. Without further limitation, an element defined by the phrase "comprising one..." does not preclude the presence of other related elements in the method or apparatus that includes that element.

[0074] The inventors have discovered that the presence of ammonium cations in a solution can deagglomerate pseudo-agglomerates of platelets. Further research revealed that ammonium cations have a significant deagglomeration effect on platelet pseudo-agglomerations caused by various factors, without requiring additional heating or cooling or a long reaction time. Only a small amount of this substance needs to be added to the solution of the sample to be tested, and platelet aggregation can be eliminated after approximately 30 seconds. Therefore, this invention provides the use of such substances to prevent and / or eliminate platelet aggregation in blood samples. Deagglomerating pseudo-agglomerates of platelets not only allows for the determination of the true platelet count in the blood sample, thus providing a reliable reference for clinical decision-making, but also eliminates the influence of larger platelet aggregation particles on the classification and counting of other blood cells, especially white blood cells.

[0075] According to the present invention, when a solution containing ammonium ions is added to a blood sample in which platelet pseudoagglomeration occurs, platelets can be rapidly deagglomerated. As shown in the following examples, various ammonium salts exhibit substantially similar deagglomeration effects on aggregated platelets at a given pH. The ammonium ion-containing compound according to the present invention may contain anions selected from the group consisting of chloride, bromide, iodide, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, hydrosulfide, thiocyanate, sulfate, bisulfate, sulfite, bisulfite, oxalate, carbonate, bicarbonate, formate, acetate, propionate, malonic acid, citrate, and combinations thereof.

[0076] To avoid being bound by theory, the mechanism of ammonium ion depolymerization of platelets remains unclear. It is speculated that it is related to the concentration of ammonium ions forming hydrated ammonia in the solution. However, while direct use of ammonia water also exhibits good depolymerization performance, it is inferior to using compounds containing ammonium ions, particularly ammonium salts such as ammonium chloride, ammonium phosphate, and ammonium halides. Therefore, it is speculated that certain types of anions in the solution may also play a role in promoting platelet depolymerization. However, when ammonium ions are absent from the solution, these anions do not have a significant depolymerization effect.

[0077] Examples of compounds containing ammonium ions include ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium nitrate, ammonium thiocyanate, ammonium bisulfite, ammonium oxalate, ammonium hydroxide, ammonium bisulfate, and ammonium bicarbonate, but are not limited to these. Preferred compounds containing ammonium ions include ammonium chloride, ammonium bromide, ammonium phosphate, and ammonium hydrogen phosphate.

[0078] According to the present invention, a composition is provided for use in preventing and / or eliminating platelet pseudoaggregation in blood samples during in vitro blood testing. The composition comprises at least one of the aforementioned ammonium ion-containing compounds. It may also comprise two or more ammonium ion-containing compounds.

[0079] The compositions comprising at least one ammonium ion mentioned herein may be in the form of the compound itself or in solution form, for example, soluble in a suitable solvent for convenient use in the purposes of this invention. Suitable solvents may be commonly used solvents that do not interfere with the detection of blood samples, such as water, alcohols (e.g., ethanol), or aqueous solutions of alcohols.

[0080] The aforementioned at least one ammonium ion-containing compound exerts a deagglomeration effect on platelets at a certain concentration in a solution containing a blood sample. In the application of this invention, the concentration of the ammonium ion-containing compound in the composition can be in the range of 1–50 mmol / L, for example, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, or 50 mmol / L. Preferably, the concentration of the ammonium ion-containing compound is in the range of 2–20 mmol / L, more preferably in the range of 5–15 mmol / L.

[0081] The inventors have discovered that ammonium ions exhibit optimal effects when the concentration is 9.5 or higher. Therefore, when the ammonium ion-containing compound undergoes depolymerization, the ambient pH is preferably 7.0 or higher, more preferably 7.5 to 11.0, and even more preferably 9.5 to 11.0.

[0082] According to the present invention, one of the aforementioned ammonium ion-containing compounds can be used, or two or more of the aforementioned compounds can be used simultaneously for platelet deagglomeration. When two or more of the aforementioned compounds are used, the total concentration of each compound conforms to the above range. As mentioned above, the causes of platelet pseudoagglomeration are diverse, and the degree of aggregation and the difficulty of deagglomeration also vary greatly. Therefore, the concentration range for the aforementioned ammonium ion-containing compounds to be effective is relatively large. For cases where deagglomeration is easier, a lower concentration can be used; conversely, for cases where deagglomeration is more difficult or the degree of aggregation is high, a higher concentration can be used. The appropriate concentration can be reasonably determined by those skilled in the art based on the following specific embodiments and actual needs.

[0083] Furthermore, the inventors unexpectedly discovered that a class of compounds containing amino groups also exhibit significant deagglomeration effects on platelet pseudoagglomeration caused by various factors. Moreover, when ammonium ion-containing compounds are used in combination with amino-containing compounds, the deagglomeration effect on aggregated platelets can be significantly enhanced.

[0084] The term "amino" as used herein refers to primary and / or secondary amino groups unless otherwise specified, but excludes tertiary amino groups.

[0085] Unless otherwise specified, the term "amino-containing compound" as used herein refers to a compound containing at least one amino group (primary amino and / or secondary amino). The compound may also optionally contain a tertiary amino group and / or an imine group, and particularly may also optionally contain an imine group.

[0086] Not wishing to be bound by theory, the depolymerization mechanism of the compounds containing amino and / or imine groups on pseudo-aggregated platelets is presumed to be that the amino / imine groups in the compounds bind to the phosphatidylserine membrane of platelets in solution and are bonded to the carbonyl ester groups (-OC(O)-) on the phosphatidylserine membrane via hydrogen bonds (see...). Figure 1The diagram illustrates the depolymerization mechanism of amino compounds. The amino groups of these compounds, bound to the phosphatidylserine membrane, block calcium ion channels, preventing the influx of calcium ions. Since platelet aggregation requires calcium ion influx, these compounds depolymerize by preventing calcium ion influx through their amino groups. As the pH gradually decreases, the amino groups on the compounds become protonated. Although the exact reason is unclear, it is speculated that the protonated amino groups no longer bind to the carbonyl ester groups on the phosphatidylserine membrane via hydrogen bonds, allowing calcium ions to flow back into the blood cells, and platelets aggregate again. Theoretically, besides tertiary amino groups, primary, secondary, or imine groups with at least one hydrogen atom attached to the nitrogen atom all possess some platelet depolymerization function.

[0087] Further research by the inventors revealed that in compounds containing amino groups, the greater the number of amino / imine groups, the better the depolymerization effect. Furthermore, the depolymerization effect of primary amino, secondary amino, and imine groups decreases in that order. Generally, compounds with stronger hydrophilicity exhibit better depolymerization effects than those with weaker hydrophilicity. Hydrophilic substituents, such as hydroxyl, carboxyl, amide, and sulfonic acid groups, have little effect on the depolymerization effect of amino / imine groups; in some cases (e.g., hydroxyl, polyhydroxyl), they may even promote depolymerization. This may be related to the fact that these groups increase the water solubility of the compound and its affinity for the phosphatidylserine membrane. Hydrocarbon groups, such as alkyl and aryl groups, have little effect on platelet depolymerization as long as they do not affect the compound's water solubility. Hydrophobic groups, such as ester groups (-C(O)-O-) and carbonyl groups (-C(O)-), can reduce the depolymerization effect to some extent.

[0088] Therefore, compounds having groups such as hydroxyl, carboxyl, or sulfonic acid groups, such as alkanolamines, amino acids, and sulfonic acids, are preferred. Furthermore, substances having multiple amino and / or imine groups are also preferred, such as guanidines and short peptides. Preferably, the amino compound has at least one primary or secondary amino group, and more preferably 1-20 primary, secondary, and / or imine groups. Preferably, the amino compound has at least one primary amino group, and more preferably 1-4 primary amino groups.

[0089] Based on the above-specified mechanism, when compounds containing amino groups depolymerize, the amino groups exist in solution in a deprotonated form. The ability of the amino / imine groups in different compounds to bind hydrogen ions in solution varies. This ability to bind / dissociate hydrogen ions can be represented by the dissociation constant pKa.

[0090] Amino compounds exist in solution in the following equilibrium:

[0091]

[0092] pKa=lg([R1R2NH]×[H +] / [R1R2NH2 + ]).

[0093] To achieve a suitable platelet disaggregation effect, the amino group of the compound should have an appropriate pKa value. For compounds with multiple amino / imine groups, the pKa of the amino group referred to herein is the first dissociation constant, i.e., the pKa of the first amino or imine group to dissociate hydrogen ions.

[0094] Typically, the amino group of the compound should have a pKa value of 1 to 16, preferably 1 to 14, and more preferably 4 to 14.

[0095] Furthermore, for the same compound, the depolymerization ability is enhanced when the pKa value of the amino group is lower than the pH value of the solution. Based on the depolymerization mechanism of the above compounds on aggregated platelets, it can be understood that as the pH gradually increases, the equilibrium between the binding and dissociation of the amino groups with hydrogen ions in the solution shifts towards dissociation. More amino groups are deprotonated, and thus more amino groups bind to the phosphatidylserine membrane of the platelets through hydrogen bonds, thereby blocking calcium inflow and achieving depolymerization. This binding further promotes the deprotonation of more amino groups, thus completing depolymerization in a shorter time without increasing the temperature.

[0096] The inventors have discovered that the depolymerization of aggregated platelets by the aforementioned amino-containing compounds is affected by pH. However, within the pH range where ammonium-containing compounds can exert their depolymerization effect, i.e., 7.0 or higher, preferably 7.5 to 11, and particularly preferably 9.5 to 11, most also exhibit good depolymerization activity. This is because a higher pH also makes it easier to conform to the rule that the depolymerization effect is enhanced when the pKa of the amino group in the aforementioned compounds is less than or equal to the pH value of the solution. Therefore, suitable types of amino-containing compounds can be selected to obtain the desired depolymerization effect.

[0097] The effective concentration of amino-containing compounds that can depolymerize pseudo-agglomerated platelets is related to the number and type of amino groups in the compound, as well as factors such as the compound's hydrophilicity. Generally, the concentration of amino-containing compounds that depolymerize platelets is in the range of 1–50 mmol / L, for example: 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, and 50 mmol / L. According to a preferred embodiment, the concentration is in the range of 2–20 mmol / L, more preferably in the range of 5–15 mmol / L.

[0098] Such amino-containing compounds can be compounds having the structure shown in formula (I), or they can be salts of it. Formula (I) is:

[0099] R1-NH-R2(I)

[0100] Wherein, R1 and R2 may be the same or different, and each is independently a group selected from the group consisting of H, -SO3H, -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 aralkyl, -C(O)-Q1, and -C(O)-O-Q2.

[0101] Wherein, Q1 is H, -NH2, 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.

[0102] 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;

[0103] Wherein, the substitution refers to being replaced by at least one group selected from the group consisting of -NP1P2, -SO3H, -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. The substitutions, wherein the -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 aralkyl are either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, halogen, -CN, -COOH, and -C(O)NH2.

[0104] 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 either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, halogen, -CN, -COOH, and -C(O)NH2.

[0105] According to one embodiment, in the compound of formula (I), R1 and R2 may be the same or different, each independently being a group selected from the group consisting of H, -SO3H, -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.

[0106] Wherein, Q1 is H, -NH2, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, or substituted or unsubstituted C7-10 aryl.

[0107] 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 aryl;

[0108] Wherein, the substitution refers to being substituted by at least one group selected from the group consisting of -NP1P2, -SO3H, -OH, -CN, -C(O)-O-P3, -O-C1-10 alkyl, -O-C6-10 aryl, -O-C7-10 alkylaryl, -O-C7-10 aryl, and -C(O)-NP1P2, wherein -O-C1-10 alkyl, -O-C6-10 aryl, -O-C7-10 alkylaryl, and -O-C7-10 aryl are either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, -CN, -COOH, and -C(O)NH2;

[0109] 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 the C1-10 alkyl, C6-10 aryl, C7-10 alkylaryl, and C7-10 aralkyl are either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, -CN, -COOH, and -C(O)NH2.

[0110] Preferably, in the compound of formula (I), R1 and R2 may be the same or different, each independently being a group selected from the group consisting of H, -SO3H, -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)-OH.

[0111] Wherein, Q1 is H, -NH2, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkylaryl, or substituted or unsubstituted C7-10 aryl.

[0112] Wherein, the substitution refers to being replaced by at least one group selected from the group consisting of -NP1P2, -SO3H, -OH, -CN, -C(O)-OH and -C(O)-NP1P2;

[0113] 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 the C1-10 alkyl, C6-10 aryl, C7-10 alkylaryl, and C7-10 aralkyl are either unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, -CN, -COOH, and -C(O)NH2.

[0114] Furthermore, in the compounds of formula (I), R1 and R2 may be the same or different, each being independently a group selected from the group consisting of H, -C(NH)-NH2, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted phenethyl and -C(O)-Q1, wherein Q1 is as defined above.

[0115] Furthermore, in the compound of formula (I), Q1 is a substituted or unsubstituted C1-6 alkyl, a substituted or unsubstituted phenyl, a substituted or unsubstituted benzyl, or a substituted or unsubstituted phenethyl;

[0116] Wherein, the substitution refers to being replaced by at least one group selected from the group consisting of -NP1P2, -SO3H, -OH, and -C(O)-NP1P2.

[0117] P1, P2, and P3 are each independently a group selected from the group consisting of H, C1-6 alkyl, phenyl, benzyl, and phenethyl, wherein the C1-6 alkyl, phenyl, benzyl, and phenethyl are unsubstituted or further substituted by at least one group selected from the group consisting of -NH2, -OH, -SO3H, -COOH, and -C(O)NH2.

[0118] The C1-16 alkyl groups described herein can be straight-chain or branched alkyl groups, preferably C1-14 or C1-12 alkyl groups, more preferably C1-10 alkyl groups, and even more preferably C1-6 alkyl groups. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-octyl, dodecyl, hexadecyl, etc., but are not limited thereto.

[0119] The C6-10 aryl group mentioned in this article can be phenyl or naphthyl.

[0120] The C7-14 alkylaryl groups described herein can be monoalkyl or polyalkyl-substituted aryl groups. Preferably, C7-10 alkylaryl groups are substituted, such as tolyl, ethylphenyl, propphenyl, butylphenyl, xylyl, methyl ethylphenyl, diethylphenyl, etc., but not limited thereto.

[0121] The C7-14 aralkyl groups described herein can be phenylalkyl groups. Preferably, C7-10 aralkyl groups are used, such as benzyl, phenethyl, phenylpropyl, phenylbutyl, etc., but are not limited thereto.

[0122] The substitution referred to herein means substitution by at least one of the defined substituents. For the amino substituent -NP1P2, according to the present invention, it is preferably polyamino-substituted, with 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, it is a preferred substituent. The number of hydroxyl groups can be 1 to 6, such as 2, 3, 4, 5, 6. Furthermore, sulfonic acid groups, carboxyl groups, and nitrile groups are also preferred.

[0123] The halogens mentioned in this article generally refer to -F, -Cl, -Br, and -I, with -F, -Cl, and -Br being preferred.

[0124] According to the following examples, preferred amino compounds are guanidines, alcoholamines (especially polyhydroxy alcoholamines, such as 1,3-diamino-2-propanol), amino acids (preferably lysine, glutamine, glycine), short peptides (such as dipeptides), and polyamino-substituted branched or straight-chain alkanes (such as 2-(aminomethyl)propane-1,3-diamine, 1,3-propanediamine, etc.).

[0125] Examples of compounds of formula (I) that can prevent and / or eliminate platelet pseudoaggregation include: sulfonic acids, such as aminosulfonic acid, aminomethanesulfonic acid, taurine, p-aminobenzenesulfonic acid, and 2,5-diaminobenzenesulfonic acid; amino acids, such as glutamic acid, glutamine, arginine, lysine, alanine, glycine, N-tris(hydroxymethyl)methylglycine, and 4-aminobutyric acid; short peptides, such as dipeptides, such as dimerlysine and dimerlyglycine; alcohols / phenolic amines (especially polyhydroxysubstituted amines), such as ethanolamine, triethanolamine, 3-amino-1-propanol, 4-amino-1-butanol, 4-hydroxybenzylamine, tyramine (4-hydroxyphenylethylamine), 1,3-diamino-2-propanol, and 3-aminobenzylamine. -1,2-Propanediol, tris(hydroxymethyl)aminomethane, p-hydroxyaniline, 3,5-dihydroxyaniline, 4-aminophenol; aromatic amines, such as m-phenylenediamine, m-phenyltriamine; alkylamines, such as ethylamine, n-propylamine, n-butylamine, tert-butylamine, 1,3-propanediamine, 2-(aminomethyl)propane-1,3-diamine; aminonitriles, such as aminoacetonitrile, triaminopropionitrile; amides, such as formamide, acetamide, carbamide, asparagine methyl ester, 2-(methylamino)butadiamide, 2-aminoacetamide, 2-aminopropionamide, glutamine; guanidines, such as guanidine, biguanide, aminoguanidine, methylguanidine, {[amino(imino)methyl]amine}-acetic acid, 1-acetylguanidine, etc., but not limited to these.

[0126] Preferred compounds of formula (I) may be hydroxyl-substituted amines, alkylamines, benzenesulfonic acid amines, amino acids and short peptides, and guanidines.

[0127] Salts of compounds containing amino groups are also within the scope of this invention. Salts of compounds containing acidic sulfonic acid groups, carboxyl groups, etc., are particularly preferred. These salts can be salts of alkali metals (such as potassium and sodium), salts of alkaline earth metals (such as magnesium), salts of ammonium cations, etc.; they can also be internal salts. Due to the effect of the aforementioned ammonium cations on platelet deagglomeration, salts of ammonium cations are especially preferred.

[0128] In the application of this invention, when the ammonium-containing compound is combined with the amino-containing compound, a good depolymerization effect can be obtained at a lower concentration. That is, when the total concentration of the two is comparable to the concentration when either is used alone, a better effect can be obtained than when either the ammonium-containing compound or the amino-containing compound is used alone. The combination of the two has a synergistic effect on platelet depolymerization. Therefore, when the two are used in combination, the total concentration can be in the range of 2 to 50 mmol / L, for example, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50 mmol / L. Preferably, the composition may include 1 to 20 mmol / L of the amino-containing compound and 1 to 20 mmol / L of the ammonium-containing compound; more preferably, the composition may include 1 to 10 mmol / L of the amino-containing compound and 1 to 10 mmol / L of the ammonium-containing compound; and even more preferably, it may include 1 to 5 mmol / L of the amino-containing compound and 1 to 5 mmol / L of the ammonium-containing compound. There is no particular limitation on the ratio of the two used. As an example, the molar ratio of the compound having an amino group to the compound containing an ammonium ion can be in the range of 1:10 to 10:1, preferably in the range of 1:4 to 4:1, and is 1:4, 1:2, 1:1, 2:1, 4:1, etc. Most preferably, the molar ratio is 1:1.

[0129] As can be seen from the following examples, a significantly improved depolymerization effect was achieved when amino-containing compounds and ammonium-containing compounds were used in combination.

[0130] This combination is particularly advantageous for in vitro blood testing applications because the concentration of the substance used for platelet disaggregation is significantly reduced. It is known that quaternary ammonium salts are known to have a destructive effect on cell membranes, especially erythrocyte membranes, and are therefore commonly used as erythrocyte lysis agents. The inventors have observed that the composition of the present invention does not have a destructive effect on erythrocytes within the above-mentioned concentration range, and the erythrocyte count remains essentially unchanged with and without the use of the composition. In particular, at the preferred concentrations of the above-mentioned combination of amino-containing compounds and ammonium-containing compounds, the composition not only achieves excellent platelet disaggregation but also has no adverse effects on blood cells, especially erythrocytes.

[0131] According to the present invention, the in vitro blood test refers to the detection of blood cells in a blood sample, such as platelets, red blood cells, and white blood cells. In the present invention, the detection can be microscopic examination or instrumental detection, preferably instrumental detection. Specifically, the detection can be, for example, using a blood analyzer to detect various blood cells and obtain corresponding detection information. The present invention does not limit the use of blood analyzers; any instrument used for in vitro blood testing can be used for the purposes of this invention.

[0132] The application of this invention is not limited to detectors; it can be used for impedance detectors, optical detectors, and other similar devices.

[0133] Taking an optical detector as an example, multiple aggregated platelets are identified as a single particle in the optical channel of a blood analyzer. Therefore, these aggregated platelets are larger in volume and have stronger fluorescence intensity than a single platelet, but are fewer in number than non-aggregated platelets. This is reflected in the detection information; see [link to relevant documentation]. Figure 2 As shown in the 3D scatter plot obtained from the Mindray hematology analyzer, aggregated platelets exhibit higher forward scattered light intensity (FS), indicating larger particle size, and also show higher fluorescence intensity (FL). Some aggregated platelets reach the volume of white blood cells, which can affect white blood cell counts. After deaggregation, the volume of platelets in the optical channel decreases, resulting in reduced fluorescence intensity and increased platelet count. See further details. Figure 2 As can be seen from the three-dimensional scatter plot, after depolymerization, the platelet particle swarm reflects that the platelet volume corresponding to forward scattered light (FL) is significantly reduced, the number of particles is increased, and the fluorescence intensity is reduced.

[0134] Similarly, with impedance detectors, aggregated platelets are treated as particles, generating an electrical signal. Because the aggregated particles are larger, they cannot be identified as platelets, leading to a falsely low platelet count. Furthermore, aggregated platelets also affect white blood cell and red blood cell counts and histogram changes. This effect was particularly noticeable in early three-part differential hematology analyzers.

[0135] According to the application of the present invention, by adding the above composition to a sample, platelet aggregation can be prevented, and already aggregated platelets can be deaggregated, thereby obtaining an accurate platelet count.

[0136] Furthermore, since there are no longer aggregated platelets, interference with white blood cell detection is also eliminated, thus providing accurate white blood cell detection information. The white blood cell detection also includes the classification and / or counting of, for example, nucleated cells, basophils, eosinophils, neutrophils, and / or lymphocytes.

[0137] The “detection information” mentioned in this article includes the raw signal (such as electrical pulses, light intensity, etc.), information obtained after processing such as histograms, scatter plots, etc., and the classification and / or counting information in the final report.

[0138] Furthermore, since the number of red blood cells in blood is far greater than that of other cell types, although platelet aggregation does affect red blood cells in optical detection, the impact is minor and has almost no effect on the counting results. However, the addition of amines and ammonium salts may cause red blood cell lysis. As can be seen from the following examples of this invention, the compounds containing ammonium ions and further compounds containing amino groups, within the concentration range defined above, do not adversely affect the sample environment (such as pH, ionic strength, etc.) nor cause damage to blood cells. Therefore, platelet deaggregation has no effect on the detection of red blood cells. This invention can be used to further classify and count mature red blood cells, reticulocytes, and nucleated red blood cells separately.

[0139] The present invention is effective for platelet aggregation caused by various reasons, therefore there are no particular limitations on the samples used for these purposes. The samples can be derived from peripheral blood, such as capillary or venous blood. The method of obtaining the samples is also not particularly limited; blood can be drawn by finger-prick puncture or venously using an anticoagulant tube. Freshly collected blood can be collected using anticoagulant tubes coated with a common anticoagulant such as EDTA, as the compositions of the present invention are also effective against EDTA-induced pseudoaggregation of platelets.

[0140] Generally, after blood collection, samples can undergo any routine processing to obtain the test sample. Such processing can include, for example, dilution, staining, lysis, etc. In the application of this invention, the composition can be added to the sample alone as a processing agent (e.g., as a platelet deagglomerating agent), or added to the sample along with the processing agent in any step of the sample processing. Therefore, the composition of this invention can be formed as a standalone processing agent; it can also be formed as a diluent, staining agent, or lysis agent with platelet deagglomeration effect by including the amino-containing compound and optionally an ammonium-containing compound in the reagent used for dilution, staining, or lysis, thereby simultaneously preventing / eliminating platelet deagglomeration in any step of the processing without hindering the processing. The reaction of the composition with platelet deagglomeration does not require additional temperature control (e.g., low temperature or heating). Platelet deagglomeration is completed in approximately 30 seconds after the processing agent containing the composition is mixed with the sample, and the sample can then be tested.

[0141] According to the present invention, the sample may be derived from mammals, preferably primates, and more preferably humans.

[0142] Therefore, the present invention also provides a method for preventing and / or eliminating platelet aggregation in an in vitro blood test, the method comprising a treatment step of preventing / eliminating platelet aggregation interference on the blood sample. The treatment step involves treating the blood sample under the conditions described above with a composition or reagent comprising the aforementioned ammonium-containing compound and optionally an amino-containing compound.

[0143] More specifically, the present invention provides an in vitro blood detection method that resists platelet aggregation interference.

[0144] The method may include treating a blood sample with a depolymerizing agent comprising at least one compound selected from those containing ammonium ions and optionally containing amino groups to prevent and / or eliminate platelet aggregation in the blood sample, and testing the blood sample treated with the depolymerizing agent using a blood analyzer to obtain at least the detection information of blood cells after eliminating interference from blood cell aggregation.

[0145] The blood cells may be platelets, red blood cells, and / or white blood cells.

[0146] The detection information, depending on the detector used, can be a pulse signal targeting particles in the sample or an optical signal. The blood analyzer can process the detection information to further obtain blood cell detection parameters such as histograms, scatter plots, classification, and / or counting.

[0147] According to one embodiment, the blood analyzer may include an impedance detector. The method may include the following steps: detecting a blood sample treated with the depolymerizing agent using an impedance detector in the blood analyzer to obtain electrical signals of particles in the blood sample, and further obtaining detection parameters for platelets and / or red blood cells.

[0148] In addition, the impedance detector is still used for detection. Before the detection, the blood sample can be further treated with a red blood cell lysis agent in addition to being treated with a depolymerizing agent. The method includes the following steps: the blood sample treated with the depolymerizing agent and red blood cell lysis agent is detected by an impedance detector in a blood analyzer to obtain the electrical signal of the particles in the blood sample, and further obtain the white blood cell detection parameters.

[0149] The three-part differential and count of white blood cells can be obtained using an impedance detector.

[0150] According to another embodiment, the blood analyzer includes an optical detector, and the method includes detecting a blood sample treated with the depolymerizing agent using the optical detector in the blood analyzer to obtain at least one scattered light intensity signal of particles in the blood sample, and further obtaining platelet detection parameters; preferably, the at least one scattered light intensity signal is a forward scattered light intensity signal. More preferably, platelet detection parameters can be obtained from both forward and lateral scattered light intensity signals.

[0151] According to another embodiment, still using the optical detector, the blood sample is further stained with a fluorescent dye before the detection, and the method includes: detecting the blood sample treated with the depolymerizing agent and fluorescent dye using an optical detector in a blood analyzer to obtain fluorescence intensity signals of particles in the blood sample and at least one scattered light intensity signal to obtain detection parameters for platelets and / or red blood cells.

[0152] Preferably, in this embodiment, the method further includes distinguishing reticulocytes and obtaining detection parameters for reticulocytes. More preferably, the at least one scattered light intensity signal is a forward scattered light intensity signal. Detection parameters for platelets and / or red blood cells can also be obtained using fluorescence intensity signals, forward scattered light intensity signals, and side scattered light intensity signals.

[0153] According to another embodiment, still using the optical detector, the blood sample is further stained with a fluorescent dye and hemolyzed with a erythrocyte lysis agent, and the method includes: detecting the blood sample treated with the depolymerizing agent, fluorescent dye and erythrocyte lysis agent using an optical detector in a blood analyzer to obtain at least two optical signals of particles in the blood sample to obtain white blood cell detection information.

[0154] Preferably, in this embodiment, the at least two optical signals are selected from fluorescence intensity signal, forward scattered light intensity signal, and side scattered light intensity signal.

[0155] In white blood cell testing, for example, the DIFF detection channel of the Mindray blood analyzer can be used to obtain four differential counts (i.e., eosinophils, neutrophils, monocytes, and lymphocytes), or further, the WNB channel of the Mindray blood analyzer can be used to obtain basophil detection information.

[0156] In the above detection method, the depolymerizing agent, fluorescent dye, and erythrocyte lysis agent can be individual reagents or arbitrarily combined into a mixed reagent. For example, the depolymerizing agent can be a diluent, staining agent, lysis agent, or staining-lysis agent with platelet depolymerization effect.

[0157] Therefore, the present invention also provides a reagent for resisting platelet aggregation interference in in vitro blood testing.

[0158] Since the volume of reagents used to process blood samples in in vitro blood tests is much larger than the volume of the blood sample itself, the processing conditions such as concentration and pH described above are generally also applicable to the antiplatelet aggregation interference reagent of the present invention.

[0159] According to the present invention, the reagent may comprise at least one of the aforementioned ammonium ion-containing compounds at a concentration of 1 to 50 mmol / L, and may further comprise at least one of the aforementioned amino-containing compounds at a concentration of 1 to 50 mmol / L. The concentration defined in this invention refers to the total concentration for two or more ammonium ion-containing compounds. Similarly, the concentration refers to the total concentration for two or more amino-containing compounds. The at least one ammonium ion-containing compound is as defined above and will not be repeated here. Its concentration is also preferably 2 to 20 mmol / L, more preferably 5 to 10 mmol / L.

[0160] The reagent further contains at least one amino compound as defined above, preferably at a concentration of 2–20 mmol / L, more preferably 5–10 mmol / L. According to a preferred embodiment, the reagent contains both an ammonium-containing compound and an amino compound, both with concentrations ranging from 1–20 mmol / L, 1–10 mmol / L, or even 1–5 mmol / L. The molar ratio can be any ratio, for example, ranging from 1:10 to 10:1, preferably 1:4 to 4:1, for the ammonium-containing compound to the amino compound.

[0161] The pH of the reagent is not particularly limited, but depends on the requirements for its use. Preferably, the pH of the reagent is at least 7.0, more preferably 7.5 to 11, and more preferably 9.5 to 11.0. The pH of the reagent can be adjusted using conventional buffers. The present invention does not particularly limit the type of buffer used for the reagent. For example, citrate buffer pairs, phosphate buffer pairs, borate buffer pairs, phthalate buffer pairs, 3-(N-morpholino)ethanesulfonic acid buffer pairs, 4-hydroxyethylpiperazine ethanesulfonic acid buffer pairs, etc., can be used, but are not limited thereto.

[0162] The reagent should have a certain osmotic pressure. The osmotic pressure can vary according to the actual requirements of the reagent. For example, for a diluent, it can be preferably 200 mOsm / L within the range of 180–240 mOsm / L; for a pyrolysis agent, it can be preferably 90 mOsm / L within the range of 70–130 mOsm / L. The reagent may include an osmotic pressure regulator. The reagent of the present invention does not have particular limitations on the osmotic pressure regulator; for example, it can be an inorganic salt, such as sodium chloride, potassium chloride, etc.; or a sugar, such as glucose, mannose, fructose, maltose, etc., but is not limited to these.

[0163] The reagent may also include surfactants. Depending on the detection purpose, the reagent may include surfactants with different functions. For example, for red blood cell detection, the reagent used as a diluent may include surfactants that cause red blood cells to spheroidize. Such surfactants include, but are not limited to, N-alkyl betaine, sodium N,N-dimethyloctadecylamine hydrochloride, sodium dodecylbenzenesulfonate, etc. As another example, for white blood cell detection, the reagent may include surfactants that alter cell membrane permeability to facilitate the passage of nucleic acid dyes through the cell membrane into the cell and their binding to nucleic acids. Such surfactants include, but are not limited to, phenoxyethanol, sodium dioctadecylamine hydrochloride, sodium N-methylammonium carboxylate, etc.

[0164] In addition, the reagent may include other necessary components as needed, such as preservatives, antibacterial agents, cell membrane protectants, chelating agents, etc. Those skilled in the art can choose to add them as needed.

[0165] According to one embodiment of the present invention, the reagent can be used as a diluent for blood samples. For example, it can be a diluent for impedance detection or a diluent for optical detection. Examples of diluent formulations include:

[0166]

[0167] According to the present invention, the reagent can also be a staining agent. Generally, because dyes are readily soluble in organic solvents, they are stored separately in the form of an organic solution in platelet analyzers. If necessary, the dye can also be added to the above-mentioned diluent to prepare a staining agent with platelet deaggregation function. The dye in the staining agent can be a nucleic acid dye. Examples of nucleic acid-specific dyes include asymmetric cyanine dyes, thiazole orange (TO), oxazole orange (YO), and acridine orange (AO), specifically PI, DAPI, and the Hoechst series (such as Hoechst 33258 and Hoechst 33342), but are not limited to these. A preferred nucleic acid dye is an asymmetric cyanine dye, such as SYBR Green. The optimal staining pH for this type of dye is generally consistent with the optimal effective pH for the compounds containing ammonium ions and compounds containing amino groups in this invention. Depending on the specific detection requirements, the staining agent may also be a mitochondrial staining agent (e.g., Janus Green B, MitoLite Red, Rhodamine 123, Mitotracker Green, Mitotracker Deep Red, Mitotracker Red, etc.) or a membrane staining agent (e.g., DiA, DiD, DiI, DiO, DiR, DiS, FDA, Alexa Fluor 488, Super Fluor 488, etc.).

[0168] Such a dye formulation can be:

[0169]

[0170] Furthermore, the reagent of the present invention can also be a lysis agent for use in the detection of leukocytes. The lysis agent used in the reagent can be any conventional erythrocyte lysis agent. As mentioned above, the amino-containing compounds in the reagent of the present invention, particularly those containing ammonium ions, have no adverse effect on erythrocytes within the defined concentration range; therefore, the type and concentration of the lysis agent in the reagent, as well as the method of use, are the same as in the prior art. Examples of erythrocyte lysis agents include quaternary ammonium salts (such as dimethylbenzylammonium chloride, dodecyltrimethylammonium chloride, dimethylbenzylalkylammonium chloride, etc.), but are not limited thereto. Such a lysis agent formulation can be:

[0171]

[0172] Again, as described above, the reagent of the present invention comprising a compound containing ammonium ions and optionally a further compound having amino groups has a low concentration of active ingredients in preventing / eliminating pseudo-platelet aggregation, does not require additional reaction conditions (such as cooling or heating), nor requires extended reaction time, and can complete the depolymerization of platelets in 30 seconds. Therefore, the compound has no adverse effect on blood detection, no influence on detection procedures and conditions, and can eliminate the interference of platelet aggregation under existing equipment and detection methods.

[0173] Examples

[0174] Example 1. Depolymerization effect of compounds containing ammonium ions on aggregated platelets

[0175] In this example, the effect of compounds containing ammonium cations on the depolymerization of aggregated platelets was determined.

[0176] Compounds containing ammonium ions (ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium thiocyanate, ammonium bicarbonate, ammonium oxalate, ammonia water) and the control compound (NaCl) were respectively added to the diluent for hematology analyzer to prepare blood sample diluents. A blood sample with aggregated platelets was obtained by treating a blood sample with ADP, a platelet aggregation inducer 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), bacteriostatic agent (6 g / L), sodium chloride (3 g / L), EDTA (0.1 g / L). The above-mentioned compounds containing ammonium ions (final concentration 0.01 mol / L) were respectively added to the diluent to prepare treatment solutions. The pH of the treatment solution was adjusted to 9.5, and the osmotic pressure was adjusted to 200 mOsm / L.

[0177] Venous blood from healthy subjects was collected into blood anticoagulant tubes for later use. Two 1ml aliquots of blood were taken, one aliquot was added to the above diluent, mixed well and the platelet count was measured in a hematology analyzer (Mindray BC-6000Plus), recorded as: PLT_O (unaggregated); the other aliquot was added with the platelet aggregation inducer ADP (final concentration 0.01 mmol / L) and mixed well. Platelets will aggregate after 5 minutes, at this time the above diluent is added, and the blood containing aggregated platelets is detected by the hematology analyzer to obtain the platelet count, recorded as: PLT_O (undepolymerized). Another 1ml of blood was taken, added with the platelet aggregation inducer ADP (final concentration 0.01 mmol / L), mixed well, and after 5 minutes, the above treatment solution was added, mixed well, and the platelet count in the treated blood was detected by the hematology analyzer, recorded as: PLT_O (depolymerized).

[0178] Generally, PLT_O (undepolymerized) < PLT_O (depolymerized) < PLT_O (unaggregated).

[0179] The term "depolymerization rate" mentioned in this article is calculated using the following formula:

[0180] Depolymerized sample: Depolymerization rate = PLT_O (depolymerization) / PLT_O (unpolymerized); or

[0181] The deagglomeration effect of the above-mentioned diluent on the samples induced by platelet aggregation can be calculated using the following formula:

[0182] Undepolymerized (control) sample: Depolymerization rate = PLT_O (undepolymerized) / PLT_O (unpolymerized).

[0183] The depolymerization rate of ammonium ion compounds at different concentrations was measured as follows: Figure 3 As shown.

[0184] from Figure 3 As can be seen, in the samples where platelet aggregation was induced by 0.01 mmol / L ADP, all compounds containing ammonium ions exhibited some depolymerization activity at a concentration of 1 mmol / L, with the optimal effect achieved at a concentration of 10 mmol / L. Specifically, at 10 mmol / L, the concentrations were: (NH4)2HPO4 99.25%, NH4Cl 95.70%, (NH4)3PO4 94.87%, NH4Br 93.48%, NH4I 92.75%, NH4HCO3 88.68%, NH4C2O4 87.67%, NH4H2PO4 86.24%, NH4SCN 85.78%, and NH4OH 83.05%. When the ammonium ion compound was replaced with NaCl, the depolymerization effect decreased to 18.90%. This indicates that it is the ammonium ions, not the anions, that play a role in platelet depolymerization. Furthermore, different anions have some influence on the depolymerization effect, but the influence is not significant.

[0185] Example 2. Effect of ambient pH on the platelet depolymerization ability of compounds containing ammonium ions.

[0186] This example investigated the effect of environmental pH on the deagglomeration effect of ammonium ion-containing compounds on aggregated platelets. Following a method similar to Example 1, 0.01 mol / L ammonium chloride was added to the diluent to prepare a treatment solution. 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 deagglomeration rate of ammonium chloride on platelets in the samples was measured, as shown in Table 1 below.

[0187] Table 1: Effect of pH on platelet depolymerization by ammonium ions in ammonium chloride

[0188] Depolymerization rate 18.62％ 31.70％ 59.61％ 94.05％ 94.62％ 95.70％ 97.72％

[0189] As shown in Table 1, when the ambient pH is above 7.5, the deagglomeration effect of ammonium ions on aggregated platelets suddenly increases to 94.05%, and remains at a high deagglomeration rate in the pH range of 7.5–11.0. This suggests that the formation of more hydrated ammonia by ammonium ions in the solution can promote platelet deagglomeration.

[0190] Example 3. Comparison of the ability of structurally similar amino-containing and non-amino-containing compounds to depolymerize platelets.

[0191] This example compares the effects of compounds with similar structures and substituents, containing and without amino groups, on platelet depolymerization. The tested compounds are shown in Table 2 below:

[0192] Table 2: Amino-containing compounds and structurally similar non-amino-containing compounds of the test subjects

[0193]

[0194]

[0195] The treatment solution was prepared using a method similar to that in Example 1, except that the compounds containing ammonium ions were replaced with the compounds in Table 2 above, and the pH of the diluent was adjusted to 14 and the osmotic pressure to 200 mOsm / L.

[0196] The platelet deagglomeration rate after ADP-induced blood treatment was tested one by one using the treatment solutions containing each compound in Table 2, as shown in Table 3 below.

[0197] Table 3. Effects of amino-containing and non-amino-containing structurally similar compounds on platelet disaggregation.

[0198]

[0199] As shown in Table 3 above, compounds containing only amino groups have a depolymerization effect on aggregated platelets, while other groups, such as sulfonic acid groups, hydroxyl groups, carboxyl groups, and carbonyl groups, have almost no depolymerization effect on aggregated platelets (comparable to the depolymerization rate of the blank control NaCl). This example confirms that the amino group is the main depolymerization agent for platelets in each compound.

[0200] Example 4. Relationship between the amount of amino groups and the ability of platelets to disaggregate.

[0201] This example compares the effects of different numbers of amino groups on platelet disaggregation in structurally similar compounds. Compounds containing different numbers of amino groups were tested using the same method as in Example 3, and the platelet disaggregation rates and pKa values ​​for each compound are shown in Table 4.

[0202] Table 4. Platelet disaggregation rate and pKa value of compounds containing different numbers of amino groups.

[0203]

[0204]

[0205]

[0206] As can be seen from Table 4 above, when the structures are similar or identical but the number of substituted amino groups differs, the depolymerization effect of the compounds on aggregated platelets increases with the increase of the number of amino groups. Among them, short peptides, polyamino-substituted alkylamines, polyamino-substituted hydroxylamines, and guanidines are more preferred.

[0207] Example 5. Relationship between aminopKa and platelet depolymerization ability under the same pH conditions

[0208] This example compares the effects of different amino groups with similar structures on platelet disaggregation under the same pH environment. Following essentially the same method as in Example 3, the difference was that different types of compounds were adjusted to a more suitable pH value. Compounds containing different numbers of amino groups were tested separately, and the platelet disaggregation rate and pKa value of each compound are shown in Table 5.

[0209] Table 5. Platelet disaggregation rates of compounds with different amino pKa values ​​at a given environmental pH.

[0210]

[0211]

[0212]

[0213] As can be seen from Table 5 above, different substituents on the amino group will affect the pKa value of the amino group.

[0214] Example 6. Relationship between ambient pH and the amino pKa of amino-containing compounds and its effect on depolymerization ability.

[0215] Example 5 shows a relationship between environmental pH and the amino pKa of amino-containing compounds. To further investigate the relationship between environmental pH and the amino pKa of amino-containing compounds, and their impact on platelet depolymerization ability, this example selected 1-acetylguanidine, a compound with a pKa of 8.33. Following a method similar to Example 1, only the environmental pH was varied between 7.0 and 10, and a series of tests were conducted. The resulting curves of pH versus depolymerization rate are shown below. Figure 4 As shown.

[0216] from Figure 4It can be seen that there is a strong correlation between environmental pH and the pKa of substances in platelet depolymerization. When pH increases, the depolymerization ability of amino-containing substances increases, with the depolymerization rate increasing from 35.23% to 97.32%. However, when pH reaches 9.5 or higher, the depolymerization rate remains essentially unchanged. This indicates that when the environmental pH exceeds the amino pKa of the compound to a certain extent, further increasing the pH has little effect on the depolymerization effect. This is consistent with the previously hypothesized depolymerization mechanism. That is, at a suitable pH, most of the amino groups of the compound exist in a deprotonated form in solution, thereby promoting or even accelerating depolymerization.

[0217] Furthermore, using a similar method, the depolymerization rate of compounds with different amino pKa values ​​on aggregated platelets was detected at pH 9.5, and the results showed ( Figure 5 Among them, some compounds with pKa values ​​between 8 and 11 were selected: N-(tris(hydroxymethyl)methylglycine) (8.1), arginine (9.0), glutamic acid (9.6), glycine (9.6), p-aminobenzenesulfonic acid (10.1), and lysine (10.7). As the amino pKa increased from 8.1 to 10.7, the depolymerization effect of each compound gradually decreased.

[0218] This example demonstrates that adjusting the pH value above the amino group's pKa value helps improve the depolymerization effect. Therefore, by adjusting the ambient pH value, the depolymerization ability of amino-containing compounds on aggregated platelets can be enhanced. Conversely, suitable amino-containing compounds can be selected based on the needs of the detection environment.

[0219] Example 7. Effect of amino-containing compound structures on the deaggregation of aggregated platelets.

[0220] To investigate the effect of compound structure on the depolymerization of aggregated platelets, compounds with representative structures were selected and tested according to the method in Example 1 at pH = 9.5. The depolymerization rates of each compound are shown in Table 6 below.

[0221] Table 6. Effects of different compounds on the disaggregation of aggregated platelets

[0222]

[0223]

[0224] As clearly shown in Table 6 above, the interaction between amino groups and cell membranes is influenced by the structure of the amino-containing substance itself. For example, diglycine has the same pKa as glycine but contains an additional secondary amino group, yet its depolymerization efficiency is still slightly lower than that of glycine. This can be attributed to the presence of an additional carbonyl group in the molecule. Furthermore, both glutamic acid and glycine have only one primary amino group with a pKa of 9.6, but glutamic acid has an additional carboxyl group, resulting in a lower depolymerization rate.

[0225] As can be seen from Table 6, carbonyl and carboxyl groups do not seem to help improve the platelet depolymerization effect of the compound, while alkyl chains have little effect on the depolymerization effect.

[0226] Furthermore, the greater the number of amino / imine groups, especially primary amino groups, the better the depolymerization effect. Arginine has a relatively large number of amino / imine groups, but its depolymerization effect is not very good, possibly because it can form cyclic lactams, which weakens the role of the amino groups.

[0227] Furthermore, hydroxyl groups appear to enhance the depolymerization effect of amino groups in compounds. For example, tris(hydroxymethyl)acetamide contains only one secondary amino group, yet its depolymerization rate is still higher than that of glycine. Tris(hydroxymethyl)acetamide, which contains a primary amino group, exhibits a better depolymerization effect on platelets. It is speculated that hydroxyl groups facilitate the compound's proximity to the platelet cell membrane, while primary amino groups are more effective at depolymerizing platelets than secondary amino groups. Of course, the higher depolymerization rate is also related to the lower pKa (8.1) of both compounds.

[0228] Furthermore, tertiary amines have almost no depolymerization effect. As can be seen from the above mechanism, amines lack hydrogen atoms capable of forming hydrogen bonds, thus hindering their function.

[0229] Example 8. The depolymerization effect of amino-containing compounds on different platelet aggregation models

[0230] Platelet aggregation can be induced by various substances, leading to blood coagulation. To investigate the deaggregation effect of amino-containing compounds on platelet aggregation induced by different factors, this embodiment, based on existing platelet aggregation pathways, selected substances that induce platelet aggregation in blood coagulation detection protocols and established multiple platelet aggregation models, including adenosine diphosphate (ADP), thrombin (THR), collagen (COL), and riscitin (RIS). This embodiment found that ADP, THR, COL, and RIS induce platelet aggregation to varying degrees with changes in concentration and time. Simultaneously, amino-containing compounds can effectively deaggregate platelets of different causes and aggregation degrees within a certain concentration and time range.

[0231] See Figure 6, which shows the relationship between the degree of platelet aggregation induced by different ADP concentrations (0.01 to 1 mM) and time. Specifically, 1 mL of venous blood was taken respectively, and ADP was added to make the final concentrations 0.01 mM, 0.25 mM, 0.5 mM and 1 mM respectively. Blood containing aggregated platelets was detected with a hematology analyzer. The hematology analyzer obtains the characteristics of various cell particles in the blood through the impedance channel and the optical channel, including quantity, size, etc. After normal blood is collected into a blood anticoagulant tube, the number of platelet particles detected by the impedance channel obtained by the hematology analyzer is recorded as (PLT_I). When platelet aggregation occurs, the volume of aggregated platelets is much larger than the volume of a single platelet, so it cannot be counted correctly by the recognition mechanism corresponding to platelets, resulting in a decrease in platelet count, which is recorded as PLT_I (after aggregation). Since such amino group-containing compounds are not added to the diluent of the impedance channel, the impedance channel reflects the degree of platelet aggregation in the aggregation model. The count before platelet aggregation is PLT_I (non-aggregated), the count after platelet aggregation is PLT_I (aggregated), and the aggregation rate = PLT_I (aggregated) / PLT_I (non-aggregated). The number of platelet particles detected by the optical channel obtained by the hematology analyzer is recorded as (PLT_O). When platelet aggregation occurs, the volume of aggregated platelets is much larger than the volume of a single platelet, so it cannot be counted correctly by the recognition mechanism corresponding to platelets, resulting in a decrease in PLT_O (non-dissociated). The amino group-containing compounds are used in the diluent of the optical channel to achieve dissociation of aggregated platelets, and PLT_O (after dissociation) after dissociation is obtained. The count before platelet aggregation is PLT_O (non-aggregated), and the count after platelet aggregation is PLT_O (non-dissociated). Similarly, PLT_O (non-dissociated) < PLT_O (dissociated) < PLT_O (non-aggregated), and the dissociation rate = PLT_O (dissociated) / PLT_O (non-aggregated). Based on the above principles, a model was established to quantitatively detect the dissociation effect of amino group-containing compounds.

[0232] It can be seen from Figure 6 that after adding ADP, platelet aggregation basically reaches the maximum within 5 minutes. Moreover, the larger the added amount of ADP, the higher the degree of platelet aggregation. When the final concentration of ADP is above 0.25 mM, the effect on the degree of platelet aggregation is generally the same. In addition, ADP at a concentration of 0.01 mM has the weakest effect on the degree of platelet aggregation, which can aggregate up to about 35% of platelets, but platelet dissociation at this concentration is also relatively slow.

[0233] With further reference to Figure 7The diagram shows the effect of amino-containing compounds on platelet depolymerization over time in blood samples induced with different ADP concentrations. Specifically, blood samples with different concentrations of aggregation inducers were treated with an amino-containing compound (1-acetylguanidine) in a diluent, following a method similar to that in Example 3. Measurements were taken 5 minutes after each sample was added to the treatment solution to obtain the depolymerization rate over time. The diluent consisted of: citric acid (0.5 g / L), surfactant phenoxyethanol (0.1 g / L), antibacterial agent (6 g / L), sodium chloride (3 g / L), EDTA (0.1 g / L), and 1-acetylguanidine (10 mmol / L), with a pH of 9.5 and an osmotic pressure of 200 mOsm / L.

[0234] from Figure 7 It can be seen that the same compound at the same concentration has a significant depolymerization effect on different platelet aggregation levels. The depolymerization effect reaches its peak immediately after the addition of the amino-containing compound, decreases over time, and then stabilizes after approximately 10 minutes. Furthermore, the lower the ADP concentration, the more stable the effect. Considering that the blood cell analyzer only requires a small amount of sample to quickly complete the test, the test can begin within 5 minutes, preferably within 2 minutes, and more preferably within 30 seconds after adding the treatment solution containing the amino-containing compound.

[0235] Therefore, by increasing the concentration of ADP, the degree of aggregation can be increased, thereby increasing the difficulty of deaggregation.

[0236] Similarly, Figure 8 and Figure 9 This is a graph showing the platelet aggregation induction test using different concentrations of THR. The method used is essentially the same as described above, except that ADP is replaced with THR. The THR concentrations were 0.1U, 0.15U, 0.2U, 0.23U, 0.25U, 0.28U, 0.30U, and 0.35U. It can be seen that the platelet aggregation effect is weak at low THR concentrations, while at high concentrations, the platelet aggregation rate increases rapidly after 1 minute. This is because after adding a certain concentration of THR, soluble fibrinogen is converted into insoluble fibrinogen, causing blood coagulation. Platelets are also encapsulated by the insoluble fibrinogen, leading to a sharp increase in the aggregation rate. Simultaneously, the depolymerization effect of 1-acetylguanidine reaches its peak immediately at all THR concentrations, but begins to decrease significantly after 1 minute. This is also related to the fact that the depolymerizing agent cannot exert its effect at this point. However, better depolymerization rates can be obtained in samples tested immediately after treatment.

[0237] Figure 10 and Figure 11This is a graph showing the platelet aggregation induction test using different concentrations of COL. The method is essentially the same as described above, except that ADP is replaced with COL. The COL concentrations are 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, and 20 mM. It can be seen that COL can rapidly induce platelet aggregation to nearly 100% at different concentrations, with lower concentrations resulting in faster aggregation. Meanwhile, the deaggregating effect of 1-acetylguanidine reaches its peak immediately at all COL concentrations except for the sample with the lowest concentration, but begins to decrease significantly after approximately 4–8 minutes. This is also because the addition of COL converts soluble fibrinogen into insoluble fibrinogen, causing blood clotting.

[0238] Figure 12 and Figure 13 This is a graph showing the platelet aggregation induction test using different concentrations of RIS. The method is essentially the same as described above, except that ADP is replaced with RIS. The RIS concentrations were 0.15 mM, 0.375 mM, 0.75 mM, 1.5 mM, and 3 mM. It can be seen that the RIS-induced platelet aggregation effect takes a relatively long time to materialize, reaching its peak after 15–20 minutes. Simultaneously, the depolymerization effect of 1-acetylguanidine reaches its peak immediately at all RIS concentrations and remains relatively stable within 10 minutes. The rapid decrease in the depolymerization rate after 15 minutes is also due to blood coagulation in the sample.

[0239] The above models of platelet aggregation induction show that aggregation inducers have different effects on the degree of platelet aggregation under different pathways. However, amino-containing compounds can immediately affect the aggregation induced by each inducer. But because soluble fibrinogen is converted to insoluble fibrinogen, causing coagulation, the disaggregation effect after coagulation is not reflected in samples with THR and COL-induced platelet aggregation. This has almost no impact on platelet classification and counting by hematology analyzers.

[0240] This indicates that the amino-containing compounds can produce good deagglomeration effects on various platelet aggregation patterns within a certain concentration and time range, and can take effect rapidly, making them easy to detect immediately without the need for additional reaction conditions (such as heating or a longer reaction time).

[0241] Example 9. Effect of combined use of amino-containing compounds and ammonium-containing compounds on platelet disaggregation.

[0242] Based on the fact that amino-containing compounds and ammonium-containing compounds may depolymerize aggregated platelets through different depolymerization mechanisms, this example determined the effect of a combination of ammonium-containing and amino-containing compounds on platelet depolymerization. Similar to Example 1, the method was followed, except that the ammonium-containing compounds were replaced with ammonium chloride, 1-acetylguanidine, a combination of ammonium chloride and 1-acetylguanidine at concentrations of 1, 2, 5, 10, 20, and 50 mmol / L, respectively, as well as NaCl as a control. In the ammonium chloride and 1-acetylguanidine combination, the concentrations of ammonium chloride and 1-acetylguanidine were equal, and the total concentration met the above requirements. Furthermore, to highlight the effect of the combination, in this example, 0.1 mmol / L ADP (i.e., 10 times the ADP concentration in Example 1) was used to induce platelet aggregation, increasing the depolymerization difficulty to study the ability of amino-containing and ammonium-containing compounds to depolymerize platelets. The measured depolymerization rates are as follows: Figure 14 As shown.

[0243] The results show ( Figure 14 In ADP-induced platelet aggregation at a final concentration of 0.1 mmol / L, the depolymerization effects of NH4Cl and 1-acetylguanidine gradually increased with increasing concentration. For example, 50 mmol / L NH4Cl alone had a depolymerization effect of 66.27%, and 50 mmol / L 1-acetylguanidine alone had a depolymerization effect of 62.14%. The combined effect of NH4Cl and 1-acetylguanidine at a total concentration of 50 mmol / L achieved a depolymerization effect of 95.48%, further enhancing platelet depolymerization. This enhancing effect was also observed at other concentrations, suggesting a synergistic effect between the two in platelet depolymerization. Therefore, when using them in combination, it is preferable to use them at lower concentrations (e.g., as low as 1–20 mM, 1–10 mM, or even 1–5 mM, respectively).

[0244] The following experiments further demonstrate the application of the reagents of the present invention, including those containing amino groups, in resisting platelet disaggregation interference in actual detection.

[0245] Test Example 1. Ammonium ion-containing compounds are used in staining solutions for the detection of platelets and red blood cells.

[0246] This test case provides a staining solution for a hematology analyzer, which can be used to exclude platelet aggregation from interfering with the detection of red blood cells and platelets. This test case, as well as the following test cases, were performed using a hematology analyzer (BC-6000Plus) from Mindray Bio-Medical Electronics Co., Ltd., Shenzhen. The following staining solution formulation was used:

[0247] Control staining solution:

[0248]

[0249] Dyeing solution A:

[0250] Based on the control staining solution, ammonium chloride was added to bring the final concentration to 0.01 mol / L.

[0251] The control staining solution and staining solution A were prepared with distilled water at 25℃, and the pH was adjusted to 9 and the osmotic pressure was 200 mOsm / L.

[0252] Take 1 ml of human venous blood sample collected in an anticoagulant tube, divide the sample into two groups, and perform analysis using the aforementioned blood cell analyzer. A control staining solution and staining solution A are used separately, mixed at a ratio of 4 μl sample to 1 ml staining solution. Add staining agent (SYBR Green, 50 mg / L) and surfactant (N-alkyl betaine, 0.01 g / L) to stain nucleated cells and spherize blood cells. The temperature is maintained at approximately 42°C. The fluorescence intensity (FL) of the treated blood sample cells is measured using lateral fluorescence at a 90° angle, the lateral scattered light intensity (SS) is measured using lateral scattered light at a 90° angle, and the forward scattered light intensity (FS) is measured using forward scattered light at an angle of 2°–5° to obtain a three-dimensional scatter plot. Take another 1 ml of the above blood sample, add platelet aggregation inducer ADP (final concentration 0.01 mmol / L), and mix well. Platelet aggregation occurs after 5 minutes. The samples were then divided into two groups and analyzed using a hematology analyzer. One group was treated with a control staining solution, and the other with staining solution A. Three-dimensional scatter plots were obtained in the same manner. As shown above, the four scatter plots obtained from this sample analysis are as follows: Figure 15 As shown.

[0253] Depend on Figure 15 It is evident that after treating blood samples induced by platelet aggregation with staining solution A, which eliminates interference from platelet aggregation, the aggregated platelets are deaggregated, and the detection of mature erythrocytes, reticulocytes, and platelets is achieved.

[0254] As shown in the figure, when staining with staining solution A, the fluorescence intensity (FL) and forward scattering intensity (FS) of platelets decreased in the 3D scatter plot, and the 3D scatter plot information of the deaggregated platelets approached that of the non-aggregated platelet sample. In the scatter plot using the control staining solution, the volume of aggregated platelets was larger than that of a single platelet, and the fluorescence intensity of the aggregated platelets was also enhanced. The proportion of reticulocytes was calculated from the 3D scatter plot by calculating the percentage of particles in the reticulocyte distribution area relative to the number of particles in the mature red blood cell and reticulocyte areas. Reticulocytes exhibited stronger fluorescence signals than normal red blood cells. The total red blood cell count measured in the non-aggregated sample was 4.46 × 10⁻⁶. 12The proportion of reticulocytes in the total red blood cells was 1.28%, and the total red blood cell count after depolymerization was 4.43 × 10⁻⁸ in the sample treated with staining agent A. 12 The total red blood cell count was not affected by aggregated platelets. The platelet count in the non-aggregated sample was 274 × 10⁻⁶. 9 The count in the non-aggregated sample was 239 × 10⁻⁶. 9 The count in the depolymerized sample was 42 × 10⁻⁶. 9 .

[0255] Meanwhile, by counting platelets, it was found that the platelet count in the sample induced by staining solution A increased, and the disaggregation rate reached 87.16%, while the disaggregation rate of the sample using the control staining solution was calculated to be 15.36%.

[0256] Test Example 2. Ammonium ion-containing compounds were used in diluents for the detection of platelets and red blood cells.

[0257] This test example provides a diluent for a hematology analyzer that can be used to exclude platelet aggregation from interfering with the detection of red blood cells and platelets. The following diluent formulation is used:

[0258] Control diluent:

[0259]

[0260] Diluent A:

[0261] Based on the control diluent, ammonium chloride was added to bring the final concentration to 0.01 mol / L.

[0262] Control diluent and diluent A were prepared with distilled water at 25℃, and the pH was adjusted to 9.5 and the osmotic pressure was 200 mOsm / L.

[0263] The test was performed using a method similar to that in Test Example 1, except that the samples were treated with the control diluent and diluent A mentioned above, respectively. Furthermore, staining agent (SYBR Green, 50 mg / L) and surfactant (N-alkyl betaine, 0.01 g / L) were added to stain nucleated cells and to spherify blood cells. The resulting four scatter plots are shown below. Figure 16 As shown.

[0264] Depend on Figure 16 It is evident that after treating blood samples induced by platelet aggregation with diluent A, which eliminates interference from platelet aggregation, the aggregated platelets are deaggregated, and the detection of mature erythrocytes, reticulocytes, and platelets is achieved.

[0265] The total red blood cell count of the non-aggregated sample was 5.72 × 10⁻⁶. 12The proportion of reticulocytes in the total red blood cells was 1.02%, and the total red blood cell count after depolymerization was 5.69 × 10⁻⁶ in the sample using diluent A. 12 The total red blood cell count was not affected by aggregated platelets. The platelet count in the non-aggregated sample was 198 × 10⁻⁶. 9 The count in the aggregated sample was 42 × 10⁻⁶. 9 The count in the depolymerized sample was 190 × 10⁻⁶. 9 .

[0266] Meanwhile, by counting platelets, it was found that the platelet count in the sample induced by staining solution A increased, and the deagglomeration rate reached 95.69%, while the deagglomeration rate of the sample using the control staining solution was calculated to be 20.79%.

[0267] Test Example 3. Impedance detection of platelets using a compound containing ammonium ions in a dilution solution.

[0268] This test case is basically the same as Test Case 2, except that the hematology analyzer used is the impedance channel of the Shenzhen Mindray Bio-Medical Electronics Co., Ltd. The obtained histogram is shown below. Figure 17 As shown.

[0269] like Figure 17 As shown, the horizontal axis of the platelet histogram represents the platelet volume (in fL), and the vertical axis represents the number of platelets. When platelets aggregate, the volume of platelets in the impedance channel increases. Under the action of the deaggregating diluent, the aggregated platelets deaggregate, and the volume of platelets in the impedance channel decreases.

[0270] Following the same formula in Test Example 1, the calculation showed that diluent A containing NH4Cl increased the number of aggregated platelets, with a deagglomeration rate of 93.17%, while the control diluent without NH4Cl had a deagglomeration rate of 18.03%.

[0271] Test Example 4. Ammonium ion-containing compounds are used in lysis buffer for the detection of leukocytes.

[0272] This test example provides a lysis buffer for use in a hematology analyzer, which can be used to exclude platelet aggregation from interfering with the detection of red blood cells and platelets. Prepare the lysis buffer according to the following formulation:

[0273] Control lysis buffer:

[0274]

[0275] Lysis solution A:

[0276] Based on the control lysis buffer, ammonium chloride was added to bring the final concentration to 0.01 mol / L.

[0277] Control lysis buffer and lysis buffer A were prepared with distilled water at 25℃, and the pH was adjusted to 9 and the osmotic pressure was 90 mOsm / L.

[0278] Following a procedure similar to Test Example 1, 1 ml of human venous blood sample collected in an anticoagulant tube was taken and divided into two groups. The samples were then analyzed using the aforementioned blood cell analyzer, with control lysis buffer and lysis buffer A used separately. A 20 μl sample was added to 1 ml of lysis buffer, followed by 20 μl of DNA staining agent (Hoechst 33342). The temperature was maintained at approximately 42°C. The fluorescence intensity (FL) of the treated blood sample cells was measured using lateral fluorescence at a 90° angle, the lateral scattered light intensity (SS) was measured using lateral scattered light at a 90° angle, and the forward scattered light intensity (FS) was measured using forward scattered light at angles of 2°–5° to obtain a three-dimensional scatter plot. Another 1 ml of the above blood sample was taken, and platelet aggregation inducer ADP (final concentration 0.01 mmol / L) was added and mixed thoroughly. Platelet aggregation occurred after 5 minutes. The samples were then divided into two groups and analyzed using a hematology analyzer. One group was treated with control lysis buffer and the other with lysis buffer A, and DNA dye was added. Three-dimensional scatter plots were obtained in the same manner. As shown above, the four scatter plots obtained from this sample analysis are as follows: Figure 18 As shown.

[0279] In the optical channel, multiple aggregated platelets are identified as a single particle. Some aggregated platelets reach the volume of a white blood cell, affecting the white blood cell count. In this test case, the white blood cell count in the non-aggregated sample was measured to be 7.49 × 10⁻⁶. 9 Of these, 4.74 × 10⁻⁶ neutrophils were present. 9 Lymphocytes 2.16 × 10 9 0.42 × 10⁻⁶ monocytes 9 Eosinophils 0.14 × 10 9 The white blood cell count after depolymerization was 8.72 × 10⁻⁶ in the sample using the control lysis buffer. 9 The number of neutrophils counted was 5.16 × 10⁻⁶. 9 Lymphocytes 2.57 × 10 9 0.61 × 10⁶ mononuclear cells 9 Eosinophils 0.32 × 10 9 The white blood cell count after depolymerization of the sample treated with lysis agent A was 7.58 × 10⁻⁶. 9 Of these, 4.79 × 10⁻⁶ neutrophils were present. 9 Lymphocytes 2.21 × 10 9 0.39 × 10⁻⁶ monocytes9 Eosinophils 0.15 × 10 9 It is evident that white blood cell count and differential are not affected by platelet aggregation after using lysis agent A, resulting in more accurate counts.

[0280] Test Example 5. Combination of amino-containing compounds and ammonium ion-containing compounds for leukocyte detection.

[0281] This test example was conducted according to the method of Test Example 4, except that lysis buffer C was prepared instead of lysis buffer A. That is, based on the control lysis buffer, an equimolar amount of 1-acetylguanidine + NH4Cl was added to make the final concentration 0.01 mol / L.

[0282] Control lysis buffer and lysis buffer C were prepared with distilled water at 25℃, and the pH was adjusted to 9 and the osmotic pressure was 90 mOsm / L.

[0283] The test was conducted following similar steps to Test Example 5, and the same 3D scatter plot was obtained. Figure 19 As shown.

[0284] In this test case, the white blood cell count of the non-aggregated sample was 6.19 × 10⁻⁶. 9 The number of neutrophils in the differential count was 3.87 × 10⁻⁶. 9 Lymphocytes 1.91 × 10 9 0.22 × 10⁻⁶ monocytes 9 Eosinophils: 0.08 × 10⁸ 9 The white blood cell count after depolymerization was measured using lysis buffer C to be 6.13 × 10⁻⁶. 9 The number of neutrophils in the differential count was 3.91 × 10⁻⁶. 9 Lymphocytes 1.88 × 10 9 0.21 × 10⁻⁶ monocytes 9 Eosinophils: 0.07 × 10⁻⁶ 9 The white blood cell count after depolymerization was 6.97 × 10⁻⁶ in the sample using the control lysis buffer. 9 The number of neutrophils counted was 4.21 × 10⁻⁶. 9 Lymphocytes 2.21 × 10 9 0.34 × 10⁻⁶ monocytes 9 Eosinophils 0.17 × 10 9 It is evident that white blood cell count and differential are not affected by platelet aggregation after using lysis agent C, resulting in more accurate counts.

[0285] Test Example 6. Staining solutions containing combinations of amino-containing compounds and ammonium-containing compounds are used for the detection of platelets and red blood cells.

[0286] This test case was conducted in essentially the same manner as Test Case 1, except that the control staining solution was prepared according to the following composition, and staining solution C was used instead of staining solution B.

[0287] Control staining solution:

[0288]

[0289] Staining solution C:

[0290] Based on the control staining solution, 1-acetylguanidine was added to bring the final concentration to 0.01 mol / L, and ammonium chloride was added to bring the final concentration to 0.01 mol / L.

[0291] It was also prepared with distilled water at 25℃, the pH was adjusted to 9, and the osmotic pressure was 200 mOsm / L.

[0292] The test was conducted using a method similar to that in Test Example 1, except that platelet aggregation in the sample was induced with ADP at a final concentration of 1 mol / L (100 times that in Test Example 1). A three-dimensional scatter plot was obtained, as shown below. Figure 20 As shown.

[0293] The total red blood cell count in the non-aggregated sample was measured to be 6.14 × 10⁻⁶. 12 The proportion of reticulocytes in the total red blood cells was 1.34%, and the total red blood cell count in the sample after platelet aggregation and disaggregation was 6.11 × 10⁻⁶. 12 The total red blood cell count was not affected by aggregated platelets. The platelet count in the non-aggregated sample was 219 × 10⁻⁶. 9 The count in the aggregated sample was 17 × 10⁻⁶. 9 The count in the depolymerized sample was 182 × 10⁻⁶. 9 .

[0294] Meanwhile, by counting platelets, it was found that the platelet count in the sample induced by dilution solution B increased, and the deagglomeration rate reached 82.73%, while the deagglomeration rate measured by the control staining solution was only 7.61%.

[0295] Test Example 7. A diluent containing a combination of amino-containing compounds and ammonium-containing compounds is used for the detection of platelets and red blood cells.

[0296] This test was conducted in essentially the same manner as Test Example 2, except that diluent C was used instead of diluent B. Diluent C was prepared by adding 1-acetylguanidine to the control diluent to achieve a final concentration of 0.01 mol / L, and adding ammonium chloride to achieve a final concentration of 0.01 mol / L. The pH of each diluent was adjusted to 9.5, and the osmotic pressure was adjusted to 200 mOsm / L.

[0297] The test was conducted using a method similar to that in Test Example 1, except that platelet aggregation in the sample was induced with ADP at a final concentration of 1 mol / L (100 times that in Test Example 1). A three-dimensional scatter plot was obtained, as shown below. Figure 21 As shown.

[0298] The total red blood cell count in the non-aggregated sample was measured to be 5.12 × 10⁻⁶. 12 The proportion of reticulocytes in the total red blood cells was 1.21%, and the total red blood cell count in the sample after platelet aggregation and disaggregation was 5.08 × 10⁻⁶. 12 The total red blood cell count was not affected by aggregated platelets. The platelet count in the non-aggregated sample was 278 × 10⁻⁶. 9 The count in the aggregated sample was 18 × 10⁻⁶. 9 The count in the depolymerized sample was 248 × 10⁻⁶. 9 .

[0299] Meanwhile, by counting platelets, it was found that the platelet count in the sample induced by diluent B increased, and the deagglomeration rate reached 89.20%, while the deagglomeration rate measured by the control diluent was 6.61%.

[0300] Test Example 8: The disaggregating effect of compounds containing ammonium ions on pseudoplatelet aggregation in clinical practice.

[0301] This test case demonstrates the disaggregation of pseudoaggregated platelets in a clinical setting. The same diluent as Diluent A in Test Case 2 was used for detection. This diluent consisted of: citric acid (0.5 g / L), surfactant phenoxyethanol (0.1 g / L), antibacterial agent (6 g / L), sodium chloride (3 g / L), and NH4Cl (0.01 mol / L). The pH of the diluent was adjusted to 9.5, and the osmotic pressure to 200 mOsm / L.

[0302] The specific steps are as follows: Blood was collected from outpatients and inpatients, anticoagulated with EDTA-K2, and 17 specimens meeting the EDTA-PTCP diagnostic criteria were included in the experimental group. These specimens showed a significant decrease in platelet count (PLT-I), platelet aggregation was observed on blood smear microscopy, and the patients were 9 males and 8 females with an average age of 51 years. Blood was drawn from patients with pseudo-platelet aggregation. Immediately after blood collection without anticoagulant, the platelets were analyzed on a hematology analyzer within 1 minute (when platelet aggregation had not yet occurred). The platelet count value obtained using this method is the true platelet count (PLT_O).

[0303] Blood was drawn from the patient using EDTA anticoagulant tubes. Two hours later (in patients with pseudo-aggregation, platelets will aggregate), the platelets were analyzed on the same hematology analyzer using a diluent containing the deaggregating agent 1-acetylguanidine. The platelet count obtained using this method is the platelet deaggregation value (PLT_O). Simultaneously, blood was drawn from the patient using EDTA anticoagulant tubes. Two hours later (in patients with pseudo-aggregation, platelets will aggregate), the platelets were analyzed on the same hematology analyzer using a diluent without the deaggregating agent NH4Cl. The platelet count obtained using this method is the platelet non-deaggregation value (PLT_O). The deaggregation effect was calculated using the following formula:

[0304] Depolymerization rate (depolymerization) = PLT_O (depolymerization) / PLT_O (truth value); or

[0305] Depolymerization rate (control) = PLT_O (not depolymerized) / PLT_O (true value).

[0306] The test results for each sample are shown in Figure 22 As shown in the figure, all pseudoaggregated platelets underwent varying degrees of disaggregation. In 13 out of 20 samples, the disaggregation rate remained at 70% after 2 hours of blood storage. In the control group, the disaggregation rate was below 20%, with the disaggregation diluent containing 10 mM ammonium chloride showing a significant effect.

Claims

1. Use of a composition in the preparation of a reagent for detecting platelet count in an in vitro blood analyzer, wherein, The composition comprises at least one ammonium ion-containing compound, and the composition further comprises at least one amino-containing compound, wherein the amino-containing compound contains at least one primary or secondary amino group. The ammonium-containing compound is selected from ammonium chloride, ammonium bromide, and ammonium iodide. The amino-containing compounds are selected from the group consisting of compounds of formula (I) and their salts: R1-NH-R2 (I) Wherein, R1 and R2 are different, each being an independent group selected from the group consisting of -C(=NH)-NH2 and -C(O)-Q1. Wherein, Q1 is a C1-6 alkyl group. The platelet count was measured in a solution with a pH of 7.5–11. The amino-containing compound has an amino group with a pKa in the range of 4 to 14. The amino-containing compound has an amino group with a pKa value lower than the pH value of the solution used to detect platelet count.

2. The use according to claim 1, wherein, The platelet count was measured in a solution with a pH of 9.5–11.

3. The use according to claim 1, wherein, In the solution used for detecting platelet count, the concentration of the ammonium ion-containing compound in the composition is 1-50 mmol / L.

4. The use according to claim 3, wherein, The concentration of the ammonium ion-containing compound in the composition is 2-20 mmol / L.

5. The use according to claim 1, wherein, In the solution used to detect platelet count, the concentration of the amino-containing compound is 1–50 mmol / L.

6. The use according to claim 5, wherein, In the solution used to detect platelet count, the concentration of the amino-containing compound is 2-20 mmol / L.

7. The use according to any one of claims 1 to 6, wherein, Blood samples used to test platelet counts are taken from the peripheral or venous blood of mammals.

8. A reagent for detecting platelet count in an in vitro blood analyzer, wherein, The reagent comprises at least one ammonium ion-containing compound at a concentration of 1-50 mmol / L, and further comprises at least one amino-containing compound at a concentration of 1-50 mmol / L, wherein the amino-containing compound contains at least one primary or secondary amino group. The ammonium-containing compound is selected from ammonium chloride, ammonium bromide, and ammonium iodide. The amino-containing compounds are selected from the group consisting of compounds of formula (I) and their salts: R1-NH-R2 (I) Wherein, R1 and R2 are different, each being an independent group selected from the group consisting of -C(=NH)-NH2 and -C(O)-Q1. Wherein, Q1 is a C1-6 alkyl group. The pH value of the reagent is 7.5~11. The amino-containing compound has an amino group with a pKa in the range of 4 to 14. The amino-containing compound has an amino group whose pKa value is lower than the pH value of the reagent.

9. The reagent according to claim 8, wherein, The reagent includes at least one compound containing ammonium ions at a concentration of 5 to 20 mmol / L.

10. The reagent according to claim 8, wherein, The pH value of the reagent is 9.5~11.

11. The reagent according to claim 8, wherein, The concentration of the amino-containing compound is 5-20 mmol / L.

12. The reagent according to any one of claims 8 to 11, wherein, The reagent further includes a buffer, an osmotic pressure regulator, and optionally at least one selected from surfactants, fluorescent dyes, and erythrocyte lysis agents.

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