Device for assessing the risk of gout attacks based on the growth rate of monosodium urate crystals

Abstract

The present invention provides a device for assessing the risk of gout attack in a subject based on the growth rate of monosodium urate crystals. In the device constructed by the present invention, there are significant differences in the growth rate of monosodium urate crystals under the induction of blood samples in different health states, so that the risk of gout attack in a subject can be assessed by the growth rate of monosodium urate crystals.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a device for evaluating the risk of gout attack based on the growth rate of monosodium urate crystals. Background Art

[0002] Acute gout is an acute arthritis that causes severe pain in the joints. Its main pathological feature is the deposition of monosodium urate crystals. These crystals are formed when serum urate concentrations are elevated and chronically deposited in the joints and tissues. The deposited crystals can directly induce joint inflammation, with clinical manifestations including sudden pain, edema, redness, swelling, and limited joint movement.

[0003] Gout is mainly divided into four stages, namely, hyperuricemia stage, acute gout stage, gout attack interval stage and chronic gout stage. Among these stages, the acute gout stage has the greatest impact on the patient's daily life. The severe pain makes it impossible for people to work or sleep. Long-term attacks can lead to structural joint damage. How to conduct risk assessment for acute gout attacks and assess individual differences in susceptibility to attacks is an important topic.

[0004] Significant progress has been made in the study of gout's pathogenesis, effects, diagnostic methods and treatment, but the risk assessment of acute gout attacks is still in its early stages. Current methods for predicting acute gout mainly use biomarkers such as uric acid, CRP and other indicators; electronic health records such as medical history and treatment patterns; imaging examinations, etc.

[0005] The above methods are all based on the results obtained from biological reactions and clinical statistical analysis, which are easily affected by individual differences. Therefore, there is an urgent need for a method or device that can assess the risk of acute gout attacks for different individuals. Summary of the invention

[0006] In order to improve the above technical problems, the present invention provides a device for assessing the risk of gout attack in a subject based on the growth rate of monosodium urate crystals. In the device constructed by the present invention, there are significant differences in the growth rate of monosodium urate crystals under the induction of blood samples in different health states, so that the risk of gout attack can be assessed by the growth rate of monosodium urate crystals.

[0007] The present invention provides a device for assessing the risk of stroke in a subject with pain based on the growth rate of monosodium urate crystals, the device comprising the following computer modules: S1) a data reading module, S2) a data processing module and S3) a data output module;

[0008] in:

[0009] S1) a data reading module reads the changes in absorbance values ​​(Y) of the sample after the serum sample of the subject is mixed with the alkaline uric acid solution at different time points (X);

[0010] S2) The data processing module performs the following operations:

[0011] S2-1) making a change curve according to the data received by the data reading module in S1); obtaining the maximum absorbance value (Top value), the minimum absorbance value (Bottom value) and the symmetry value S of the curve according to the curve;

[0012] S2-2) Obtain the Hill Slope value (the maximum slope value of the curve) and the corresponding time point EC at which the Hill Slope appears by means of the parameter fitting equation 50 value;

[0013] S2-3) The calculation results (HillSlope value and / or EC) of S2-2) are used. 50 Value) to judge the risk of gout attack;

[0014] S3) The data output module outputs the risk of gout attack based on the judgment result of S2-3).

[0015] According to an embodiment of the present invention, in the S1) data reading module, the blood sample of the subject is a venous blood sample.

[0016] According to an embodiment of the present invention, in S1) the data reading module, the blood sample of the subject is a pre-treated blood sample; preferably, the pre-treatment step specifically comprises: subjecting the blood sample to a procoagulant treatment, centrifuging, and taking serum;

[0017] Preferably, the centrifugation conditions are 1500r-2500r, 5min-10min; for example, 2000r, 8min.

[0018] According to an embodiment of the present invention, S1) in the data reading module, the alkaline uric acid solution is a sodium hydroxide solution of uric acid;

[0019] Preferably, the concentration of the sodium hydroxide solution is 0.5 mol / L-1.0 mol / L, for example 0.6 mol / L, 0.667 mol / L, 0.7 mol / L, 0.8 mol / L.

[0020] According to an embodiment of the present invention, in S1) the data reading module, the concentration of uric acid in the alkaline uric acid solution is 30 mg / mL-40 mg / mL, for example, 32 mg / mL, 33.3 mg / mL, 35 mg / mL, 38 mg / mL, 40 mg / mL.

[0021] According to an embodiment of the present invention, S1) in the data reading module, the volume ratio of the serum sample to the alkaline uric acid solution is 5-20:1, preferably 8-15:1, such as 9:1, 10:1, 11:1, 12:1, 13:1, 14:1;

[0022] Preferably, the serum sample is mixed with the alkaline uric acid solution in a cell culture plate (eg, a 384-well plate).

[0023] According to an embodiment of the present invention, S1) in the data reading module, the change of absorbance value (Y) at different time points (X) is read by an absorbance monitor (e.g., a multifunctional cell imaging microplate detector);

[0024] Preferably, the detection wavelength is 520nm-540nm, preferably 525nm-535nm, for example 530nm;

[0025] Preferably, the detection temperature is 28°C-32°C, for example 30°C;

[0026] Preferably, the absorbance value is detected once every 1 min-10 min; for example, the absorbance value is detected once every 5 min;

[0027] Preferably, the detection is continued for 1 h-5 h, such as 2 h, 3 h, 4 h.

[0028] According to an embodiment of the present invention, an absorbance monitor is used to monitor the overall absorbance change during serum-induced crystal growth, thereby obtaining the crystal growth amount at different time points. The absorbance value is positively correlated with the crystal growth amount, thereby quantitatively measuring the crystal growth amount at different time points.

[0029] According to the embodiment of the present invention, in the S2) data processing module (S2-2)), the parameter fitting equation is a five-parameter fitting equation;

[0030] Preferably, the formula of the parameter fitting equation is:

[0031] Denominator=(1+(2^(1 / S)-1)*((EC 50 / X)^HillSlope))^S,

[0032] Numerator = Top–Bottom,

[0033] Y=Bottom+(Numerator / Denominator);

[0034] in:

[0035] Y is the absorbance value;

[0036] X is the time point corresponding to the absorbance value;

[0037] Top is the maximum absorbance value of the curve (i.e., MAX{Y});

[0038] Bottom is the minimum absorbance value of the curve (i.e., MIN{Y});

[0039] S is the symmetry of the curve, and its value is S∈[0,1] (close to 1, the fitting curve is approximately symmetrical, and close to 0, the curve is more asymmetrical);

[0040] HillSlope is the Hill slope (used to measure the maximum slope value of the curve, used to describe the maximum rate of crystal growth);

[0041] EC 50 , the corresponding time point when the Hill slope appears (used to describe the time point when the crystal reaches the maximum growth rate).

[0042] According to an embodiment of the present invention, in S2) the data processing module, the fitting method is asymmetricsigmoidal.

[0043] According to the embodiment of the present invention, using HillSlope, EC 50 Two indicators are used to measure the growth characteristics of monosodium urate crystals. When the HillSlope is larger, the fastest rate of crystal growth is faster; when EC 50 The smaller the time, the earlier the fastest crystal growth rate occurs, the faster the crystal enters the optimal growth point, and the faster the crystallization is completed.

[0044] According to an embodiment of the present invention, in S2) a data processing module,

[0045] If the HillSlope value is <5.10 OD / min (e.g., 0 < HillSlope value <5.10 OD / min, e.g., HillSlope value is 0.5 OD / min, 1.0 OD / min, 1.5 OD / min, 2.0 OD / min, 2.5 OD / min, 3.0 OD / min, 3.39 OD / min, 3.5 OD / min, 4.0 OD / min, 4.5 OD / min, 5.0 OD / min), the risk of gout attack is low;

[0046] If 5.1OD / min≤HillSlope value≤7.15OD / min (e.g., HillSlope value is 5.3OD / min, 5.5OD / min, 5.8OD / min, 6.0OD / min, 6.3OD / min, 6.5OD / min, 6.8OD / min, 7.0OD / min), the risk of gout attack is moderate;

[0047] If the Hill Slope value is >7.15 OD / min (for example, the Hill Slope value is 7.5 OD / min, 8.0 OD / min, 8.5 OD / min, 9.0 OD / min, 10.0 OD / min, 11.0 OD / min, 12.0 OD / min, 13.0 OD / min, 15.0 OD / min), the risk of gout attack is high.

[0048] According to an embodiment of the present invention, in step S2),

[0049] If EC 50 Value>80min (e.g. EC 50 The value is 85min, 90min, 95min, 98min, 100min, 110min, 120min), the risk of gout attack is low;

[0050] If 63min≤EC 50 Value ≤ 80min (e.g. EC 50 If the value is 65min, 68min, 70min, 73min, 75min, 78min, 80min), the risk of gout attack is medium;

[0051] If EC 50 Value < 63min (e.g. EC 50 The value is 60min, 50min, 40min, 30min, 20min, 10min, 5min), the risk of gout attack is high.

[0052] According to the embodiment of the present invention, if the HillSlope value, EC 50 If the prediction results of the two values ​​are inconsistent, the prediction result with higher risk shall prevail.

[0053] The present invention also provides a device combination for assessing the risk of gout attack in a subject based on the growth rate of monosodium urate crystals, wherein the device combination comprises the above-mentioned computer module and an absorbance monitor (eg, a multifunctional cell imaging microplate detector).

[0054] Beneficial effects of the present invention:

[0055] ① The present invention provides a device for evaluating the risk of gout attack in a subject based on the growth rate of monosodium urate crystals. The device constructs a monitoring scheme for crystal growth rate under body fluid induction, which can quickly and easily record the overall growth amount of monosodium urate crystals at each time point.

[0056] ② The equipment provided by the present invention can objectively describe the differences in the growth rates of monosodium urate crystals induced by the body fluids of patients in different health conditions without the need for complex instruments and monitoring methods.

[0057] ③ The equipment provided by the present invention can quantify the growth rate of crystals, for example, by comparing the growth rate difference of crystals induced by serum of healthy people and gout patients, exploring new potential factors for gout attacks; by comparing the growth rate difference of crystals induced by serum of high-risk and low-risk gout patients, developing assessment indicators and tools for the risk of gout attacks; by comparing the effect of gender differences on crystal growth rate, exploring the reasons for gender differences in gout attacks, etc. It can also be used to monitor the growth rate of other crystals, and develop new research ideas and methods for the occurrence and development of crystalline diseases (stones, pseudogout, cholesterol crystals in the blood vessel walls of patients with coronary atherosclerosis, etc.).

[0058] ④ The device provided by the present invention eliminates the error caused by the difference in blood uric acid values ​​between different specimens, and the device provided by the present invention is not interfered by serum background uric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a flow chart of the implementation of the risk assessment of gout attacks based on the growth rate of monosodium urate crystals of the present invention.

[0060] Figure 2 The invention provides a five-parameter fitting equation and a curve quantification schematic diagram thereof.

[0061] Figure 3 is a graph showing the relationship between the growth rate of uric acid crystals induced by serum and the blood uric acid value in the embodiment of the present invention; A is the relationship between HillSlope and uric acid concentration, B is EC 50 The relationship between the value and the uric acid concentration value.

[0062] Figure 4 : is a graph showing the growth rate of uric acid crystals induced by serum in the embodiment of the present invention and the condition of gout attack; wherein A is the absorbance value of crystal growth under different conditions, and B is the EC under different conditions. 50 value, C is the HillSlope value under different conditions, D is the absorbance value under different conditions at the end of the test, E is the ROC curve for distinguishing gout from health and hyperuricemia based on Hillslope, and F is the ROC curve based on EC 50 ROC curve for distinguishing gout from health and hyperuricemia based on the measurement standard.

[0063] Figure 5 The growth rate of serum-induced uric acid crystals varies among individuals in patients with metabolic diseases and other joint diseases except gout.

Claims

1. A device for assessing the risk of gout attack based on the growth rate of monosodium urate crystals, the device comprising the following computer modules: S1) a data reading module, S2) a data processing module and S3) a data output module; in: S1) a data reading module reads the changes in absorbance values ​​(Y) of the sample after the subject's serum sample is mixed with the alkaline uric acid solution at different time points (X); S2) The data processing module performs the following operations: S2-1) making a change curve according to the data received by the data reading module in S1); obtaining the maximum absorbance value of the curve, the minimum absorbance value of the curve, and the symmetry value S of the curve according to the curve; S2-2) Obtain the Hill Slope value and the corresponding time point EC at which the Hill Slope appears by means of the parameter fitting equation 50 value; S2-3) The HillSlope value and EC calculated by S2-2) 50 Value, to judge the risk of gout attack; S3) the data output module outputs the risk of gout attack according to the judgment result of S2-3); S1) In the data reading module, the alkaline uric acid solution is a sodium hydroxide solution of uric acid; The concentration of the sodium hydroxide solution is 0.5 mol / L-1.0 mol / L; the concentration of uric acid in the alkaline uric acid solution is 30 mg / mL-40 mg / mL; the volume ratio of the serum sample to the alkaline uric acid solution is 5-20:1; S2) In the data processing module, the formula of the parameter fitting equation is: Denominator=(1+(2^(1 / S)-1)*((EC 50 / X)^HillSlope))^S, Numerator = Top–Bottom, Y=Bottom+(Numerator / Denominator); in: Y is the absorbance value; X is the time point corresponding to the absorbance value; Top is the maximum absorbance value of the curve; Bottom is the minimum absorbance value of the curve; S is the symmetry degree of the curve, and its value is S∈[0,1]; HillSlope is the slope of the hill; EC 50 The value is the corresponding time point where the Hill slope appears.

2. The device according to claim 1, characterized in that S1) In the data reading module, the blood sample of the subject is a venous blood sample; and / or, S1) in the data reading module, the blood sample of the subject is a pre-treated blood sample; And / or, S1) in the data reading module, the volume ratio of the serum sample and the alkaline uric acid solution is 8-15:

1.

3. The device according to claim 2, characterized in that S1) In the data reading module, the pre-processing step is specifically: subjecting the blood sample to a coagulation-promoting treatment, centrifugation, and collection of serum.

4. The device according to claim 1 or 2, characterized in that The absorbance value (Y) changes at different time points (X) were read by an absorbance monitor.

5. The device according to claim 4, characterized in that The detection wavelength is 520nm-540nm; and / or, the detection temperature is 28°C-32°C; And / or, the absorbance value is detected every 1 min to 10 min; And / or, continuous detection for 1h-5h.

6. The device according to claim 1 or 2, characterized in that S2) In the data processing module, If the HillSlope value is <5.10 OD / min, the risk of gout attack is low; If 5.1OD / min≤HillSlope value≤7.15OD / min, the risk of gout attack is moderate; If the HillSlope value is greater than 7.15 OD / min, the risk of gout attack is high.

7. The device according to claim 1 or 2, characterized in that S2) In the data processing module, If EC 50 If the value is >80min, the risk of gout attack is low; If 63min≤EC 50 If the value is ≤80min, the risk of gout attack is medium; If EC 50 If the value is less than 63min, the risk of gout attack is high.

8. The device according to claim 1 or 2, characterized in that If HillSlope value, EC 50 If the prediction results of the two values ​​are inconsistent, the prediction result with higher risk shall prevail.

9. A device combination for assessing the risk of gout attack based on the growth rate of monosodium urate crystals, the device combination comprising the device according to any one of claims 1 to 8 and an absorbance monitor.

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